Acta Biomaterialia 126 (2021) 63–91

Contents lists available at ScienceDirect

Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actbio

Review article

Natural medicine delivery from biomedical devices to treat bone

disorders: A review
Susmita Bose∗, Naboneeta Sarkar, Dishary Banerjee
W. M. Keck Biomedical Materials Research Laboratory, School of Mechanical and Materials Engineering, Washington State University, Pullman, WA 99164,
United States

a r t i c l e i n f o a b s t r a c t

Article history: With an increasing life expectancy and aging population, orthopedic defects and bone graft surgeries are
Received 27 September 2020 increasing in global prevalence. Research to date has advanced the understanding of bone biology and
Revised 18 February 2021
defect repair mechanism, leading to a marked success in the development of synthetic bone substitutes.
Accepted 20 February 2021
Yet, the quest for functionalized bone grafts prompted the researchers to find a viable alternative that
Available online 28 February 2021
regulates cellular activity and supports bone regeneration and healing process without causing serious
Keywords: side-effects. Recently, researchers have introduced natural medicinal compounds (NMCs) in bone scaffold
Biomaterials that enables them to release at a desirable rate, maintains a sustained release allowing sufficient time for
Drug delivery tissue in-growth, and guides bone regeneration process with minimized risk of tissue toxicity. According
Natural medicinal compounds to World Health Organization (WHO), NMCs are gaining popularity in western countries for the last two
Vitamins decades and are being used by 80% of the population worldwide. Compared to synthetic drugs, NMCs
Bone disorders
have a broader range of safety window and thus suitable for prolonged localized delivery for bone regen-
Bone tissue engineering
eration. There is limited literature focusing on the integration of bone grafts and natural medicines that
provides detailed scientific evidences on NMCs, their toxic limits and particular application in bone tis-
sue engineering, which could guide the researchers to develop functionalized implants for various bone
disorders. This review will discuss the emerging trend of NMC delivery from bone grafts, including 3D-
printed structures and surface-modified implants, highlighting the significance and potential of NMCs for
bone health, guiding future paths toward the development of an ideal bone tissue engineering scaffold.

Statement of significance

To date, additive manufacturing technology provids us with many advanced patient specific or de-
fect specific bone constructs exhibiting three-dimensional, well-defined microstructure with intercon-
nected porous networks for defect-repair applications. However, an ideal scaffold should also be able
to supply biological signals that actively guide tissue regeneration while simultaneously preventing post-
implantation complications. Natural biomolecules are gaining popularity in tissue engineering since they
possess a safer, effective approach compared to synthetic drugs. The integration of bone scaffolds and
natural biomolecules exploits the advantages of customized, multi-functional bone implants to provide
localized delivery of biochemical signals in a controlled manner. This review presents an overview of
bone scaffolds as delivery systems for natural biomolecules, which may provide prominent advancement
in bone development and improve defect-healing caused by various musculoskeletal disorders.
© 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction highest contributor to global disability. Approximately 1 in 3 peo-

1.1. Current trends in bone tissue engineering scaffolds
With worldwide incidences of hip and knee osteoarthritis ex-
ceeding 300 million cases, musculoskeletal diseases became the
∗
Corresponding author.
E-mail address: sbose@wsu.edu (S. Bose).
ple across the world is living with chronic, painful musculoskele-
tal conditions [1]. Trauma, back pain, and arthritis are a few of
the most common reasons that necessitate prolonged hospital-
ization. Osteosarcoma, one of the most frequently occurring pri-
mary bone cancers, also accounts for about 4.3 and 3.4 per million
cases worldwide among young males and females (age group 0-
24 years), respectively [2]. The prevalence of bone disorders and
associated hospital visits surpass that of other major health con-

https://doi.org/10.1016/j.actbio.2021.02.034
1742-7061/© 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
S. Bose, N. Sarkar and D. Banerjee
ditions such as cancers, respiratory and circulatory diseases. The
“gold standard” for bone replacement is autologous grafts. Yet,
this strategy has limitations since it involves donor site morbid-
ity and invasive surgical procedures. The biocompatible allogenic
grafts are also accompanied by the risks of immunological reaction
and transmission of diseases including human immunodeficiency
virus (HIV) and hepatitis virus transferal. With increasing advances
in technology, the whole expanse of bone grafting has broadened
to include xenografts and synthetic materials. Engineered osseous
tissue is not only based on the principles governing cellular and
molecular biology, but also steered by the fundamentals of bio-
engineering and biomechanics.
The field of orthopedic biomaterials is dynamic, and the neces-
sity and properties of implants are governed by the site of implan-
tation and load-bearing expectations. While considering biomateri-
als for large segmental bone defects at load-bearing sites, metallic
implants such as titanium (Ti) and titanium alloys are mostly pre-
ferred due to their desirable mechanical properties, corrosion re-
sistance and good biocompatibility. Yet, over the years metallic im-
plants have been challenged by several drawbacks including poor
osseointegration, toxic metal-ion leaching, and stress-shielding —
all of which particularly hinder their long-term clinical success.
To lower the incidents of implant failure due to stress shielding,
porous metallic implants have been designed that could signifi-
cantly reduce the elastic modulus of the implants and provide ad-
equate mechanical signals for bone in-growth through the pores.
Besides, several strategies such as, surface modification of metal
with bioactive ceramics or addition of trace elements like zinc (Zn),
iron (Fe), magnesium (Mg), and silicon (Si) have been proposed to
stimulate osseointegration, better bone formation and promote os-
teoconductivity [3].
Ceramic biomaterials have been a popular choice for tissue
engineering scaffolds since they closely mimic the chemical and
structural composition of extracellular matrix of bone. Among
these are calcium phosphate ceramics, including hydroxyapatite
(HA) and tricalcium phosphate (TCP). Other than the compositional
similarities to the bone mineral, calcium phosphate ceramics also
confer a high degree of osteoconductivity and can have tailored
resorption rate based on clinical needs, such as craniomaxillofa-
cial and spinal fusion applications [4]. HA has a slower degra-
dation rate than TCP, often taking >1 year and thus a popular
choice for bioactive coating on metal implants. Additionally, by tai-
loring Ca:P (calcium-to-phosphorous ratio) or introducing designed
porous architecture, the degradation rate of the ceramic scaffolds
can be altered to suitably match the bone growth rate. Despite
their desirable properties, the clinical application of ceramic scaf-
folds is limited to low load bearing sites due to their inherent
brittleness.
Another promising material often used for bone tissue engi-
neering application are polymers, including both synthetic and nat-
urally sourced. Although natural polymers such as collagen, fibrin
or chitosan offer optimum cellular adhesion, they lack control over
properties and occasionally possess immunogenicity. Synthetic
polymers, such as FDA-approved polylactic acid (PLA), polyglycolic
acid (PGA) are biocompatible, biodegradable and also provide finer
control over chemical and physical properties. Yet, in most cases,
these properties are offset by their poor mechanical strength and
stiffness, limiting their clinical application in bone sites with a low
expectation of stress. Despite advances with modern manufactur-
ing technique, bone defect repair is often complicated due to the
various challenges posed by clinical scenarios such as osteoporosis,
osteomyelitis, and osteosarcoma. Various growth factors or small
molecules are utilized to guide bone regeneration in the context of
compromised wound healing abilities or extensive musculoskeletal
disorders.
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Acta Biomaterialia 126 (2021) 63–91
1.2. Challenges in current treatment approach for bone regeneration
Modern therapeutics take a direct approach to the treatments
of musculoskeletal problems. This may encompass a series of tests
and scans to identify the cause of the trouble, followed by diag-
nosis. Minor injuries are treated by administering pain-relieving
oral drugs and immobilizing the affected area using slings, casts,
or bone plates and screws. Further treatments include physiother-
apy for strengthening the surrounding muscles and speeding up
the healing process to regain normal activity of bone. Oral dosages
of the drugs get absorbed in the gastrointestinal tract of the hu-
man body and then into the bloodstream. Thus, due to the in-
direct nature of the oral drug absorption, patients are often pre-
scribed higher drug dosages, which lead to various side effects in-
cluding obesity, higher blood pressure, increased appetite, insom-
nia, cataracts, or glaucoma [6].
More complex and severe injuries necessitate orthopedic surg-
eries like total joint replacements followed by the administration
of steroids and painkillers to alleviate inflammation and relieve
pain. For severe cases of arthritis, the total joint replacements
(TJR) provide reduced pain and improved mobility. According to
the Swedish joint registry, 10-year survival rate of cementless total
hip arthroplasty is 85%, the rate decreases to 70% at 15 years [5].
Although TJR surgery is one of the most successful clinical pro-
cedures performed today, a number of possible complications can
result in early implant failure. Such complications, including asep-
tic loosening, secondary infections, and osteolysis are increasing in
prevalence. Early implant failure is especially inhibiting for younger
patients, requiring extensive revision surgeries.
Current engineering scaffolds used for bone replacements are
challenged by lack of osteoinductivity, gradual implant loosening,
risks associated with primary and secondary infections, and mis-
match in mechanical properties [7]. To overcome these drawbacks,
osteoinductive proteins are combined with osteoconductive carri-
ers to allow timed release of the biomolecules and enhance bone
formation. In some cases, bioactive surfaces of scaffold materials
help to accelerate osseous tissue integration and improve the life
of the implants. Cellular attachment and adhesions are directly
influenced by the topographical nature of the scaffolds [8]. Vari-
ous therapeutic molecules of interest have been immobilized on
the implant surfaces to improve the bioactivity of the scaffolds.
Orthopedic implants loaded with bone growth factors have been
demonstrated to enhance osteogenesis and improved cellular func-
tionalities. One of the most commonly used growth factors for or-
thopedics are bone morphogenic proteins (BMP), especially BMP-
7 and BMP-2, and transforming growth factor-beta (TGF-β ) [9].
These growth factors are covalently attached or adsorbed to mod-
ify the surface of the implant and directly impact the bone remod-
eling procedure improving osseointegration and new bone or os-
teoid formation. However, inherent limitations of growth factors
including short half-life, poor stability, burst release, and related
adverse effects such as ectopic bone formation hindered their clin-
ical success. To overcome these challenges and develop appropri-
ate delivery system, researchers have tried to optimize their dosage
and rate of release. Growth factor-based delivery systems have also
shown rapid osseous tissue regeneration; however, due to insuffi-
cient vascularization, even the short-term induced bone formation
does not guarantee life-long success of the implants [10].
All orthopedic scaffolds behave as foreign materials once im-
planted in the physiological system and hence, they are susceptible
to microbial infections. According to the American Joint Replace-
ment Registry 2017 annual report, prosthetic joint infection is the
highest leading cause for early-linked revisions (<3 months) for to-
tal knee arthroplasty (TKA) (43.9%) and the second most common
reason for total hip arthroplasty (THA) (21.5%) revisions [11]. PJI or
prosthetic joint infection can occur at any time after the surgery
S. Bose, N. Sarkar and D. Banerjee
and it is classified into early (< 3 months), delayed (3 months- 2
years), and late (> 2 years). Early infection is caused due to direct
perioperative inoculation during surgery or within following 2-4
days by highly virulent bacterium, delayed is caused due to peri-
operative inoculation of less virulent bacterium within 2-10 weeks,
and late is predominantly caused due to hematogenous remote in-
fections. Technological advances have ensured the sterility of the
implantation site and provide physical barriers like laminar airflow
chambers and closed bodysuits. Local resistance to infection has
been ensured by the impregnation of antibiotics into the implant
system [12]. Nevertheless, the adverse effects of the antibiotics on
the mechanical strength of the bone cement and the promotion of
resistant strains of bacteria are still considered as major concerns
[13]. Moreover, the impregnated antibiotics are typically released
over two weeks and the long-term microbial invasion limits the
potential of this strategy.
1.3. Natural medicinal compounds and bone implants: era of
integration
The use of natural medicines dates back to the beginning of
civilization. Until the 17th century, natural medicines contributed
to the development and discovery of several life-saving drugs that
became mainstays of human pharmacotherapy for healing various
clinical anomalies. The practice of natural medicine began to di-
minish with the discovery of synthetic drugs in the late 1800s,
which soon became the standard approach for disease treatment.
However, in the past few decades, adverse effects associated with
the long-term use of synthetic medicines led to a surge in public
interest and acceptance of herbal medicine. Currently, almost 80%
of the world’s population relying on them for treatment of various
health challenges. Natural medicinal compounds take a holistic ap-
proach, which tries to work with the bone’s natural healing sys-
tem. Reports since the beginning of the 1990s demonstrate the de-
mand for alternatives of modern medicine leading to the inclusion
of natural medicines in the confinement of synthetic medicine.
The rising popularity of natural medicines also led to the devel-
opment of the Office of Alternative Medicine by the National In-
stitutes of Health in 1992 [15]. The safe and effective practices of
natural medicine in tandem with the conservation of the indige-
nous principles pose to be another challenge. Knowledge about the
genesis of these natural medicines alongside clinical practices, risk
management, and research can be a useful to understand their po-
tential in tissue engineering. Clinical studies on 148 patients with
bone and soft tissue tumors for 15 years show the efficacy of this
integration tool. The combination of the subcutaneous implantable
delivery system with Chinese herbs led to the treatment of the tu-
mors with alleviated side effects of the gastrointestinal tract, heart,
kidneys, and urinary system, with a synergistic strengthening ef-
fect on the immune system [16]. Another study on 3D printed
calcium phosphate scaffolds, incorporated with Indian spice, cur-
cumin, showed improvement of new bone formation and blood
vessel formation, 12 weeks from implantation in a rat distal femur
model [17]. Fig. 1 presents the integration of the natural medicinal
compounds into the bone implants for prolonged delivery of drugs
at localized injury sites.
Therefore, possible solutions for musculoskeletal disorders may
involve functionalization of the scaffolds with a quintessen-
tial blend of osteogenic, angiogenic and antibacterial natural
biomolecules. Traditional medicine addresses a series of bone dis-
orders that have plagued the world over the years and are dis-
cussed in this review. The integration of modern synthetic bone
grafts with active natural medicinal compounds of optimal dosages
may bear the answer to the long-lasting side effects and encourage
improved quality of health, vitality, and longevity. Fig. 2 presents
an overview of various bone grafts and how they can be integrated
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Acta Biomaterialia 126 (2021) 63–91
with natural medicinal compounds to achieve localized delivery of
drugs to repair bone defects associated with musculoskeletal dis-
orders such as osteoporosis, osteosarcoma, osteomyelitis and os-
teoarthritis.
2. Overview of three most common bone disorders
2.1. Osteoporosis
Osteoporosis, characterized by low bone mineral density, and
micro-architectural deterioration of skeletal tissue, has become one
of the most common metabolic bone disorders [18]. One in three
women and one in twelve men are affected by osteoporosis, which
leads to an increased risk of fragility fracture [19]. Older men
and women are considered to be at more risk, as bone resorp-
tion tends to increase due to a low level of parathyroid hormone
and calcitonin. Certain lifestyles and diet also govern the possibil-
ity of developing osteoporosis; such as; excessive alcohol intake,
lack of physical exercise, cigarette smoking, high protein consump-
tion, diet with low green and leafy vegetables and fruits, and low
calcium and vitamin D intake [20]. Osteoporosis and related frac-
tures are estimated to affect over 8.9 million fractures annually,
worldwide and an annual cost reaching 32 billion in Europe and
19 billion in the US [21]. Osteoporosis is a “silent” disease as it
is difficult to diagnose at an early stage. The major clinical indica-
tion of osteoporosis is a fracture, which occurs at a late stage when
massive bone loss has already occurred.
In recent years, several anti-osteoporosis drugs have become
available for the treatment of osteoporosis and osteoporosis-
related fractures. However, over time, the safety and efficacy of
anti-osteoporosis drugs have become a major concern for the pa-
tients as well as medical advocates [22]. Bisphosphonates (Alen-
dronate, Risedronate, Ibandronate, and Zoledronic acid), are a class
of osteoporotic drugs, which are the most commonly prescribed to
osteoporotic patients [23]. They act by binding at bone remodeling
sites, inhibiting the osteoclastic resorption, and hence suppress-
ing bone resorption [24]. It was approved by the Food and Drug
Administration (FDA) and came to market in 1995 and instantly
became a money-making drug by bringing nearly $3.2 billion by
2005. This success was the reason behind the development of
several anti-osteoporosis drugs, with even more convenient doses
such as ibandronate, which can be taken orally once a month or
intravenously once every three months [25]. However, within a
year of its launch, various cases of adverse effects, including chest
pain, stomach, esophagus injuries, difficulty swallowing and heart-
burn, were reported. Within a short period, manufacturers had to
change the label of the alendronate to update on its safety and
side effects. However, growing evidence has linked bisphospho-
nates to a long list of serious and worrisome side effects, such as
chronic kidney disease, hypocalcemia, ocular complications, erosive
esophagitis, and ulceration leading to esophageal cancer, atrial fib-
rillation, osteonecrosis and incapacitating bone, joint, and muscle
pain [26]. Prolonged bisphosphonate therapy causes an oversup-
pression of bone remodeling due to its osteoclast-inhibiting ability,
which leads to lower bone turnover rate, microdamage in bone and
atypical femoral fracture. A study reported that 13.5% of women
with osteoporosis who received alendronate experienced a fracture
compared to 18% of those who took a placebo. For people suffer-
ing from bone loss due to long-term steroid use, bisphosphonate
improved the BMD of the lower spine by 4% but it does not re-
duce the number of spinal fractures. Some studies even showed
that long-term use would increase the risk of a certain type of
fracture [27].
The long-lasting side effects of the bisphosphonates coupled
with the low benefits to the osteoporotic fractures lead researchers
to find safer alternatives. Several over-the-counter medicines in-
S. Bose, N. Sarkar and D. Banerjee Acta Biomaterialia 126 (2021) 63–91

Fig. 1. Integration of natural medicinal compounds and bone grafts: Localized delivery of natural medicinal compounds from bone scaffolds can be a promising alternative
to conventional bone grafts that provides controlled delivery of biochemical cues to the target sites without inducing adverse effects on healthy bone. (A) Biological sources
of various natural medicinal compounds. (B) bone cement injection in vertebroplasty. (C) Evolution of dental implants over the years, from titanium implant with zirconia
top, one-piece zirconia implant to current two-piece ceramic implants. (D) synthetic 3D printed cranial implants, patient specific jaw reconstruction. (E) Cemented joint
prosthesis, that uses a quick-setting bone cement that bonds with patient’s natural bone. Cementless or press-fit implants can be coated with HA which enhance bone
growth onto the porous coating surface. (F) (i) The 3D pelvis was segmented by the thresholding process in the CAD software. The extent of tumor was outlined on each
axial CT image and its tumor volume was extracted (red in color). A 3D bone tumor model was created for the surgical planning. (ii) Surgeons performed the virtual
resections by defining the locations and orientations of the resection planes. (iii) The virtually resected tumor was extracted. As the bone was deformed by the tumor, the
mirrored 3D image from the normal side of the hemipelvis was used to duplicate the core shape of the implant. (iv) The flanges and the acetabular cup were added for
better implant stability. The final implant design had a normal acetabular contour and the components for implant fixation. Reprinted from [14] CC BY. (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of this article.)

cluding calcium and vitamin D supplements are being used to help
prevent fractures by reducing bone resorption and therefore de-
creasing the chance of fracture. Doctors suggest 20 0 0 international
units of vitamin D supplements and 1,200 mg of calcium for adult
osteoporotic patients.
2.2. Osteoarthritis
Osteoarthritis is the most prevalent degenerative joint disease,
associated with chronic pain, stiffness, joint locking, or joint dis-
ability [28]. Worldwide, 300 million people are estimated to be af-
fected by osteoarthritis [29]. Obesity, aging, heredity, and post joint
injury-related trauma are recognized to be the main etiologies for
osteoarthritis. The pathological symptoms include the destruction
of articular cartilage, hypertrophy of bone, formation of osteophyte,
and inflammation in the synovium [30].
Since obesity is one of the major risk factors of osteoarthri-
tis, treatment generally starts with nonpharmacologic therapy or
various weight loss exercises [31]. Pharmacological treatment for
mild osteoarthritis starts with acetaminophen since it is inexpen-
sive, effective, and has lesser gastrointestinal side effects than non-
steroidal anti-inflammatory drugs (NSAIDs) [32]. When the symp-
toms are moderate to severe, NSAIDs such as ibuprofen, naproxen,
diclofenac are prescribed. NSAIDs have severe side effects such as

66
S. Bose, N. Sarkar and D. Banerjee Acta Biomaterialia 126 (2021) 63–91

Fig. 2. Natural medicinal compounds for the treatment of various musculoskeletal disorders: Natural medicines with distinct biological activities can be optimized for
effective dosage concentration and desired release kinetics. Then it can be incorporated within tissue engineering grafts to repair bone defects caused by surgical intervention
due to various musculoskeletal disorders including osteoporosis, osteosarcoma, osteomyelitis, osteoarthritis, and rheumatoid arthritis. Localized delivery of natural medicines
in an optimized dosage might promote bone defect repair, lower the dosage frequency of systemic drug administration and minimize tissue toxicity caused by high dosages
of synthetic drugs.

high blood pressure, gastrointestinal bleeding, and renal dysfunc-
tion [33]. Cox-2 inhibitors such as celecoxib have a lower gas-
trointestinal side effect, however, are expensive and elevate the
risk of cardiovascular disease [34]. Low dosages of opioids are
only given to a patient when the acetaminophen or NSAID ther-
apy fails to exert any effectand patients are closely monitored due
to their potential for addiction and abuse [35]. Corticosteroids or
hyaluronic acid injections are the next options for treating os-
teoarthritis, which along with a local anesthetic, such as lidocaine,
provide an immediate and short relief that lasts for 1-2 months
[36]. Surgeries are the last resort for patients who do not re-
spond with non-pharmacological or pharmacological therapy. The
most common surgical treatment is the total joint replacement
of the hip, knee, and shoulder. Various implants and prosthetic
devices are available in the market and the national joint reg-
istry has estimated that these hip replacements have a survival
rate of 89.4% at 15 years, 70.2% at 20 years, and 57.9% at 25
years [37].

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S. Bose, N. Sarkar and D. Banerjee
2.3. Rheumatoid arthritis
Rheumatoid arthritis (RA) is a chronic autoimmune disease as-
sociated with inflammation in synovial joints, which ultimately re-
sults in damage and progressive disability [38]. It is more prevalent
among women below 50 years of age with a female to male ratio
of 3:1. Although RA is rare in men below 45, they start develop-
ing symptoms with increasing age. The clinical symptoms include
arthralgia, swelling, disability in motion, which also leads to pre-
mature deaths [39].
Current treatment of RA involves diagnosis, administration of
disease-modifying anti-rheumatic drugs (DMARD), NSAIDs, and
glucocorticoids as prescribed by the rheumatologist [40]. Early
diagnosis based on patient history, physical examination, blood
tests, and radiograph help in lower joint damage, better func-
tional stability, reduced radiologic progression, cost-effectiveness,
and DMARD-free remission. Delayed diagnosis and initiation of
DMARD therapy often lead to bony erosions and narrower joint
space, however, it relies on different healthcare systems in differ-
ent countries as well as on the patient and physician [41]. Oral cor-
ticosteroids and NSAIDs have their adverse effects, leaving doctors
hesitant about their prescriptions.
Abundant options for pharmacological treatment and little in-
formation on their safety and efficacy make RA treatment chal-
lenging. Till today, about 60-90% of patients suffering from chronic
pain and not satisfied with the conventional treatment opt for al-
ternative treatments for RA. Over the years, patients have resorted
to alternative measures including spa therapy, mineral baths, chi-
ropractic, and acupuncture, which have short term benefits for
osteoarthritic patients [42]. Glucosamine and chondroitin supple-
ments, and topical analgesic made from capsaicin, an active ingre-
dient of chili peppers, are also popularly used as adjunct therapies
to treat osteoarthritic pain [43]. This emerging interest in alterna-
tive medicines coupled with tremendous adverse effects associated
with synthetic medicines suggest the need for thorough research
towards the safety and efficacy of herbal medicines. Fig. 3 demon-
strates three chief parameters for a natural medicinal drug delivery
system that determines the overall clinical success of a bone graft.
This includes desired bioavailability of poorly absorbed hydropho-
Fig. 3. Bioavailability, controlled release kinetics and tissue materials interaction
are three important criteria for a clinically successful localized drug delivery sys-
tem. Poor bioavailability of naturally sourced biomolecules often creates a barrier
in their practical application. In such cases, various drug delivery carriers have been
employed to control the release kinetics and subsequently enhance the bioavailabil-
ity. A controlled release of biomolecules from bone grafts at a desired concentration
improves host tissue and implant materials interaction and provides biological func-
tionality to the scaffold for treating different musculoskeletal disorders.
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Acta Biomaterialia 126 (2021) 63–91
bic biomolecules, controlled release kinetics of biomolecules from
bone grafts to achieve desired concentration at localized site and
improved host-tissue implant materials interaction with minimized
cytotoxicity.
3. Natural medicinal compounds and nutraceuticals for bone
health
3.1. Acemannan from aloe vera
The history of aloe vera usage dates back to 1750 BC when
in the Mesopotamian civilization it had been mentioned for its
medicinal values. Aloe vera is a perennial plant characterized by
fleshy spear-shaped leaves with saw-like teeth (also called rind),
which is filled with a transparent, gelatinous, and semisolid gel.
The aloe vera gel contains around 99% of water and numerous
compounds with predominant medicinal values. Although geo-
graphic variations witness deviations in the constituents of aloe
vera gel, most of it is composed of aloin and aloe resin, contribut-
ing around 80 % of the dry weight of the gel. The pharmacology of
the variety of constituents has been surprisingly diverse. Ranging
from anti-microbial properties to usage as a laxative and wound
healing applications, the potential medicinal values of aloe vera
have been the subject of research for the past few years. [44].
Primary and secondary infections pose a crucial cause of pre-
mature in vivo bone-implant failures [45]. Much effort has been
invested in the fabrication of scaffolds with the tunable release of
anti-microbial drugs or with higher oral dosages of contemporary
antibiotic drugs like ciprofloxacin and ofloxacin [46]. In this regard,
the anti-microbial properties of aloe vera have also been well rec-
ognized. A variety of studies have compared the antimicrobial ef-
fects of this plant with silver sulfadiazone and have successfully
calculated the inhibitory and lethal dosage of the plant [47]. Ef-
fective properties of aloe vera have been found against microbes
like E. coli, S. faecalis, P. aeruginosa. Most prominently, aloe vera
has been shown to inhibit skin microbes better than silver sulfa-
diazone in agar diffusion plates. Recently, researchers have shifted
their attention to the saccharide concentration like galactose, ara-
binose, acetylated mannose (acemannan), and xylose in the aloe
vera. Amongst these, acemannan, a water-soluble mannan polymer
with an acetyl group, received a significant clinical prominence
as an anti-viral or immune potentiating agent for the treatment
of acquired immune deficiency syndrome (AIDS) [48]. A combina-
tion of scaffolds with tunable delivery of aloe vera may hold the
key to circumvent the concerns of antibiotic incorporation to pre-
vent microbial infection. Acemannan has shown vital effects on fe-
line leukemia and fibrosarcomas and is presently used as an ap-
proved drug in veterinary sciences [49,50]. In a 12-week clinical
trial study involving 44 cats, a dosage of 2 mg/kg of acemannan
injection has shown to be effective for the treatment of leukemia
[50]. Another study showed enhanced rat bone marrow stromal
cell proliferation, vascular endothelial growth factor (VEGF), and al-
kaline phosphatase (ALP) activity by the treatment of acemannan
along with higher bone mineral density and accelerated healing
[51,52], shown in Fig. 4. Acemannan enhances the activity of sev-
eral cytokines including interleukin (IL)6 and tumor necrosis fac-
tor (TNF)-ɑ, which are associated with osteoid formation and bone
mineralization. Aloe vera also helps in alleviating osteoclast activ-
ity by influencing the NF-κ B pathway, crucial for bone regenera-
tion in total joint replacement orthopedic surgeries [53]. The bone
formation and mineralization pathways influenced by acemannan
are summarized in Fig. 5. Efficacy of acemannan in osteoid forma-
tion and improved osseointegration at the interface of load-bearing
implants has also been demonstrated in a rat distal femur model
as early as 5 weeks, as shown in Fig. 6 [54].
S. Bose, N. Sarkar and D. Banerjee Acta Biomaterialia 126 (2021) 63–91

Fig. 4. Efficacy of acemannan in bone formation in vitro and in vivo. (A) Acemannan significantly enhanced periodontal ligament cell ALPase activity after 72 h of incubation
at concentrations of 2 and 4 mg/mL. [∗ denotes statistical difference with the untreated group; P < 0.05, n = 3] (B-C) Acemannan increased periodontal ligament cell mineral
deposition at 9 and 18 day. The 0.5, 1, 2 and 4 mg/mL acemannan-treated groups had significantly higher mineralization as shown by more intensely stained areas by
alizarin-red staining compared to the untreated group [∗ , # Compared with the untreated group at the 9th and 18th day of incubation, respectively, P < 0.05, n = 9] (D)
Enhanced bone mineral density (BMD) was also noted in a dog tooth model 4 weeks after extraction[∗ , # Significant difference compared to the untreated socket; p < 0.05,
n = 7] (E). Histopathology of the tooth socket demonstrates numerous thick and dense bone trabeculae bridges in the acemannan treated group compared to large unfilled
zones in the control group after Hematoxylin and Eosin (H&E) staining bar 200 μm (a1, b1) and 50 μm (a2, b2). Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced
with permission [51]. Reproduced with permission from Springer [52].

However, higher doses of aloe vera juice or latex had been ob-
served to interfere with a variety of different drugs [55], increasing
the potentials of cardiac glycoside and also the toxicity of antiar-
rhythmic drugs. The over usage of aloe vera juice also aggravates
the potassium loss caused by corticosteroids. If consumed in con-
junction with glyburide drugs for diabetes, aloe vera causes hypo-
glycemia.
3.2. Curcumin from turmeric
Curcumin, the chief active component of turmeric, is derived
from the rhizome of a perennial herb named Curcuma longa of Zin-
giberaceae family [56]. The origin of curcumin dates back nearly
40 0 0 years to India, where it was mainly used as a traditional
medicinal agent and culinary spice. Turmeric contains more than
100 components, among which the chief components are a volatile
oil known as tumerone, and three other principal curcuminoids.
Among them, curcumin (77%) is the most abundant, followed by
dimethoxycurcumin (17%) and bisdemethoxycurcumin (3%) [57].
Curcumin is widely researched among scientists because of its
diverse biological and therapeutic properties. A host of in vitro
and in vivo studies showed curcumin’s ability to possess anti-
inflammatory, anti-microbial, anti-arthritic, and anti-cancer prop-
erties [58].
Curcumin regulates various molecular targets including a tran-
scription factor, nuclear factor kappa beta (NF-κ B), which is also

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S. Bose, N. Sarkar and D. Banerjee Acta Biomaterialia 126 (2021) 63–91

Fig. 5. Possible mechanism of action of acemannan-mediated bone remodeling. Acemannan stimulates the production of cytokines IL-6 and TNF- α :, which allows pre-
osteoblasts to form osteoblast and express RANKL. Osteoblast-derived RANKL and M-CSF bind to RANK and c-FMS receptor on monocytes and differentiate them to mature
osteoclasts. [IL-6: Interleukin-6, TNF-α : tumor necrosis factor-alpha, RANK: receptor activator of nuclear factor kappa B, RANKL: receptor activator of nuclear factor kappa B
ligand, M-CSF: macrophage colony stimulating factor, c-FMS: Colony-stimulating Factor-1 Receptor.

Fig. 6. The efficacy of acemannan released from HA coated Ti implants on in vitro osteoblast cells and in vivo bone regeneration in rat distal femur model. (A) Aloe vera
gel and it’s chief active component, acemannan. (B) After 1 day of incubation, osteoblast cell density increased significantly in presence of acemannan compared to HA
coating. However, the cell viability decreased when acemannan concentration was more than 500 μg/ml of FBS enriched DMEM. (C) Controlled release of acemannan from
TCP matrix has been achieved in physiological pH for 14 days. (D) CT scan and SEM micrographs of HA-coated Ti64 implants shows the presence of acemannan improved
the osteoid formation, evident from the enhanced reddish orange color around the vicinity of the implant. SEM micrographs also showed no gaps at the interface of the
acemannan implants. The combined presence of chitosan with acemannan further mineralized the new osteoid formation around the implant, shown by the greenish blue
color, leading to improved early stage osseointegration. Osseous tissue had been observed to interlock with the implant in the SEM micrographs, leaving no visible gaps.
(E) Histomorphometric assay performed using the ImageJ software from the histology micrographs of Ti6Al4V implants. Presence of acemannan significantly improved the
osteoid formation. Reprinted with permission from [54]. Copyright (2019) American Chemical Society. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)

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S. Bose, N. Sarkar and D. Banerjee
Fig. 7. Mechanism of action of curcumin via NFκ B pathway shows curcumin blocks phosphorylation and degradation of NF–κ B inhibitor (Iκ B), which leads to formation of
inactive NF–κ B-Iκ B conjugate. Suppression of NF-κ B positively influence osteoblast differentiation and prevents osteoclastic bone resorption. Adapted with permission from
[58]. Copyright (2019) American Chemical Society.
found to be associated with the bone remodeling process, as
shown in Fig. 7 [59]. Curcumin inhibits phosphorylation and degra-
dation of NF–κ B inhibitor (Iκ B) and thereby prevents the forma-
tion of inactive NF–κ B-Iκ B conjugate [60]. Downregulation of NF-
κ B pathway positively impacts bone cell differentiation and pre-
vents osteoclastic resorption. Besides, curcumin suppresses in vitro
and in vivo cytokine TNF-α expression, which is observed in ele-
vated levels among postmenopausal women and is considered to
be correlated with osteoporosis [61].
Oral administration of curcumin has been shown to re-
duce osteoclast-induced bone matrix degradation in the insulin-
dependent diabetic rat model. A study showed incubation with
50 μM curcumin for 2 hours prevented RANKL-activated NF-κ B
expression in a murine monocyte RAW 264.7 cells. It prevented
RANKL- and TNF-induced osteoclastogenesis and pit formation by
inhibiting Iκβ phosphorylation and degradation. It was further
demonstrated that 10 μM of curcumin for a day was able to reduce
the resorption pits by 80% compared to control [62]. Chen et al.
demonstrated that curcumin inhibits dexamethasone-induced os-
teoporosis in rats in a dose-dependent manner as shown in Fig. 8
[63]. Hie et al. demonstrated oral administration (120 mg/d) of
curcumin for 14 days has osteoclast-inhibitory effects in insulin-
dependent diabetes mellitus rats [64]. It regulated and maintained
the mRNA levels of cathepsin K, and TRAP, a higher level of which
are associated with bone matrix degradation. It also helped to de-
crease the urinary deoxypyridinoline level, which forms after col-
lagen degradation. Furthermore, the rats supplemented with cur-
cumin demonstrated decreased osteoclast levels compared to the
normal. However, curcumin did not show any effect on ALP ex-
pression and osteocalcin level in diabetic rats.
Curcumin has anti-inflammatory activity and thus alleviates the
inflammatory markers IL-6, IL-8, 5-lipoxygenase, and COX-2 ex-
pression [65]. It selectively inhibits COX-2 instead of COX-1 and
therefore protects against gastrointestinal damages caused by non-
steroidal anti-inflammatory drugs. Additionally, curcumin reduces
the formation of reactive oxygen species (ROS), which degrades
cartilage [66]. More clinical studies are needed to confirm cur-
cumin’s ability to prevent or suppress osteoarthritis.
The cellular uptake of curcumin is higher in malignant cells
compared to the healthy tissues, which suggests the selective cyto-
toxicity of curcumin towards tumor cells rather than normal cells.
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Acta Biomaterialia 126 (2021) 63–91
A study reported 10 μM of curcumin prevents MG-63 osteosarcoma
cell viability by 50%, however they enhanced healthy osteoblast
cell viability to 80% [67]. Moran et al reported that curcumin pre-
vents MG-63 osteosarcoma growth or mineralization because of
its ability to inhibit NO synthase expression and NO production,
which is a regulatory parameter for osteosarcoma viability [68].
However, a low level of NO is necessary for osteoblast growth and
bone metabolism, a high amount of it induces cytotoxicity in os-
teoblast cells.
Though some studies reported curcumin’s ability to inhibit the
mineralization of osteoblast, it neither induced apoptosis nor de-
creased the calcium content in the distal femur model in diabetic
rats. These results imply that curcumin reduced osteoclast activity
without causing cytotoxicity towards healthy osteoblast. A study
investigated the effect of oral administration of curcumin on post-
menopausal osteoporosis model induced by ovariectomized female
rats [69]. Curcumin decreased the serum estradiol levels and in-
creased cancellous bone formation in normal rats. A study from
our group developed porous 3D printed TCP scaffolds loaded with
curcumin to improve the osteogenic potential of bone graft scaf-
folds. Results in Fig. 9 showed improved in vitro osteoblast cell vi-
ability and proliferation as well as increased in vivo mineralized
bone formation in the presence of curcumin compared to control
[70].
There has been no incidence of adverse effects or toxicity for
turmeric even at high doses. Curcumin has been administered at a
dosage as high as 8g/d and still did not show toxic effects on hu-
mans. FDA has asserted curcumin to be considered as GRAS (Gen-
erally Recognized as Safe) [71].
3.3. Allicin from garlic
The history of garlic use for the treatment of a variety of ail-
ments predates the written history. The Sanskrit documents men-
tion the usage of garlic for medicine in India for more than 50 0 0
years ago, whilst the Chinese have been using it for at least 30 0 0
years [72].
Garlic (Allium sativum L.), a common spice used for cooking,
has been receiving attention from researchers due to its multi-
faceted pharmacological activities including anti-osteoporotic prop-
erties. It is a perennial plant that belongs to the lily family and is
S. Bose, N. Sarkar and D. Banerjee Acta Biomaterialia 126 (2021) 63–91

Fig. 8. (A) Effects of curcumin on the bone metabolism of rats with dexamethasone (DXM)-induced osteoporosis. Rats with DXM-induced osteoporosis exhibited significantly
decreased osteocalcin levels and increased collagen-type I fragments (CTX) serum levels. The administration of curcumin reversed these changes [##P<0.01 compared to the
control group, ∗ ∗ P<0.01 compared to the DXM group]. (B) Effects of curcumin on the Wnt signaling pathway in primary osteoblasts. (top) The mRNA expression levels of
Wnt, β -catenin, low-density lipoprotein receptor-related protein 5 (LRP5), sclerostin (SOST) and Dickkopf-1 in DXM-stimulated primary osteoblasts [##P<0.01 compared
to the control group, ∗ ∗ P<0.01 compared to the DXM group]. (middle) The phosphorylation of glycogen synthase kinase-3β (GSK-3β ) in dexamethasone (DXM)-stimulated
primary osteoblasts. β -catenin trafficking in DXM-stimulated primary osteoblasts. (bottom) DXM significantly inhibited the Wnt signaling pathway, and curcumin prevented
the inhibition (Scale bar, 50 μm). (C) Histological study using H&E staining in rat femur showing effects of curcumin in dexamethasone (DXM)-induced osteoporosis. DXM
caused obvious damage to the femurs of rats and curcumin attenuated the damage (Scale bar, 100 μm). Reprinted from [63] CC BY.

cultivated worldwide. There are few studies that reported the ben-
eficial effect of garlic on bone health. The main active ingredient
of garlic is a sulfur compound named alliin, which is hydrolyzed
by alliinase enzyme, forming allicin. Allicin produces various active
compounds such as allyl methyl trisulphide, diallyl disulfide, and
ajoene, which stimulate bone metabolism. Garlic also contains sev-
eral monoterpenes such as citral, geraniol, and linalool as well as
flavonoids such as quercetin and rutin [73].
The antimicrobial effects of garlic have been well documented
in literature proving the historic nature of its usage. Studies by
Huddleston et al. in 1944 demonstrated the efficacy of garlic
against the growth of bacteria including Staphylococcus, and Strep-
tococcus even at low concentrations [74]. The antimicrobial ef-
fects of garlic have been compared with commercially used an-
tibiotics like streptomycin, penicillin, and tetracycline and demon-
strated successful usage of garlic in treating conditions resistant to
antibacterial drugs [74], making it an ideal candidate to be im-
mobilized with engineered scaffolds to prevent primary and sec-
ondary infection. Garlic administration has also demonstrated the
reduction of anaerobes and coliforms [75]. A clinical study pre-
sented the antibacterial treatment of Helicobacter pylori, by ame-
liorating the average antibody titer and hence a slowdown in the
progression of peptic ulcers [76]. Antifungal and antiviral activities
have also been demonstrated by the administration of garlic in a
variety of in vitro, in vivo and clinical studies [77]. Although allicin
is the most effective ingredient for the antifungal activity of gar-
lic, even aqueous extracts of garlic in a hundred times dilution has
been observed to be effective against fungal skin infections includ-
ing capitis in vivo. Antiviral effects of garlic against influenza and
the common cold virus have also been extensively studied in vivo,
in vitro, and also in clinical studies [78].
Garlic has been reported to be used as one of the earliest
“performance enhancement” agents to Olympic athletes [79]. A
study reported that garlic oil recovered the occurrence of low
bone density in ovariectomized rats. Significant changes in pri-
mary and secondary markers, such as serum ALP, serum tartrate-
resistant acid phosphatase (TRAP) activity, urinary excretion of cal-
cium, phosphate, hydroxyproline and urinary calcium to creatinine
ratio were reported [80]. Another group in a study suggested gar-
lic has estrogen-like properties and therefore drives away osteo-
progenitor cells from adipocyte to osteoblast differentiation [81].
Osteoclast release serum tartrate-resistant acid phosphatase (TRAP)
and produce free radicals, which in turn produce free radicals that
initiate bone resorption [82]. It is reported that garlic reduces the
level of TRAP and thereby slows down the process of bone resorp-
tion. Another study from the same group showed garlic oil supple-
mented diet suppressed the effect of ovariectomy in rats such as
low BMD, and low tensile strength [83]. Garlic oil has also been
shown to have enhanced recovery percentage of the urinary tract,
bone density, serum content compared to lovastatin, and 17 β -
estradiol in an ovariectomized model of rat [84].
Alongside, remarkable enhancement of immune responses like
increment of pathogen attacking activity of T cells, neutrophils and
macrophages have also been observed in animal models from fresh
garlic or any commercial products containing allicin [85]. Hip-
pocrates prescribed garlic as a treatment of cancer in 400 BC, and
the effects of garlic on cancer could be exhibited by the improve-
ment of the immune system [86].
No toxic dosage of garlic has been identified for most individu-
als, but the difficulty in effectively detoxify allicin and other sulfur-
containing compounds from human bodies lead to heartburn or ir-
ritation in the digestive tract [87]. Garlic interferes with platelet

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S. Bose, N. Sarkar and D. Banerjee Acta Biomaterialia 126 (2021) 63–91

Fig. 9. Effect of curcumin released from calcium phosphate matrix for osteoblast in vitro proliferation and in vivo bone regeneration (a) Osteoblast (hFOB) cell viability
of after day 3,7 and 11 days of culture by MTT assay (n=9). Curcumin with PCL-PEG showing statistically significant difference compared to the untreated TCP control.
[∗ ∗ ∗ denotes P value < 0.0 0 01 compared to control, “ns” denotes not statistically significant compared to control]. (b) SEM images for osteoblast cell culture of control,
control+curcumin+PCL/PEG samples after 7 days of culture. Regardless of the time point, polymeric curcumin coated samples exhibiting much higher cell proliferation
compared to samples without polymer (c) Optical microscopy images of tissue-implant sections after von Willebrand Factor (vWF) and modified Masson Goldner trichrome
staining showing angiogenesis and osteoid like new bone formation respectively after 6 weeks of surgery in rat distal femur model (d) Histomorphometric analysis using
ImageJ showing enhanced osteoid like new bone formation in curcumin coated TCP scaffolds compared to control TCP scaffolds after 6 weeks of surgery. [∗ ∗ ∗ denote P values
< 0.0 0 01 compared to control]. Reprinted from [70] with permission from Elsevier.

aggregation [88] and hence should not be used with anticoagulant
drug administrations.
3.4. Gingerol from ginger
Ginger, the rhizome of an erect perennial herb, is indigenous
to the South Asian peninsula but is now cultivated widely all
over the tropics. Chinese records confirm the usage of ginger for
anti-inflammatory purposes since early fourth century BC, whereas
Ayurveda documents its use in musculoskeletal diseases more than
2500 years ago [89].
Similar to the other medicinal plants, ginger is made up of
more than 100 different compounds [90]. Volatile oils extracted
from the ginger constitute sesquiterpenes such as zingiberol, zin-
giberene, and other phenolic compounds such as gingerol, shogaol,
and a small quantity of vitamin A. These have been identified
to be the pharmacologically active component of the root. More-
over, around 4.5mgs of salicylate has been identified in 100gms
of the fresh ginger root [91]. Ginger has been shown to inhibit
cyclooxygenase and lipoxygenase activity, and in turn, prevented
leukotriene synthesis and carrageenan-induced hind paw edema in
an in vivo anti-inflammatory study in rats [92,93]. Double-blinded,
placebo-controlled studies involving 261 patients with knee OA
showed 63% of patients who were administered ginger extracts
found relief compared to the 50% under placebo within 6 weeks.
The patients who consumed ginger extracts showed improvement
in OA condition in terms of walking, standing, and less frequent
dependence on pain medications. The same study, however, re-
ported mild gastrointestinal adverse effects (59:21 patients) after
intaking ginger extract compared to the group receiving placebo
[94]. A double-blind crossover clinical trial showed that a thera-
peutic dosage of 170mg of ginger three times daily showed sta-
tistically significant efficacy over placebo in the initial treatment
period, however, a significant difference was not observed by the
end of the study [95]. The efficacy of bone protection effects of
crude ginger extract, normalized to 26 mg/kg/day containing gin-
gerols, polar compounds, and essential oils, was explored in an
ovariectomy-induced bone loss rat experimental model. It was ob-
served that the crude extract of ginger was more efficient to pre-
serve bone mineral density, suggesting its osteoclast-suppression
activity along with its anti-inflammatory effects by influencing the
NF-κ B pathway [96]. Another study demonstrated the potency of
gingerol extract on the tumor necrosis factor (TNF-ɑ)-suppressed
osteoblast differentiation, as demonstrated in Fig. 10 [97].
In a clinical study, 7 patients receiving temporary relief from
commercial pain relief drugs were suggested to intake 0.1 to 1
gram of powdered ginger as daily food consumption. Even with
the difference in dosage, substantial improvement in joint move-
ments and pain relief was reported by all patients [98]. A follow-up
study to this experiment with twenty-eight patients with rheuma-
toid arthritis revealed significant improvement in pain and joint
mobility after administration of 0.5 to 4 grams of powdered gin-

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S. Bose, N. Sarkar and D. Banerjee
Fig. 10. The potency of gingerol extract (0-50 mM) on collagen production and ALP mRNA expression with and without stimulation of tumor necrosis factor (TNF)-α. Data
are reported as mean±SE for 3 independent experiments. #P,0.05 compared to 0 mM; ∗ P,0.05 compared to control (Con) (Student’s t-test). Results suggest that gingerol
enhances TNF-α suppressed osteoblast like cell activity and differentiation, revealing beneficial effects of gingerol on treatment of bone disorders. Reprinted from [97] CC BY.
ger per day for a span of 3 months to 2.5 years [99]. Fresh ginger
contains more amounts of gingerols, which are effective against
inflammation and hence presumed to be more potent than dried
and powdered ones [100]. Anticancer activities of ginger have been
demonstrated by the inhibition of tumor formation in carcinogenic
experimental models [101].
The anti-emetic efficacy of ginger in alleviating chemotherapy-
induced nausea and vomiting has been demonstrated in osteosar-
coma patients receiving high dosages of anticancer drugs. In 60
chemotherapy cycles of alternate doxorubicin and cisplatin, bone
cancer patients were randomly administered with ginger extracts
or placebo tablets in addition to commercial drugs like dexam-
ethasone and ondansetron. 56% of patients receiving ginger extract
experienced moderate to severe nausea compared to 94 % of the
control group [102].
Ginger has been demonstrated to not possess any toxic ef-
fects when administered in optimum dosages. Acute toxicity ex-
periments carried in mice models revealed no mortality or cyto-
toxicity up to 7 days at a dosage of 2.5 gm per kg, where an
increment of the mortality rate to 10 and 30 percent was ob-
served on dosage from 3 to 3.5 gms per kg respectively [103].
Most clinical studies that exhibit the efficacy of ginger on pain
relief and bone formation uses around 1 gm of powdered gin-
ger, which is relatively lower than the daily consumption of gin-
ger spice in many South Asian countries. Hence, around 2 to 4
gms of dry powdered ginger are considered safe for daily con-
sumption and are believed to be effective against osteoporosis and
osteoarthritis [104].
3.5. Vitamin B6, B9 (folic acid) and vitamin B12
B vitamins are comprised of eight water-soluble vitamins that
play an essential part in the growth, development, and other cel-
lular functions in our body, primarily acting as coenzymes for var-
ious physiological reactions. Among them, vitamin B6 (pyridox-
ine), vitamin B9 (Folate or folic acid) and vitamin B12 (cobal-
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Acta Biomaterialia 126 (2021) 63–91
amine) have established their prominent roles in maintaining bone
health. These three B vitamins are involved in the metabolism of a
sulfur-containing amino acid, homocysteine, a key product identi-
fied for accelerated bone loss and increased fracture risks in post-
menopausal women [105]. Homocysteine is a breakdown product
of methionine, one of the essential amino acids used for protein
synthesis. Increased levels of homocysteine have the potential to
induce osteoporosis if it is retained in the body for a long pe-
riod. A prospective study involving women with higher levels of
homocysteine revealed twice as much as the risk of non-vertebral
fractures compared to women with lower levels of homocysteine
[106]. Since no association of enhanced homocysteine level and
bone mineral density at the femoral heads, stems, or spine was ob-
served, the non-vertebral fracture risk was attributed to the poor
bone quality. Fig. 11 demonstrates the mechanism of action of folic
acid, vitamin B6 and B12 in the metabolism of homocysteine, lead-
ing to lower risks of fracture and improved bone quality. Folic
acid induces the remethylation of homocysteine to methionine and
hence, substantially reduces the levels of homocysteine [107]. Vi-
tamin B12 has also been associated with a reduction of homocys-
teine levels. A double-blinded study involving stroke patients with
increased levels of homocysteine, a dosage of 5 mg of folic acid
augmented with 1500 micrograms of vitamin B12 was shown to
reduce incidences of hip fractures by more than 75% compared to
the placebo control groups [108]. In another study involving 161
menopausal women, low levels of folate, and not homocysteine
or vitamin B12 were linked to osteoporosis and low bone min-
eral density [109]. Yet another clinical study involving 652 women
and 615 men to demonstrate the effects of vitamin B12 on homo-
cysteine levels, showed that high homocysteine with low vitamin
B12 levels was associated with low broadband ultrasound attenu-
ation, expressions of high bone turnover markers, and eventually
higher risks of fractures [110]. Pernicious anemia has also been ex-
plored as one of the factors leading to osteoporotic fractures [111].
Studies on the effects of vitamin B12 on ALP expresssed by human
bone marrow progenitor cells attribute to the suppression of os-
S. Bose, N. Sarkar and D. Banerjee
Fig. 11. Mechanism of action of vitamin B6, vitamin B9 (Folic Acid) and vitamin B12 in reducing osteoclast activity. Vitamin B6, B9 and B12 directly influence homocysteine
metabolism. Upregulation of homocysteine can be correlated to the reactive oxygen species (ROS) formation and NF-κB activation, and subsequent osteoclastic activity.
Adapted from [107] with permission from Elsevier.
teoblast differentiation, leadingto the risk of osteoporotic fractures
[112]. Vitamin B6 influences homocysteine metabolism by convert-
ing it to cystathionine and further to cysteine. Reports demon-
strate that vitamin B6 supplementation in patients with genetic
disorders like homocystinuria helps to reverse the elevated lev-
els of homocysteine [113]. In vivo animal model studies present
the effectiveness of vitamin B6 towards faster healing time [114].
Low vitamin B6 level has emerged as a common ailment, even
amongst healthy individuals. Diminished levels of vitamin B6 also
indicate impaired cartilage growth, defective bone regeneration,
lower bone density eventually causing osteoporosis in animal mod-
els [115]. Progesterone secretion is also influenced by the role of
vitamin B6.
3.6. EGCG from green tea
Epigallocatechin gallate (EGCG) is the most abundant catechin
found in green tea, one of the most popular beverages consumed
all over the world. Green tea is produced from the leaves and buds
of an evergreen shrub (Camellia sinensis), which was originated in
China thousands of years ago. Both black and green tea are de-
rived from the same plant but vary in their production method.
Steaming of freshly cut tea leaves leads to the production of green
tea, a rich source of polyphenols, fluoride, and vitamin K with
the potential anticancer and antioxidant properties. Various exper-
imental and population-based studies have reported the efficacy of
green tea in bone health and osteoporosis prevention where it has
been demonstrated to inhibit osteoclastic bone resorption, particu-
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Acta Biomaterialia 126 (2021) 63–91
larly in the prevention of age-related bone loss and stimulate os-
teoblast activity [116–118]. Numerous mechanisms of action have
been proposed to explain the biological activity of green tea in-
cluding its antioxidant, anti-inflammatory, anti-osteoclast activity
[119]. A clinical study with 171 post-menopausal women adminis-
tered with green tea polyphenols showed substantial improvement
in bone turnover markers within as early as 30 days [120]. Stimula-
tory effects of EGCG on enhanced osteogenic properties of murine
mesenchymal stem cells have also been studied [121]. A minimum
of 250 mg of green tea, with higher contents of epigallocatechin-
3-gallate polyphenols per day, is recommended for the bene-
fits of bone health, with no reported toxicity or adverse side
effects.
3.7. Rutin from onion and leek
Onion and leek are also reported to have a beneficial effect on
bone [122]. Rutin, the active compound is a glycosylated product
of quercetin, which is abundantly found in onion [123]. A report
says consumption of onion at a dose of 1g/day reduces in vitro os-
teoclastic bone resorption and in vivo bone loss in an osteoporotic
rat model [122]. Another study reported increased femoral strength
and trabecular bone density along with decreased bone resorption
after the consumption of rutin [124]. It also increases plasma os-
teocalcin concentration in rats, implying increased bone formation
[125]. However, later studies reported that leeks and wild garlic,
which contain a low amount of rutin, also hinder bone resorption.
Therefore, it is reported that not rutin but γ -L-glutamyl-trans-S-
S. Bose, N. Sarkar and D. Banerjee
1-propenyl-L-cysteine-sulfoxide is the active ingredient in onion,
which shows its effect in bone resorption [126]. However, another
researcher reported the inhibitory effects of quercetin and rutin on
osteoclastogenesis [127]. The study shows decreased in vitro bone
resorption due to reduced osteoclast formation and viability. This
result attributes to the fact that rutin inhibits RANK protein on os-
teoclast progenitor cells as well as induces apoptosis in mature os-
teoclast by activating caspase 3 and caspase 8.
3.8. Soy proteins and isoflavones from soy
Soybeans, often referred to as “miracle crops”, are being con-
sumed in Asian countries for centuries due to their high-quality
protein content. The history of soy dates back as early as 5,0 0 0
years ago when farmers used to grow it on the windy planes of
China. Cultivation and consumption of soy spread across the globe
and finally reached America before World War II, where a soy meal
gained its popularity as a low-cost, high protein food [128]. Soy-
bean contains 39.6% high-quality proteins, which are comparable
to those sourced from animals. Therefore, soyfood soon became
the low-cost, vegan substitute for milk and animal proteins. Peo-
ple with lactose intolerance can also consume soymilk as it is the
most nutritious alternative for cow’s milk.
Soy and foods containing soy, being a rich source of phytoe-
strogens considered to be beneficial to bone health. A study found
that Southeast Asian women show comparatively higher BMD and
reduced bone turnover compared to the other region of the world
due to their consumption of a higher phytoestrogen-rich diet than
the rest of the world [129]. The isoflavones are chemically simi-
lar to mammalian estrogen and therefore have the ability to attach
to the estrogen receptor resulting in ER-β -mediated inhibition of
cell growth and proliferation. It also inhibits the NFκ B pathway,
which attributes its anti-tumor efficacy against non-hormonal can-
cer. Besides, soy is associated with its beneficial effects on bone
health and inhibits fractures related to osteoporosis by downreg-
ulating markers of osteoclastic bone resorption and increasing os-
teoblast differentiation markers like ALP, a mechanism shown in
Fig. 12 [130,131]. A recent paper from our group reported that
genistein, daidzein and glycitein, three chief isoflavones can be in-
tegrated within the 3D printed bone scaffold matrix with designed
porosity to achieve multimodal biological activity [131]. Controlled
release of drugs from the multifunctional scaffolds enhanced os-
teoblast proliferation, differentiation, however, prevented osteosar-
coma growth and attachment. Besides, the scaffold modulated neu-
trophil recruitment 24 hours after surgical implantation in a rat
distal demur model, suggesting its potential anti-inflammatory ac-
tivity which may promote early wound healing and subsequent
bone regeneration as shown in Fig. 13.
Reduced estrogen levels in postmenopausal women signifi-
cantly increase the osteoporosis-related fracture risk including ver-
tebral and hip fractures. Estrogen therapy remained the only FDA-
approved treatment for preventing osteoporosis-related fractures
among postmenopausal women. Hormonal therapy is effective to
prevent one-third of the fracture risk, however at the expense
of many side effects. Therefore, the consumption of soy foods
among postmenopausal women gained immense interest due to
the earlier research confirming the estrogen-mimicking effects of
isoflavones [132].
In vivo results also confirmed the evidence of improved bone
mineral density with the consumption of soy. Soy-rich diet re-
sults in low occurrences of osteoporosis and related bone fractures
in Japan [133]. A randomized, double-blinded, placebo-controlled
study reported that a dose of 54 mg of genistein was effective
in the treatment of bone loss in the spine and femoral neck
among postmenopausal women [134]. In another clinical trial with
a longer period, 62 post-menopausal women were given soy-based
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diets containing 60 mg of isoflavones along with 25 g of proteins
for one year. Results showed an increased presence of bone for-
mation markers in blood and urine compared to the control sub-
jects [135]. Another study showed a daily intake of 35g soya for
12 weeks in 15 perimenopausal women, which resulted in a re-
duced level of urinary deoxypyridinoline and an increased level of
ALP [136]. Deoxypyridinoline is the degradation product of mature
collagen and therefore indicates bone resorption, whereas ALP de-
notes osteoblastic activity and is considered as a bone formation
marker. Another study investigated the effect of isoflavones, ad-
ministered with soy protein, casein on the skeleton of orchidec-
tomized rats [137]. After a dose of isoflavones, the weight loss in
rats after orchidectomy was much lesser. However, there are few
studies, which revealed that the presence of calcium is necessary
to exhibit any pharmacological effect of isoflavones. Various short-
term and long-term studies on postmenopausal Asian women re-
ported the beneficial effect of the consumption of soy food on re-
duced fracture risks and improved bone turnover and bone mineral
density [138,139]. Increasing the dose of isoflavones did not show
a pronounced increase in bone calcium content compared to the
lower dose, which suggests a balanced dose of 50-100 mg/day of
isoflavone can be effective to promote bone health.
3.9. Vitamin K
Since its discovery in 1929, Vitamin K has received widespread
attention due to its involvement in the normal blood coagulation
process. It consists of two vitamers, vitamin K1, which is abun-
dantly found in green leafy vegetables such as broccoli, cabbage,
spinach, lettuce, etc.; and vitamin K2, which is synthesized by in-
testinal bacteria and can also be derived from dairy products such
as cheese [140].
In the last couple of decades, there has been an emerging
interest in vitamin K due to its potential benefit towards bone
metabolism. Vitamin K is essential for the conversion of glutamate
to gamma carboxyglutamate (Gla protein), 80% of which is present
in osteocalcin, a bone protein synthesized in bone-forming cells.
Osteocalcin has three gamma carboxyglutamate residues that at-
tract Ca ions and incorporate them into the hydroxyapatite crys-
tals. During this process, a small fraction of carboxylated osteocal-
cin can be found in blood circulation, which indicates bone for-
mation, and therefore osteocalcin is considered an important bone
turnover marker. Vitamin K deficiency causes an increased level of
undercarboxylated osteocalcin (ucOC), a protein devoid of biologi-
cal activity, and is observed in osteoporotic patients [140]. A study
from our group showed that vitamin K2 increased the proliferation
and viability of osteoblast cells compared to control while it in-
hibited attachment, proliferation, and viability of the osteosarcoma
cells (MG-63). Vitamin K2 showed synergistic chemopreventive ef-
fects when administered with curcumin. The in vivo study indi-
cated that vitamin K2 increases and further enhance osteointegra-
tion in rat distal femur model when incorporated with curcumin
[141]. The results and underlying mechanism of action of vitamin
K are illustrated in Fig. 14.
Increased calcium retention has been found in ovariectomized
rats supplemented with vitamin K and humans on a vitamin K-rich
diet, suggesting vitamin K positively influences calcium balance, a
mineral involved in bone metabolism [142]. Few in vitro and in
vivo data show vitamin K positively influences bone metabolism
in the presence of vitamin D [143]. Reports have also confirmed
a reduced level of circulating serum vitamin K1 in osteoporotic
patients, compared to control [144]. A couple of studies showed
low BMD and increased chance of hip fractures are related to a
higher level of ucOC, which is an indication of low vitamin K in-
take [145,146]. Studies reported dietary intake of vitamin K-rich
food items such as lettuce or natto is linked to reduced hip frac-
S. Bose, N. Sarkar and D. Banerjee Acta Biomaterialia 126 (2021) 63–91

Fig. 12. The graphical presentation shows the dietary sources of soy isoflavones, percentage constituents of isoflavones found in soy foods and similarity of isoflavones
to mammalian estrogen. The chemical structure of primary three isoflavones, genistein, daidzein and glycitein are also presented. (A) The acid dissociation constant or pKa
values for genistein, daidzein and glycitein are 6.51, 6.48 and 6.92, respectively, indicating that these isoflavones are easily deprotonated at physiological pH of 7.4, resulting in
overall higher resonance stability and solubility in physiological pH (B) The possible mechanism of action of soy isoflavones towards chemoprevention and anti-inflammatory
activity is demonstrated through NFκ B inhibition pathway. (C) Estrogen-mimicking activity of soy isoflavones result in its ability to bind to the estrogen receptor and
subsequent ER-β -mediated inhibition of cell growth and proliferation. (D) Isoflavones also suppress osteoclastogenesis through the upregulation of OPG expression, which in
turn inhibits RANK-RANKL interactions. Reprinted from [131] with permission from Elsevier.

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S. Bose, N. Sarkar and D. Banerjee Acta Biomaterialia 126 (2021) 63–91

Fig. 13. (A) FESEM images showing the effects of soy isoflavones loaded 3DP tri calcium phosphate (TCP) bone tissue engineering scaffolds for in vitro osteoblast cell
proliferation in a flow perfusion bioreactor at day 5. Combination of soy isoflavones, genistein, daidzein and glycitein in 5:4:1 ratio show presence of higher osteoblast
proliferation compared to control. Interestingly, more cells could be seen near the designed pores of the 3DP scaffolds indicating the importance of flow transport through
the porous channel. (B) MTT assay (n = 3) showing the effects of soy isoflavones loaded 3DP TCP bone tissue engineering scaffolds for in vitro osteoblast cell proliferation in
a flow perfusion bioreactor at day 5 and 10.) [∗∗ denotes P-value <0.0 0 01, statistically significant difference between control and test sample]. [Control: 3D TCP: 3D printed
porous TCP scaffold] (C) ALP assay (n = 3) showing the effects of soy isoflavones loaded 3DP TCP bone tissue engineering scaffolds for in vitro osteoblast cell differentiation in
a flow perfusion bioreactor at day 10. [∗∗ denotes P-value <0.0 0 01, statistically significant difference between control and test sample]. [Control: 3D TCP: 3D printed porous
TCP scaffold]. (D) Optical microscopy images of decalcified tissue-implant specimens after H&E staining showing inflammatory cell recruitment after 24 hours of surgery in
rat distal femur model. Blue and black arrow show neutrophil recruitment and presence of osteocytes, respectively. (E) FESEM images showing the effects of soy isoflavones
loaded 3DP TCP bone tissue engineering scaffolds for in vitro MG-63 cell proliferation in a static condition at day 3, 7 and 11. Layers of bone cancer cells can be seen on
TCP scaffold alone, where the presence of genistein exhibits lower cell proliferation suggesting its chemopreventive ability. Although daidzein and glycitein did not have
a pronounced effect on MG-63 cells on day 7, the combined effect of three isoflavones showed significant chemopreventive effects in all the time points. Reprinted from
[131] with permission from Elsevier. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

tures in the Asian population [147]. However, the effect of vitamin
K antagonists, which are prescribed as oral anticoagulants showed
contradictory results [148].
As mentioned earlier, some studies reported dietary intake of
vitamin K decreases the level of ucOC, urinary Ca excretion, and
improves bone turnover profile. Sokoll et al. found that the dietary
intake of vitamin K in a concentration of 420 μg/d showed a re-
markable reduction in circulating ucOC level [149]. Another study
showed dietary administration of 80 μg/d of vitamin K1 was able
to lower the level of ucOC in postmenopausal women. This study
has been validated by several other reports, which used varied
populations and different dosages of vitamin K [150]. Urinary cal-
cium excretion has also been lowered in postmenopausal women
receiving a dose of 1mg vitamin K1 or 45 mg vitamin K2 per
day [151]. It is noteworthy that the presence of vitamin D along
with vitamin K did not show any enhanced effect in osteocalcin
level.
In 1991, the first study was published correlating the effect of
vitamin K on BMD, which shows vitamin K reduces bone den-
sity loss in patients on hemodialysis with the low-turnover bone
disease [152]. It is interesting to note that the effect of vita-
min K2 on bone density is more pronounced than that of vita-
min D. A placebo-controlled study with 546 osteoporotic patients
showed patients receiving 45 mg/day of vitamin K2 for 1 year
had higher BMD than control [153]. Studies showed vitamin D
and vitamin K2 work synergistically to increase BMD among post-
menopausal women compared to patients only receiving either vi-
tamin D3 or K2 [154]. A well-controlled clinical trial on the ef-
fect of dietary intake of vitamin K1 along with vitamin D and cal-
cium on BMD shows higher BMD than the control [155]. Studies
also showed daily intake of vitamin K2 increases BMD and re-
duces fractures by a factor of almost 2.5-fold among osteoporotic
patients [156].
Dietary intake of vitamin K did not exhibit any adverse effect
suggesting a broad safety range of this compound [157]. A daily
dose of vitamin K2 as high as 90 mg for 2 years did not cause
any detrimental effect in patients, as shown in a double-blinded,
placebo-controlled study [158]. Institute of Medicine also men-
tioned, "A search of the literature revealed no evidence of toxic-
ity associated with the intake of either the phylloquinone (vitamin
K1) or menaquinone (vitamin K2) form of vitamin K”.
3.10. Vitamin A
Vitamin A has one of the earliest mentions in medical history,
where ancient Egyptians identified it as a cure for night blindness
more than 3500 years ago. However, it was not discovered until
1912 when an English biochemist named Frederick Gowland Hop-
kins, found an unknown factor present in milk, and later received
the Nobel prize for the discovery [159]. Vitamin A is a fat-soluble
vitamin, found in foods from animal sources such as dairy prod-
ucts, fish and liver meat as well as in sweet potatoes, carrots, dark
leafy greens, winter squashes, lettuce, dried apricots, cantaloupe,
bell peppers, fish, liver, and tropical fruits. Vitamin A is an essen-
tial factor to maintain the immune system, for growth and devel-
opment and good vision. It can be found in two different forms;
the principal ones being retinol and provitamin A (beta carotene).
Retinol can be found in the small intestines of animals, where it
is converted from retinyl palmitate, which is an ester, primarily
found in Vitamin A. The carotenes alpha-carotene, beta carotene,
and gamma carotene contain retinyl group, which is essential for
vitamin activity [160].
Both osteoblast and osteoclast cells have nuclear receptors of
retinoic acid, which is a metabolite of vitamin A or retinol. There-
fore, vitamin A is a crucial factor in the bone remodeling process.
It is reported that either too high (hypervitaminosis A) or too low

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S. Bose, N. Sarkar and D. Banerjee Acta Biomaterialia 126 (2021) 63–91

Fig. 14. (A) Dietary sources of vitamin K and its mechanism of action: vitamin K is essential for the conversation of glutamate to Gla protein, an important constituent
of osteocalcin, a bone protein. Presence of carboxylated osteocalcin in blood indicates bone formation. (B) In vivo Masson Goldner staining by curcumin, vitamin K2, and
curcumin + vitamin K2 loaded HA-coated Ti implant showing the gap between the implant and the surrounding bone tissue after 5 days of implantation at rat distal
femur model. The new bone formation or osteoid tissue is stained with red/orange; mineralized bone tissue is stained with green/blue, and the implant can be seen in
black. (B) Histomorphometric analysis using ImageJ software showing percentage osteoid or new bone tissue formation and percentage total bone formation within 250 μm
radius of implant after 5 days. Drug-loaded implant exhibited statistically significant difference in osteoid formation and total bone formation after 5 days indicating better
osseointegration ability compared to the untreated HA coated Ti control (∗ P < 0.001 compared to control, ∗ ∗ P < 0.05 compared to control). Reprinted with permission from
[141]. Copyright (2020) American Chemical Society. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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S. Bose, N. Sarkar and D. Banerjee
(hypovitaminosis A) vitamin A has an adverse effect on bone tis-
sue [161]. High level of vitamin A develops increased bone re-
sorption, which leads to a higher chance of fragility fractures. A
cross-sectional study on 175 Swedish women reported that high
dietary intake of vitamin A results in low BMD and a higher risk
of hip fractures [162]. Also, deficiency of vitamin A leads to in-
sufficient osteoclast formation, which in turn forms excessive de-
position of periosteal bone due to uncontrolled osteoblast activity
[163]. Therefore, to ensure safety and efficacy vitamin A should be
consumed within recommended levels.
One of the earliest studies on the effect of vitamin A and
bone growth showed vitamin A deficiency resulted in an abnor-
mal growth in basioccipital bone and spine due to altered osteo-
clast and osteoblast activity [164]. However, as soon as vitamin
A-rich diet is re-administered to the animals, normal osteoblast-
osteoclast cellular activity has been restored in order to reinstate
the normal shape of the dysplastic bones. Few in vitro studies
showed 24 hours of retinol incubation inhibited collagen synthe-
sis in embryonic chick calvaria [165]. Other studies reported that
a high concentration of vitamin A resulted in disintegration and
loss in metachromasia and shrank in mouse fetuses [166]. Hy-
pervitaminosis A also hindered bone development in fetuses from
pregnant rats [167]. Excess consumption of vitamin A relates to
poor bone growth, loss of bone mineral content, and increased
risk of fractures [168,169]. The effect of retinoids on osteoclast
cultures had erratic results. Few showed retinoic acid stimulates
osteoclastic bone resorption and inhibits osteoblast cell prolifera-
tion [170] Moreover, some studies showed carotenoid stimulates
proliferation and differentiation of osteosarcoma cells as well as
inhibits parathyroid hormone-mediated osteoclastogenesis in rat
bone marrow [171]. A study showed children treated with syn-
thetic retinoids for more than 4 years exhibited abnormal ossi-
fication and calcification [172]. It is also interesting to note that
fractures associated with vitamin A are mostly dependent on the
retinol level. However, beta-carotene levels do not influence vita-
min A-related fractures.
In conclusion, the effect of vitamin A on the skeletal health of
humans is highly dose-dependent due to its narrow therapeutic
window. Excessive as well as inadequate dietary intake of vitamin
A deteriorates bone health. Further studies are required to estab-
lish a safe range to avoid any adverse effects or undesired interac-
tions due to vitamin A toxicity.
3.11. Vitamin C
Vitamin C deficiency or scurvy has been identified in as early
as 1500 B.C. This infamous disease has been associated with vari-
ous adverse medical conditions including gingivitis, bone pain, im-
paired bone growth, slower wound healing, and pseudoparalysis.
The first clinical trial by Dr. James Lind in the 18th century demon-
strated reduced symptoms of scurvy after consumption of citrus
fruits. Finally, in 1931, scientist Albert Szent-Gyorgyi isolated hex-
uronic acid, the chief compound found in citrus fruits, which was
later renamed ascorbic acid or vitamin C [173]. Since its discov-
ery, vitamin C is recognized to have an essential role in the growth
and development of connective tissue. Although animals are a poor
source for vitamin C, it is abundantly found in citrus fruits, green
and red peppers, strawberries, tomatoes, Indian gooseberry, turnip,
broccoli, and other leafy vegetables.
Vitamin C is present in the human body in the form of ascor-
bate. This molecule acts as cofactors for several enzymatic reac-
tions in our body and therefore considered as an essential require-
ment for normal physiological functions. It is reversibly oxidized
into an ascorbyl radical and then to dehydroascorbate and thus
acts as an antioxidant. Oxidative stress is considered as one of
the possible causes of osteoporosis. Vitamin C, due to its inherent
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Acta Biomaterialia 126 (2021) 63–91
antioxidant properties prevents the formation of reactive oxygen
species and thereby prohibits osteoclast activity [174].
In the past few decades, vitamin C has been extensively re-
searched due to its widespread pharmacological properties towards
a number of diseased conditions including cancer, cardiovascular
diseases, and the common cold. It has also been considered to be
beneficial for bone health [175]. Vitamin C is a crucial factor for
collagen cross-linking, which constitutes around 90% of the bone
matrix. In fact, the severe clinical condition of scurvy is caused
due to the weak collagen structure in the bone. Vitamin C con-
tains antioxidants, which reduce oxidative stress in the bone. Be-
sides, it is known to reduce the adverse effects of smoking, which
increases the chance of hip fracture by generating free radicals and
increasing bone resorption. A study reported that the consumption
of vitamin E and C significantly reduced the risk of hip fractures
among smokers [176]. The study concludes that vitamins contain-
ing antioxidants are beneficial against oxidant-mediated bone frac-
tures among smokers. Results from a study performed in our group
exhibit the inhibition of osteosarcoma cellular growth and attach-
ment on the vitamin C loaded sample surface compared to control
TCP samples. Fig. 15 also shows a significant increase in osteoblast
cell proliferation, attachment, and ALP density suggesting the pos-
itive influence of vitamin C on bone forming cells [177,178].
One of the earliest experiments in 1940 investigating the effect
of vitamin C on bone health in guinea pigs reported that vitamin
C deficiency resulted in impaired bone matrix formation and mat-
uration compared to control [179]. Rats can synthesis ascorbic acid
in their body. Therefore, genetically impaired Shionogi rats (ODS),
which cannot produce ascorbic acid by themselves were selected
for a study, and a vitamin C-deficient diet was given to them for
five weeks. Rats with ascorbic acid-deficient diet showed a signif-
icant reduction in bone formation with delayed fracture healing
compared to the group that received vitamin C supplements [180].
Framingham osteoporosis study conducted among elderly
adults showed a lower risk of hip and non-vertebral fractures with
dietary as well as supplemental intake of vitamin C [181]. No sig-
nificant result was found on women. Results showed a high dosage
of total vitamin C intake of about 313 mg/d lowered the hip frac-
ture risk by 44% compared to those who took 94 mg/day of vi-
tamin C. Again, subjects taking 260 mg/d vitamin C exhibited a
69% lower risk of hip fractures compared to subjects who did not
take any supplements. However, the dosage of vitamin C was much
higher than the US recommended daily allowance of 90 mg/d for
men and 75 mg/d for women.
A study using a mouse model showed vitamin C deficiency re-
sulted in impaired bone formation, lowered osteoblast differentia-
tion, and bone density as well as the formation of fractures [182].
Chronic deficiency of vitamin C significantly lowers osteoblast pro-
liferation and chondrocyte differentiation. Another study reported
inadequate intake of vitamin C shows reduced plasma osteocal-
cin level and in vivo bone formation [183]. Other studies reported
insufficiency in vitamin C intake results in osteoporosis and loss
of BMD [184]. Vitamin C supplements suppress bone resorption
in postmenopausal women [185]. A study reported that the post-
menopausal women taking vitamin C supplements had a 6.7%
higher average BMD compared to the control group [186]. Addi-
tionally, a daily dosage of 159 mg of vitamin C resulted in signif-
icantly reduced chances of hip fracture among older patients who
had ever smoked [187]. Vitamin C is also reported to be beneficial
by lowering the risk of fracture in postmenopausal women with a
history of smoking or estrogen therapy [188].
Vitamin C is considered safe and well-tolerated with the up-
per limit set at 2 g daily for adults. There are few cases where
high dosages of vitamin C have been associated with increased
risk of kidney stone, iron overload, and hemolysis in patients with
S. Bose, N. Sarkar and D. Banerjee Acta Biomaterialia 126 (2021) 63–91

Fig. 15. (A) Possible mechanism of action of vitamin C on inhibition of tumor cells and proliferation of osteoblast cells. The oxidized form of vitamin C or dehydroascorbate
(DHA) is taken up by the cells via glucose transporters, which is also upregulated in tumor cells compared to normal cells. Therefore, tumor cells automatically uptake
more glucose along with DHA compared to normal cells. Inside the cell, DHA is again reduced back to vitamin C, where it gets accumulated and acts as a pro-oxidant that
produces oxidative stress. Generation of free radicals or reactive oxygen species (ROS) and hydrogen peroxide (H2O2) causes cellular damage, suppresses tumor growth and
subsequently results in vitamin C-mediated cell death. (B) MTT osteosarcoma cell viability assay showing presence of vitamin C has significantly decreased osteosarcoma cell
viability by almost 2.5-fold at day 7 [∗ denotes P < 0.05, statistically significant difference compared to untreated HA coated Ti control]. (C) SEM images of osteosarcoma
cell culture on vitamin C loaded HAp coated cpTi samples at day 3 and day 7 showing significantly reduced osteosarcoma cell proliferation in samples with 25 mM vitamin
C. (D) Osteoblast cell viability and ALP activity on control and vitamin C loaded scaffolds after 3, 7, and 11 days of culture showing a pronounced increase in osteoblast
cell proliferation in the presence of vitamin C compared to control. Data are presented as mean ± standard deviation (∗ P < 0.05 compared to untreated TCP control). (E)
Osteoblast cellular morphology on scaffolds after 7 days of culture showing a clear distinction between vitamin C scaffolds with the control. Layers of osteoblast cells can be
seen in scaffolds containing vitamin C compared to control. Reprinted from [177,178] with permission from Elsevier.

glucose-6-phosphate dehydrogenase deficiency, however, the re-
ports are inconsistent [189].
3.12. Vitamin D
The discovery of vitamin D dates back to the early 20th century
when Dr. Elmer McCollum was conducting his experiments on fat-
soluble minerals at Johns Hopkins University. Dr. McCollum and his
coworkers tried to treat rickets in rats by feeding them heated, ox-
idized cod liver oil. Surprisingly cod liver oil, with inactive vitamin
A still continued to treat rickets, which was then attributed to the
presence of an unknown essential mineral, referred to as vitamin
D. The era of isolating nutritional forms of vitamin D came to an
end when the chemical structures along with synthesis methods
were performed by Windaus, leading to a Nobel prize-winning en-
deavor in 1928. Gradually, the works of several researchers led to
the demonstration of the necessity of vitamin D on calcium ab-
sorption [190], and its direct influence on bone mineralization as
well as osteoclast-mediated bone resorption [191]. Since then, vi-
tamin D has been established as an essential mineral required for
the maintenance of a healthy bone structure for the human body.
To date, sunlight has been recognized as the best source of vita-
min D as ultraviolet B rays (UVB) react with the cholesterol present
in our skin to produce vitamin D. The only non-animal source of
vitamin D is mushroom, which converts ergosterol into vitamin D
on exposure to UV light. This source is sufficient to sustain veg-
ans over the winter months; however, more research is required
to establish the stability and shelf life of the vitamins from these
mushrooms [192]. The photoproduced vitamin D from the skin is
consecutively metabolized in the liver and the kidneys to form 1,25
dihydroxy vitamin D, shown in Fig. 16. Although the significance of
vitamin D has been unappreciated for a long period, recent studies
show a direct correlation between the amount of 1,25 dihydroxy
vitamin D in the serum with bone density, which also corresponds
to a lower risk of fractures [193]. Also, 1,25 dihydroxy vitamin D
has been shown to influence the biosynthesis of osteocalcin and
matrix Gla protein [194]. Vitamin D increases the calcium absorp-
tion in the intestine, which in turn reduces bone resorption medi-
ated by parathyroid hormones. In the United States, especially in
the Northern States, most of the children depend on fortified milk
for their vitamin D source. However, as the consumption of dairy
products lowers in adolescence, the absence of adequate vitamin
D affects the calcium absorption in the body. Although multiple
studies confirmed low to no correlation between calcium and vi-
tamin D in the physiological system [195,196], most of these stud-
ies were conducted with lower vitamin D dosages, inadequate to
raise the amounts of hydroxyvitamin D in the serum. Other stud-
ies in a randomized controlled trial showed the efficacy of vitamin
D in reducing hip or non-vertebral fractures in elderly menopausal
women, even by intake of an inadequate dosage of vitamin D
[197]. It has also been observed to improve muscle strength

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S. Bose, N. Sarkar and D. Banerjee
Fig. 16. (A) Mechanism of action of vitamin D3 (VD3) mediated bone formation and mineralization and inhibition of osteoclast activity. (B) Osteoclast resorption pit assay
showing clear resorption pits on HA and HA loaded with polycaprolactone/ polyethylene glycol (PCL/PEG) samples however little to no resorption pits could be found on
samples loaded with VD3 indicating reduced osteoclast activity. (C) SEM images with hFOB after 7 days of culture showing no cytotoxic effects with PCL/PEG or VD3/PCL/PEG
loading. Reprinted from [199] CC BY-NC 3.0.
and balance [198], thereby diminishing the chances of collapsing
as well.
A study from our group demonstrated vitamin D3 loaded cal-
cium phosphate bone tissue engineering scaffolds promote in vitro
osteoblast cell proliferation and suppresses osteoclast activity. The
controlled release kinetics of vitamin D3 was obtained by a com-
bination of hydrophilic and hydrophobic polymer (PEG: PCL at 65:
35 molar ratio) coating on calcium phosphate scaffolds [199,200].
The recommended daily allowance of vitamin D is 600 IU for
healthy adults; yet the deficiency of vitamin D remains a major
concern across the world. Functional indicators, like the levels of
parathyroid hormones, point to 80 nmol/L of vitamin D as suffi-
cient, but many evidence and clinical trial-based reports demon-
strate a higher dosage for vitamin as effective, especially for the
prevention of cancer [201,202]. Vitamin D has been shown to in-
tercalate with retinol protein and the DNA exon, influencing the
metabolism and cell division in both healthy and cancerous cells
[203].
Vitamin A has been demonstrated to slow down the vitamin D-
mediated calcium absorption in the bone, impeding bone forma-
tion [204]. Low amounts of vitamin D interfere with osteocalcin
expression mediated by vitamin K [205].
4. Localized delivery of natural medicinal compounds from
bone tissue engineering scaffolds
Utilization of natural medicinal compounds and essential nutri-
ents shown in Figs. 17 and 18 for the treatment of a variety of bone
disorders may deem to be a cost-effective and healthy preventive
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Acta Biomaterialia 126 (2021) 63–91
choice since it possesses less adverse effects compared to conven-
tional synthetic medicines. These compounds which are used as
regular cooking spices like curcumin, garlic, and ginger and others
available from natural sources have been used regularly by Asian
countries for treating a range of bone disorders. However, for ef-
fective utilization of natural medicinal compounds in the treatment
of bone-related complications, intensive research is now dedicated
on the integration of herbal medicine and additively manufactured
bone implants for controlled and sustained release of the drug
molecule from scaffold that fulfill the therapeutic needs in the pa-
tient’s body without causing adverse effects. To deepen our un-
derstanding, more investigations are required to focus on the de-
sign and development of drug loaded bone implants, optimize the
drug-scaffold interaction, and obtain an effective localized deliv-
ery of drugs maintaining the minimum effective concentration in
targeted tissue. The combination of advanced modern bone tissue
engineering technologies and natural medicinal compounds with
osteogenic, antitumor, antimicrobial, and anti-inflammatory activi-
ties opened up a whole new era of research envisioning advanced
treatment opportunities and solutions for enhancing bone growth,
inhibiting osteoclastic bone resorption, and preventing other bone-
related complications.
Various natural medicinal compounds incorporated within bone
tissue engineering scaffolds have been proposed and tested for
their ability to repair critical-sized defect, however successful bone
regeneration depends on two key factors - controlled release of
drug from the scaffold matrix and desired drug-material inter-
action. Among various NMCs, curcumin is one of the most re-
searched plant-derived compounds in bone tissue engineering due
S. Bose, N. Sarkar and D. Banerjee
Fig. 17. Natural medicines, their active constituent and effects on bone health.
to its multipronged therapeutic benefits and wide range of benefi-
cial applications. A recent study investigating the use of curcumin-
loaded biodegradable polyurethane scaffolds for cartilage regener-
ation revealed enhanced in vitro proliferation and differentiation of
chondrocytes [206]. Another interesting approach combined bioac-
tive graphene oxide and zinc-curcumin complex in electro-spun
nanofiber to investigate its ability for bone regeneration [207].
The result demonstrated that zinc-curcumin composite nanofibers
enhanced osteogenic performance, as well as induced antibacte-
rial properties to the nanofiber. Apart from incorporating NMCs
in bone graft materials, few studies also made an effort to uti-
lize NMCs in form of bone fillers, films, granules or sponges for
different surgical needs. Two studies have tested the efficacy of
soy-based degradable bone fillers for their potential in in vivo and
in vitro applications [208,209]. The results concluded that soy-
based granules induce in vitro osteoblast differentiation, reduce
macrophage population and features new trabecular bone regen-
eration after 8 weeks of implantation. As of yet, aloe vera has
most significantly been used for skin regeneration and healing pur-
poses. However, a recent effort using a combination of commercial-
ized collagen sponge (Hemospon®), 8% aloe vera and mesenchymal
stem cells from human dental pulp enhances non-critical bone de-
fects and significantly lowers (p<0.05) acute inflammatory reaction
after 30 days of implantation [210]. Table 1 summarizes the in-
tegration of potential natural medicinal compounds and synthetic
bone substitutes for treating various musculoskeletal disorders.
5. Drug delivery strategies for natural medicinal compounds
With the increased understanding of bone biology and bone
pathogenesis, bone tissue engineering field has begun to move be-
yond the development of conventional bone grafts. The emerging
trends in tissue engineering and drug discovery have led to the
development of advanced bone scaffolds with multipronged ther-
apeutic potential which not only mimics the structural hierarchy
of bone but also effectively delivers biochemical cues to the tar-
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Acta Biomaterialia 126 (2021) 63–91
get site [216]. Motivated primarily by a desire for enhanced bone
regeneration, these multifunctional scaffolds can provide therapeu-
tic solutions to various orthopedic disorders by controlling the ap-
propriate localized release of bioactive molecules. A variety of lim-
itations in conventional clinical therapies for metabolic or non-
metabolic bone disorders including osteoporosis, osteomyelitis or
bone infection, osteoarthritis, and bone cancer have driven the
urge to find alternative strategies involving targeted drug deliver-
ies. To overcome the limitations of systemic toxicity, adverse ef-
fects on other organs or tissues, as well as low targeting efficiency
of conventional treatment regimens, localized delivery of natural
medicinal compounds is a safer and effective solution for treating
orthopedic diseases.
The delivery schemes have ranged from the direct embed-
ding of natural medicinal compounds within the biodegradable
porous scaffolds to more sophisticated stimuli-responsive drug
delivery where biomolecules are encapsulated within micro or
nanoparticles. Owing to their high surface reactivity, and pore con-
nectivity, biodegradable porous ceramics have demonstrated the
ability to incorporate therapeutic molecules within their struc-
ture by a variety of techniques including adsorption, physical en-
trapment, and covalent bonding. The drug release kinetics for
ceramic-based drug delivery is primarily influenced by diffusion
and scaffold resorption rate [217]. Polymeric bone grafts also ex-
hibit unique advantages as drug delivery carriers due to their
controlled biodegradability, biocompatibility, and ease in process-
ing. However, poor mechanical strength and toxic degradation
by-products remain some of the major concerns associated with
biodegradable polymers as drug delivery systems for bone regen-
eration [218]. Given that natural medicinal compounds often pos-
sess poor stability and low bioavailability, direct administration
of these biomolecules within ceramic or polymer scaffolds does
not always guarantee controlled and desired release kinetics. In
such cases, micro- or nanoencapsulation of natural medicinal com-
pounds is pursued which has shown to improve the bioavail-
ability and clinical efficacy. In recent years, various drug deliv-
S. Bose, N. Sarkar and D. Banerjee
ery carriers including micelle, liposomes, nanoparticles, nanotubes,
mesoporous silica are being extensively investigated to improve
bioavailability of poorly soluble drug molecules, to control release
kinetics, to allow efficient drug-loading, and most importantly to
target the diseased site [219–221]. Besides, significant advances
have been accomplished in recent days to accomplish spatiotempo-
ral delivery of bioactive molecules, where tissue-engineered bone
grafts can be patterned with either spatially compartmentalized or
time-dependent drug delivery cascade sequence for treating bone
diseases [222,223].
6. Critical concerns and future directions
6.1. Safety concerns related to natural medicinal compounds
Natural medicinal compounds have found profound usage over
the years for the treatment of a variety of bone disorders in tan-
dem with bone health promotion and enhancement of quality and
span of life. These prevalent usages of these NMCs have been de-
rived from the innate advantages of a mixture of multiple active
compounds present in NMCs over a single component drug, foster-
ing the multi-dimensional potential for bone disorders. Yet, a "pas-
sionate ambivalence" fueled by contemporary medicine, has shown
negative pronouncements towards the NMCs’ appropriate and safe
usage, efficacy, and quality assurance. Therefore, several critical is-
sues, discussed in this section of the review – toxicity, quality con-
trol, stability, and shelf life and standardization need to be care-
fully analyzed for the successful clinical translation of NMCs for
treating bone diseases.
6.1.1. Critical need for dose optimization
The holistic approach of NMCs to healthcare makes it an at-
tractive candidate for the treatment of bone disorders and leaving
a long-lasting pseudo belief on human minds that they are safe
Fig. 18. Essential nutrients and their effects on bone health.
84
Acta Biomaterialia 126 (2021) 63–91
and harmless. Albeit many deaths or hospitalizations have been re-
ported as an effect of over-the-counter contemporary drugs, such
due to NMCs are so rare that the United States National Poison
Control does not even have a category dedicated to NMCs [224].
Although the safety and efficacy of several NMCs have been in-
vestigated and demonstrated in vitro and in vivo in small ani-
mal models [225] through systematic research, however as stated
by Paracelsus, ‘All substances are poisons; there is none, which is
not a poison. The right dose differentiates a poison and a remedy’
[226]. This generates the demand for extensive studies on NMCs
to determine their efficacy and safety. The herbal medicinal plants
also tend to vary significantly depending on weather conditions,
soil, growing conditions, and species, necessitating the need for
precise investigations. Furthermore, the problem could be more
complicated when NMCs are used in combination with synthetic
drugs [227], or other biologics arising from the pathogens, toxins,
or pesticides used during the growing conditions [228]. Most of
the peer-reviewed literature on herbal medicine does not have a
complete safety profile in animal or human models, keeping the
therapeutic index of the extracts unknown to the end-users. Still,
it is noteworthy to mention, NMCs are relatively safe and reports
of toxicity are rare compared to that of synthetic drugs. Often, the
toxicity of the NMCs is misrepresented and is caused by the or-
ganic solvent present in the plant extracts, as for the case for the
reported hepatoxicity for the alcohol extraction of Bupleurum Chi-
nese, a traditional Chinese herb mostly known for alleviating the
symptoms related to hepatitis, liver cirrhosis, and flu [229].
6.1.2. Isolation and stability
Wild herbal medicinal plants contain multiple active compo-
nents, many of which have not been identified yet, with virtually
no chemical fingerprinting available for most such herbs. The in-
herent advantage of multiple active compounds in NMCs hence
poses a concern in the identification, separation, and isolation of
S. Bose, N. Sarkar and D. Banerjee Acta Biomaterialia 126 (2021) 63–91

Table 1
Localized delivery of natural medicinal compounds (NMCs) from various bone grafts for musculoskeletal disorders.

Substrate Fabri cation process NMCs Release profile Biological activity Ref

PCL nanofibers Electro -spinning Curcumin In aqueous medium, the MC3T3-E1 pre-osteoblast proliferation was moderated with [211]
fibers released 18% of increased loading of curcumin and was 50% lower on the
the encapsulated drug in fibers containing 5% curcumin at day 10 than the control.
3 days and 60% in 9 days. Osteogenesis by ALP expression and mineral deposits by
FTIR and Alizarin red staining were markedly higher for 1%
curcumin compared to neat polymer but lower for 5%
curcumin.
TCP scaffolds Binder-jet 3D printing Curcumin PCL, PEG and PLGA MTT cell viability assay and morphological characterization [70]
polymeric system are by FESEM showed highest cell viability with samples
used to enhance coated with curcumin-PCL-PEG. The presence of curcumin
curcumin’s in TCP results in enhanced bone formation after 6 weeks.
bioavailability, provide Complete mineralized bone formation increased from 29.6%
higher release, and to 44.9% in curcumin-coated scaffolds compared to pure
enable continuous TCP.
release of curcumin for
22 days.
TCP scaffolds Binder-jet 3D printing Curcumin In vitro curcumin release Presence of liposomal curcumin results in a 96% decrease [58]
from the liposome is in in vitro osteosarcoma cell proliferation and viability after
carried over 60 days, 11 days of incubation, compared to control. Results from in
showing 30% release of vitro osteoblast cell culture demonstrate that liposomal
the drug. curcumin promotes cell attachment, proliferation, viability,
and differentiation.
HA nano-particles Sol-gel method Curcumin N/A The synthesized HA nanoparticles possessed antibacterial, [212]
antifungal, antioxidant, and anticancer activities.
PLGA micro Single emulsion solvent Curcumin The drug release kinetic Sustained curcumin release from the scaffold inhibited the [213]
spheres in eva-poration study showed that the overproduction of ROS in mesenchymal stem cells caused
collagen/HA Cur release from the by diabetic serum. Cur-loaded scaffold alleviated the
composite composite scaffolds negative effects of diabetic serum on the proliferation,
lasted up to 30 days. migration, and osteogenic differentiation of mesenchymal
stem cells. When implanted into bone defects in type 2
diabetic rats, the Cur-loaded scaffold showed a greater
bone formation capability compared to the pure scaffold.
Se-doped HA Co-precipita-tion and Curcumin Exhibited controlled Hemolysis and cytotoxicity tests confirmed the nanorods [214]
nanorods hydrothermal route release of 1.38% of the with good blood compatibility and cell compatibility.
initially loaded curcumin
in the physiological
buffers over 159 h.
Acemannan sponge Freeze-drying Acemannan N/A MicroCT revealed a significant increase in bone surface and [215]
bone volume, and tissue mineral density compared with
the control group (p < 0.05). Histologically, the
acemannan-treated groups had denser bone matrix
compared with the control group.
HA-coated Plasma-spray coating Acemannan Chitosan coating is used Improved osteoid formation from 34% in control HA to [54]
titanium alloys to control the burst ∼49% with the incorporation of acemannan in doped
release of the coatings reveals the efficacy of acemannan on early stage
acemannan from the osteogenesis, 5 weeks after implantation in the rat distal
calcium phosphate femur model.
matrix. The results show
controlled release of
acemannan from the
chitosan coatings.
TCP scaffolds Binder-jet 3D printing Soy isoflavones After 16 days, genistein, Presence of geinstein, daidzein and glycitein in 5:4:1 ratio [131]
daidzein and glycitein result in 90% reduction in cellular viability after 11 days,
exhibit 72.5, 100 and compared to control. The scaffolds show increased cellular
13.75% release in pH 7.4, viability, proliferation, and differentiation, compared to the
and 25.1, 23.3 and 2.97% control. Neutrophil recruitment at the inflammation site is
release in pH 5.0, demonstrated using H&E staining suggesting the
respectively. immunomodulatory potential of soy isoflavones at 24 h
post-surgical specimens from rat distal femur model.
HA-coated Plasma-spray coating Vitamin C After 21 days, the Presence of high dosages 25 mM vitamin C shows a [178]
titanium alloys implants loaded with statistically significant decrease in osteosarcoma cell
400 and 800 μg of viability after 3 days, while both 5 mM and 25 mM vitamin
vitamin C have shown a C reduce cellular viability by 2.5 folds compared to the
cumulative release of control after 7 days. The presence of vitamin C shows no
62.7 and 74.1% in pH obvious signs of cytotoxicity towards osteoblast cell-line at
5.0, whereas, 50.9% and day 3 and day 7, as confirmed by the MTT assay and layers
53.1% of release are of hFOB cellular morphology on the surface of the implants
observed from the by FESEM.
implants loaded with
400 and 800 μg of
vitamin C in pH 7.4
respectively.
(continued on next page)

85
S. Bose, N. Sarkar and D. Banerjee Acta Biomaterialia 126 (2021) 63–91

Table 1 (continued)

Substrate Fabri cation process NMCs Release profile Biological activity Ref

TCP scaffolds Uniaxially pressing Vitamin C PCL coating prevents the Presence of vitamin C demonstrates enhanced attachment, [177]
burst release of the drug proliferation, and viability of human osteoblast cells in TCP
from TCP scaffolds and scaffolds. Osteoblast cell viability shows two folds increase
results in a more in the presence of vitamin C, regardless of the time point.
controlled and sustained Moreover, an increase in ALP density has been shown in
release of vitamin C over day 11 suggesting the possible influence of vitamin C on
60 days osteoblast differentiation.
TCP scaffolds Binder-jet 3D printing Vitamin D Mixed polymer system Little to no resorption pits were identified with vitamin [199]
of PCL and PEG is D3/PCL/PEG loaded HA samples compared to control and
utilized to control polymer loaded indicating a reduction in osteoclast activity.
release of vitamin D3 FESEM images showed more osteoblast cell morphology on
from additively samples with vitamin D3 compared to control TCP and TCP
manufactured porous with PCL/PEG as well as no cytotoxicity assessed through
TCP scaffolds with MTT assay.
interconnected porosity.
HA-coated Plasma-spray coating Vitamin K HA coating enables The dual release of curcumin and vitamin K2 showed little [141]
titanium alloys sustained release of to no osteosarcoma cell attachment with 95% and 92%
curcumin and vitamin lower osteosarcoma cell viability compared to control, at
K2 in both physiological days 7 and 11, respectively. Vitamin K2 promoted
pH and acidic osteoblast viability and proliferation to almost 9- fold at
microenvironment. day 11. Enhanced in vivo osseointegration ability of
curcumin- and vitamin K2- incorporated HA-coated
Ti6Al4V implant after 5 days of femoral epicondyle defect
surgery in rat distal femur model.

the active components required for the treatment of a particular
disease. Guided fractionation techniques are used to isolate the ac-
tive compounds, like gingerol and allicin, and should be investi-
gated in vivo before proceeding to clinical trials. The isolation of
individual component in NMC also seems to be a cost-extensive
procedure and poses a burden for their commercialization. The
stability of the isolated extracts forms yet another unique chal-
lenge for many NMCs. For example, aqueous insoluble curcumin
and sparingly soluble allicin have a low shelf life and undergo
degradation at room temperature. This generates the need for the
development of an appropriate delivery vehicle with tuned in con-
trolled delivery rates to increase the bioavailability of these NMCs
in the physiological microenvironment.
6.1.3. Standardization
To ensure successful translation, bench to bedside, the plants
should be grown in controlled growing conditions and all the
plants should be derived from standardized taxonomic records.
Extensive studies on the seed preservation, location, and
weather conditions of the cultivation are needed for an efficient
understanding of their influence on the extracts. Since the phy-
tochemical profiles and the biological efficacy of the extracts are
dependent on the growing conditions and will vary significantly
depending on the weather conditions and geographical locations
like drought, flood, a careful evaluation of the influence of these
parameters should be conducted for quality control and stan-
dardization, ensuring their appropriate usage and approval by the
drug policymakers [230]. Alongside, the composition, isolation, and
dosage of the different plant extracts should be standardized, con-
trolled, and free of any potential toxins.
6.2. Clinical applications and future directions for NMC delivery from
bone scaffolds
However, current research has reformed that view and came to an
understanding that early inflammatory response and macrophage
recruitment at the injury site are beneficial to healing as long as
it is highly regulated. Therefore, in order to utilize the controlled
delivery of NMC for enhanced bone regeneration rate and faster
wound healing, the inflammatory process needs to be carefully
modulated rather than suppressed completely. Another challenge
in bone tissue engineering that is being currently addressed by
many researchers, is the potential complications from bone infec-
tions that severely jeopardize natural bone healing. Antibiotic re-
sistance, tissue toxicity, and delayed bone regeneration are some
of the drawbacks of systemic antibiotic therapy, which signifies the
need for localized NMC delivery from bone grafts for treating or
preventing infection without negatively impacting the bone heal-
ing and regeneration. In fact, plant-based medicines have a modest
track record as antimicrobial agents for treating life-threatening in-
fectious diseases before the advent of modern antibiotics [232]. Fi-
nally, as the tissue engineering field moves towards clinical transla-
tion, adequate and appropriate vascularization must be considered
for successful bone formation. Although the close interconnection
between angiogenesis and osteogenesis has long been recognized,
the generation of blood vessel network or production of angiogenic
factors in tissue engineering constructs were not given enough at-
tention [233]. One promising avenue of drug delivery involves the
generation of angiogenic signals by controlled NMC delivery, which
eventually enhances the rate of bone regeneration and improves
tissue functionality. In the end, clinical translation is the key and
researchers need to move beyond the initial scientific discovery
[234]. With the increased complexity and added natural medicines
for drug delivery, challenges to receive FDA approval and commer-
cialization of the bone grafts become more difficult. Therefore, re-
searchers need to consider the potential translation of such tissue
engineering constructs for clinical use.

While the most-studied applications of NMC delivery from im- 7. Summary

planted bone scaffolds have long been focused on promoting bone
regeneration, emerging trends in tissue engineering provide more
insights on the modulation of inflammation, infection control, and
vascularization. Earlier views on inflammation and bone healing
postulated that prolonged inflammation due to the surgery and
implantation of bone grafts delays the wound healing time and is
therefore considered to be detrimental for bone regeneration [231].
Recent advancements in additive manufacturing paired with the
surging clinical demand for biomedical implants contributed to-
wards the vast success of patient specific synthetic bone grafts
within a short span. Yet, the synthetic implants are challenged
with brief clinical success and limited life-time due to implant-
failure, demanding revision surgeries, especially in younger pa-

86
S. Bose, N. Sarkar and D. Banerjee
tients. Modification of the material chemistry by incorporating
comparatively safer natural biomolecules by chemical or physical
adsorption may directly influence the cellular activity and holds
the potential to be used for future implant developments. With op-
timized dosage, and proper knowledge on active ingredients, toxic
limits, possible interactions and particular applications, researchers
will be able to design and develop new generation scaffolds to al-
low successful bone regeneration and prevent adverse reactions at
the site of implantation. Improved bone healing capacity, osseoin-
tegration and tailored biological efficacy can be expected out of
these functionalized orthopedic implants by the development of
advanced strategies of drug delivery or controlled release of nat-
ural medicinal compounds in a time-dependent and controlled en-
vironment, offering hope for the future generation bone grafts.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgments
Authors would like to acknowledge financial support from the
and National institute of dental and craniofacial research (NIDCR)
of the NIH under Award number R01 DE029204-01 (PI: S. Bose)
and National Institute of Arthritis and Musculoskeletal and Skin
Diseases (NIAMS) of the National Institutes of Health (NIH) under
Award number R01 AR066361 (PI: S. Bose). The content is solely
the responsibility of the authors and does not necessarily repre-
sent the official views of the National Institute of Health.
References
[1] G. Peat, R. McCarney, P. Croft, Knee pain and osteoarthritis in older adults:
a review of community burden and current use of primary health care, Ann.
Rheum. Dis. 60 (2) (2001) 91–97.
[2] A. Luetke, P.A. Meyers, I. Lewis, H. Juergens, Osteosarcoma treatment–where
do we stand? A state of the art review, Cancer Treat. Rev. 40 (4) (2014)
523–532.
[3] S. Bose, D. Ke, H. Sahasrabudhe, A. Bandyopadhyay, Additive manufacturing
of biomaterials, Prog. Mater Sci. 93 (2018) 45–111.
[4] S. Bose, S. Vahabzadeh, A. Bandyopadhyay, Bone tissue engineering using 3D
printing, Mater. Today 16 (12) (2013) 496–504.
[5] N.P. Hailer, G. Garellick, J. Kӓrrholm, Uncemented and cemented primary total
hip arthroplasty in the Swedish Hip Arthroplasty Register, Acta Orthop. 81
(2010) 34–41.
[6] I.R. Reid, Efficacy, effectiveness and side effects of medications used to pre-
vent fractures, J. Intern. Med. 277 (6) (2015) 690–706.
[7] M. Roy, GA. Fielding, H. Beyenal, A. Bandyopadhyay, S. Bose, Mechanical, in
vitro antimicrobial, and biological properties of plasma-sprayed silver-doped
hydroxyapatite coating, ACS Appl. Mater. Interfaces 4 (3) (2012) 1341–1349.
[8] S. Tarafder, W.S. Dernell, A. Bandyopadhyay, S. Bose, SrOand MgOdoped
microwave sintered 3D printed tricalcium phosphate scaffolds: Mechanical
properties and in vivo osteogenesis in a rabbit model, Journal of Biomedical
Materials Research Part B: Applied Biomaterials 103 (3) (2015) 679–690.
[9] S. Tarafder, S. Banerjee, A. Bandyopadhyay, S. Bose, Electrically polarized
biphasic calcium phosphates: adsorption and release of bovine serum albu-
min, Langmuir 26 (22) (2010) 16625–16629.
[10] T.M. De Witte, L.E. Fratila-Apachitei, A.A. Zadpoor, N.A. Peppas, Bone tissue
engineering via growth factor delivery: from scaffolds to complex matrices,
Regener. Biomater. 5 (4) (2018) 197–211.
[11] J. Parvizi, T.L. Tan, K. Goswami, C. Higuera, C. Della Valle, A.F. Chen, N. Shohat,
The 2018 definition of periprosthetic hip and knee infection: an evi-
dence-based and validated criteria, J. Arthroplasty 33 (5) (2018) 1309–1314.
[12] JL. Paris, N. Lafuente-Gómez, M. Victoria Cabañas, J. Román, J. Peña, M. Val-
let-Regí, Fabrication of a nanoparticle-containing 3D porous bone scaffold
with proangiogenic and antibacterial properties, Acta Biomater. 86 (2019)
441–449.
[13] J.K. Ellington, M. Harris, M.C. Hudson, S. Vishin, L.X. Webb, R. Sherertz, In-
tracellular Staphylococcus aureus and antibiotic resistance: implications for
treatment of staphylococcal osteomyelitis, J. Orthop. Res. 24 (1) (2006) 87–93.
[14] K.C. Wong, S.M. Kumta, N.V. Geel, J. Demol, One-step reconstruction with a
3D-printed, biomechanically evaluated custom implant after complex pelvic
tumor resection, Comput. Aided Surg. 20 (1) (2015) 14–23.
[15] M. Ekor, The growing use of herbal medicines: issues relating to adverse re-
actions and challenges in monitoring safety, Front. Pharmacol. 4 (2014) 177.
87
Acta Biomaterialia 126 (2021) 63–91
[16] K. Bi, Da-W Yang, X.-F. Yang, J.-M. Wen, D.-F. Li, L.-R. Wang, T.-M. Cong,
D. Song, Yu-H Qiu, Xu-B Chai, X.-H.L.Y.-J. Zeng, Combination of western
medicine and traditional Chinese medicine for treatment of malignant bone
and soft tissue tumors, Orthoped. Rheumatol. 10 (4) (2018), doi:10.19080/
OROAJ.2018.10.555793.
[17] S. Bose, N. Sarkar, D. Banerjee, Effects of PCL, PEG and PLGA polymers on cur-
cumin release from calcium phosphate matrix for in vitro and in vivo bone
regeneration, Mater. Today Chem. 8 (2018) 110–120.
[18] KN. Tu, JD. Lie, C. King Victoria Wan, M. Cameron, AG. Austel, JK. Nguyen,
K. Van, D. Hyun, Osteoporosis: a review of treatment options, Pharm. Thera-
peut. 43 (2) (2018) 92.
[19] JM. Kling, BL. Clarke, NP. Sandhu, Osteoporosis prevention, screening, and
treatment: a review, J. Women’s Health 23 (7) (2014) 563–572.
[20] M.K. Karlsson, C. Lindén, C. Karlsson, O. Johnell, K. Obrant, E. Seeman, Exer-
cise during growth and bone mineral density and fractures in old age, Lancet
North Am. Ed. 355 (9202) (20 0 0) 469–470.
[21] R. Burge, B. Dawson-Hughes, D.H. Solomon, J.B. Wong, A. King, A. Toste-
son, Incidence and economic burden of osteoporosis-related fractures in the
United States, 20 05–2025, J. Bone Miner. Res. 22 (3) (20 07) 465–475.
[22] JA. Kanis, P. Delmas, P. Burckhardt, C. Cooper, D. obot Torgerson, Guidelines
for diagnosis and management of osteoporosis, Osteoporos. Int. 7 (4) (1997)
390–406.
[23] C. McGreevy, D. Williams, Safety of drugs used in the treatment of osteoporo-
sis, Therapeut. Adv. Drug Saf. 2 (4) (2011) 159–172.
[24] S. Khosla, JP. Bilezikian, DW. Dempster, E. Michael Lewiecki, PD. Miller,
RM. Neer, RR. Recker, E. Shane, D. Shoback, JT. Potts, Benefits and risks of
bisphosphonate therapy for osteoporosis, J. Clin. Endocrinol. Metabolism 97
(7) (2012) 2272–2282.
[25] R. Keen, Osteoporosis: strategies for prevention and management, Best Pract.
Res. Clin. Rheumatol. 21 (1) (2007) 109–122.
[26] M. McClung, ST. Harris, PD. Miller, DC. Bauer, K. Shawn Davison, L. Dian,
DA. Hanley, DL. Kendler, C.K. Yuen, E. Michael Lewiecki, Bisphosphonate ther-
apy for osteoporosis: benefits, risks, and drug holiday, Am. J. Med. 126 (1)
(2013) 13–20.
[27] TD. Rachner, S. Khosla, LC. Hofbauer, Osteoporosis: now and the future,
Lancet North Am. Ed. 377 (9773) (2011) 1276–1287.
[28] C. Chen, J.S. Di, W. Zhao, T. Wang, L. Han, JL. Hamilton, H.-.J. Im, Osteoarthri-
tis: toward a comprehensive understanding of pathological mechanism, Bone
Res. 5 (1) (2017) 1–13.
[29] J. Samuels, S. Krasnokutsky, SB. Abramson, A tale of three tissues, Bull. NYU
Hosp. 66 (2008) 244–250.
[30] C.G. Helmick, D.T. Felson, R.C. Lawrence, S. Gabriel, R. Hirsch, C.K. Kwoh,
M.H. Liang, H.M. Kremers, M.D. Mayes, P.A. Merkel, S.R. Pillemer, Estimates
of the prevalence of arthritis and other rheumatic conditions in the United
States: Part I, Arthritis Rheumatism 58 (1) (2008) 15–25.
[31] A. Anandacoomarasamy, L. March, Current evidence for osteoarthritis treat-
ments, Therapeut. Adv. Musculoskeletal Dis. 2 (1) (2010) 17–28.
[32] T. Towheed, L. Maxwell, M. Judd, M. Catton, MC. Hochberg, GA. Wells, Ac-
etaminophen for osteoarthritis, Cochrane Database Syst. Rev. 1 (2006).
[33] H. Jick, Risk of upper gastrointestinal bleeding and perforation associated
with individual non-steroidal anti-inflammatory drugs, Lancet North Am. Ed.
343 (8900) (1994) 769–772.
[34] Y.F. Chen, P. Jobanputra, P. Barton, S. Bryan, A. Fry-Smith, Gl Harris, R.S. Taylor,
Cyclooxygenase-2 selective non-steroidal anti-inflammatory drugs (etodolac,
meloxicam, celecoxib, rofecoxib, etoricoxib, valdecoxib and lumiracoxib) for
osteoarthritis and rheumatoid arthritis: a systematic review and economic
evaluation, NIHR Health Technology Assessment programme: Executive Sum-
maries, NIHR Journals Library, 2008.
[35] W. Zhang, R.W. Moskowitz, G. Nuki, S. Abramson, R.D. Altman, N. Arden,
S. Bierma-Zeinstra, K.D. Brandt, P. Croft, M. Doherty, M. Dougados, OARSI rec-
ommendations for the management of hip and knee osteoarthritis, Part II:
OARSI evidence-based, expert consensus guidelines, Osteoarthritis Cartilage
16 (2) (2008) 137–162.
[36] RR. Bannuru, NS. Natov, IE. Obadan, LL. Price, CH. Schmid, TE. McAlindon,
Therapeutic trajectory of hyaluronic acid versus corticosteroids in the treat-
ment of knee osteoarthritis: a systematic review and meta-analysis, Arthritis
Care Res. 61 (12) (2009) 1704–1711.
[37] N. Sodhi, MA. Mont, Survival of total hip replacements, Lancet North Am. Ed.
393 (10172) (2019) 613.
[38] J.S. Smolen, D. Aletaha, I.B. McInnes, Rheumatoid arthritis, Lancet 388 (2016)
2023–2038.
[39] American College of Rheumatology Subcommittee on Rheumatoid
Arthritis Guidelines, Guidelines for the management of rheuma-
toid arthritis: 2002 update, Arthritis Rheumatism 46 (2) (2002) 328–
346.
[40] J.S. Smolen, R. Landewé, F.C. Breedveld, M. Buch, G. Burmester, M. Dougados,
P. Emery, C. Gaujoux-Viala, L. Gossec, J. Nam, S. Ramiro, EULAR recommenda-
tions for the management of rheumatoid arthritis with synthetic and biolog-
ical disease-modifying antirheumatic drugs: 2013 update, Ann. Rheum. Dis.
73 (3) (2014) 492–509.
[41] C. Wang, Complementary and alternative medicine and osteoarthritis, Int. J.
Integr. Med. 1 (2013).
[42] TE. McAlindon, MP. LaValley, JP. Gulin, DT. Felson, Glucosamine and chon-
droitin for treatment of osteoarthritis: a systematic quality assessment and
meta-analysis, JAMA 283 (11) (20 0 0) 1469–1475.
S. Bose, N. Sarkar and D. Banerjee
[43] RP. Riemsma, JR. Kirwan, E. Taal, J.J. Hans, Patient education for adults with
rheumatoid arthritis, Cochrane Database Syst. Rev. 2 (2003).
[44] B.K. Vogler, E. Ernst, Aloe vera: a systematic review of its clinical effective-
ness, Br. J. Gen. Pract. 49 (447) (1999) 823–828.
[45] S. Sakka, P. Coulthard, Implant failure: etiology and complications, Med. Oral.
Patol. Oral. Cir. Bucal. 16 (1) (2011) e42–e44.
[46] C. Castro, E. Sanchez, A. Delgado, I. Soriano, P. Nunez, M. Baro, C. Evora,
Ciprofloxacin implants for bone infection. In vitro–in vivo characterization,
J. Control. Release 93 (3) (2003) 341–354.
[47] L. Langmead, D.S. Rampton, Complementary and alternative therapies
for inflammatory bowel disease, Aliment. Pharmacol. Ther. 23 (3) (2006)
341–349.
[48] M. Rodriguez-Bigas, N.I. Cruz, A. Suarez, Comparative evaluation of aloe vera
in the management of burn wounds in guinea pigs, Plast. Reconstr. Surg. 81
(3) (1988) 386–389.
[49] J.B. Kahlon, M.C. Kemp, R.H. Carpenter, B.H. McAnalley, H.R. McDaniel,
W.M. Shannon, Inhibition of AIDS virus replication by acemannan in vitro,
Mol. Biother. 3 (3) (1991) 127–135.
[50] G.K. King, K.M. Yates, P.G. Greenlee, K.R. Pierce, C.R. Ford, B.H. McAnalley,
I.R. Tizard, The effect of Acemannan Immunostimulant in combination with
surgery and radiation therapy on spontaneous canine and feline fibrosarco-
mas, J. Am. Anim. Hosp. Assoc. 31 (5) (1995) 439–447.
[51] P. Chantarawaratit, P. Sangvanich, W. Banlunara, K. Soontornvipart, P. Thun-
yakitpisal, Acemannan sponges stimulate alveolar bone, cementum and peri-
odontal ligament regeneration in a canine class II furcation defect model, J.
Periodontal Res. 49 (2) (2014) 164–178.
[52] S. Boonyagul, W. Banlunara, P. Sangvanich, P. Thunyakitpisal, Effect of ace-
mannan, an extracted polysaccharide from Aloe vera, on BMSCs proliferation,
differentiation, extracellular matrix synthesis, mineralization, and bone for-
mation in a tooth extraction model, Odontology 102 (2) (2014) 310–317.
[53] L. Zhang, I.R. Tizard, Activation of a mouse macrophage cell line by aceman-
nan: the major carbohydrate fraction from Aloe vera gel, Immunopharmacol-
ogy 35 (2) (1996) 119–128.
[54] D. Banerjee, S. Bose, Effects of Aloe vera gel extract in doped hydroxyapatite–
coated titanium implants on in vivo and in vitro biological properties, ACS
Applied Bio Mater. 2 (8) (2019) 3194–3202.
[55] A.D. Klein, N.S. Penneys, Aloe vera, J. Am. Acad. Dermatol. 18 (1988) 714–720.
[56] R. Rohanizadeh, Yi Deng, E. Verron, Therapeutic actions of curcumin in bone
disorders, BoneKEy Rep. 5 (2016).
[57] P. Anand, AB. Kunnumakkara, RA. Newman, BB. Aggarwal, Bioavailability of
curcumin: problems and promises, Mol. Pharm. 4 (6) (2007) 807–818.
[58] N. Sarkar, S. Bose, Liposome-encapsulated curcumin-loaded 3D printed scaf-
fold for bone tissue engineering, ACS Appl. Mater. Interfaces 11 (19) (2019)
17184–17192.
[59] A.C. Bharti, N. Donato, S. Singh, B.B. Aggarwal, Curcumin (diferuloyl-
methane) down-regulates the constitutive activation of nuclear factor–κ B
and Iκ Bα kinase in human multiple myeloma cells, leading to suppres-
sion of proliferation and induction of apoptosis, Blood 101 (3) (2003) 1053–
1062.
[60] Y.X. Xu, K.R. Pindolia, N. Janakiraman, R.A. Chapman, S.C. Gautam, Curcumin
inhibits IL1 alpha and TNF-alpha induction of AP-1 and NF-kB DNA-binding
activity in bone marrow stromal cells, Hematopathol. Mol. Hematol. 11 (1)
(1997) 49–62.
[61] AC. Bharti, Y. Takada, BB. Aggarwal, Curcumin (diferuloylmethane) inhibits
receptor activator of NF-κ B ligand-induced NF-κ B activation in osteoclast
precursors and suppresses osteoclastogenesis, J. Immunol. 172 (10) (2004)
5940–5947.
[62] K. Ozaki, Y. Kawata, S. Amano, S. Hanazawa, Stimulatory effect of curcumin
on osteoclast apoptosis, Biochem. Pharmacol. 59 (12) (20 0 0) 1577–1581.
[63] Z. Chen, J. Xue, T. Shen, S. Mu, Q. Fu, Curcumin alleviates glucocorticoid-in-
duced osteoporosis through the regulation of the Wnt signaling pathway, Int.
J. Mol. Med. 37 (2) (2016) 329–338.
[64] M. Hie, M. Yamazaki, I. Tsukamoto, Curcumin suppresses increased bone re-
sorption by inhibiting osteoclastogenesis in rats with streptozotocin-induced
diabetes, Eur. J. Pharmacol. 621 (1) (2009) 1–9.
[65] M. Mathy-Hartert, I. Jacquemond-Collet, F. Priem, C. Sanchez, C. Lambert,
Y. Henrotin, Curcumin inhibits pro-inflammatory mediators and metallopro-
teinase-3 production by chondrocytes, Inflamm. Res. 58 (12) (2009) 899.
[66] Ho-J Moon, W.-.K. Ko, S. Wook Han, D-Su Kim, Yu-S Hwang, H.-.K. Park,
IlK Kwon, Antioxidants, like coenzyme Q10, selenite, and curcumin, inhibited
osteoclast differentiation by suppressing reactive oxygen species generation,
Biochem. Biophys. Res. Commun. 418 (2) (2012) 247–253.
[67] R. Chang, L. Sun, TJ. Webster, selective cytotoxicity of curcumin on osteosar-
coma cells compared to healthy osteoblasts, Int. J. Nanomed. 9 (2014) 461.
[68] JM. Moran, R. Roncero-Martin, FJ. Rodriguez-Velasco, JF. Calderon-Garcia,
P. Rey-Sanchez, V. Vera, ML. Canal-Macias, JD. Pedrera-Zamorano, Effects of
curcumin on the proliferation and mineralization of human osteoblast-like
cells: implications of nitric oxide, Int. J. Mol. Sci. 13 (12) (2012) 16104–16118.
[69] D.L. French, J.M. Muir, C.E. Webber, The ovariectomized, mature rat model of
postmenopausal osteoporosis: an assessment of the bone sparing effects of
curcumin, Phytomedicine 15 (12) (2008) 1069–1078.
[70] S. Bose, N. Sarkar, D. Banerjee, Effects of PCL, PEG and PLGA polymers on cur-
cumin release from calcium phosphate matrix for in vitro and in vivo bone
regeneration, Mater. Today Chem. 8 (2018) 110–120.
[71] J. Epstein, IR. Sanderson, TT. MacDonald, Curcumin as a therapeutic agent:
88
Acta Biomaterialia 126 (2021) 63–91
the evidence from in vitro, animal and human studies, Br. J. Nutr. 103 (11)
(2010) 1545–1557.
[72] H Koch, L Lawson (Eds.), Garlic: the Science and Therapeutic Application of
Allium Sativum L. and Related Species, Williams & Wilkins, Baltimore, 1996.
[73] E. Block, The chemistry of garlic and onions, Sci. Am. 252 (1985) 114–
119.
[74] J.C. Harris, S.L. Cottrell, S. Plummer, D. Lloyd, Antimicrobial properties of Al-
lium sativum (garlic), Appl. Microbiol. Biotechnol. 57 (3) (2001) 282–286.
[75] S.B. Vohora, M. Rizwan, JA. Khan, Medicinal uses of common Indian vegeta-
bles, Planta Med. 23 (1973) 381–393.
[76] B.A. Salih, FM. Abasiyanik, Does regular garlic intake affect the prevalence
of Helicobacter pylori in asymptomatic subjects? Saudi Med. J. 24 (2003)
842–845.
[77] P.V. Venugopal, TV. Venugopal, Antidermatophytic activity of garlic (Allium
sativum) in vitro, Int. J. Dermatol. 34 (1995) 278–279.
[78] ND. Weber, DO. Andersen, JA. North, BK. Murray, LD. Lawson, BG. Hughes,
In vitro virucidal effects of Allium sativum (garlic) extract and compounds,
Planta Med. 58 (05) (1992) 417–423.
[79] R.S. Rivlin, Historical perspective on the use of garlic, J. Nutr. 131 (3) (2001)
951S–954S.
[80] J. Banu, E. Varela, G. Fernandes, Alternative therapies for the prevention and
treatment of osteoporosis, Nutr. Rev. 70 (1) (2012) 22–40.
[81] M. Mukherjee, A.S. Das, D. Das, S. Mukherjee, S. Mitra, C. Mitra, Role of oil
extract of garlic (Allium sativum Linn.) on intestinal transference of calcium
and its possible correlation with preservation of skeletal health in an ovariec-
tomized rat model of osteoporosis, Phytother. Res. 20 (5) (2006) 408–415.
[82] M. Roy, G.A. Fielding, A. Bandyopadhyay, S. Bose, Effects of zinc and stron-
tium substitution in tricalcium phosphate on osteoclast differentiation and
resorption, Biomater. Sci. 1 (1) (2013) 74–82.
[83] M. Mukherjee, A.S. Das, S. Mitra, C. Mitra, Prevention of bone loss by oil ex-
tract of garlic (Allium sativum Linn.) in an ovariectomized rat model of os-
teoporosis, Phytother. Res. 18 (5) (2004) 389–394.
[84] M. Mukherjee, A.S. Das, D. Das, S. Mukherjee, S. Mitra, C. Mitra, Effects of
garlic oil on postmenopausal osteoporosis using ovariectomized rats: com-
parison with the effects of lovastatin and 17β -estradiol, Phytotherapy Res.:
Int. J. Devoted Pharmacol. Toxicol. Eval. Nat. Prod. Deriv. 20 (1) (2006) 21–27.
[85] M. Patya, MA. Zahalka, A. Vanichkin, A. Rabinkov, T. Miron, D. Mirelman,
M. Wilchek, HM. Lander, A. Novogrodsky, Allicin stimulates lymphocytes and
elicits an antitumor effect: a possible role of p21 ras, Int. Immunol. 16 (2)
(2004) 275–281.
[86] V. Petrovic, A. Nepal, C. Olaisen, S. Bachke, J. Hira, C.K. Søgaard, Z. Bartsova,
Anti-cancer potential of homemade fresh garlic extract is related to increased
endoplasmic reticulum stress, Nutrients 10 (4) (2018) 450.
[87] A.M. Baruchin, A. Sagi, B. Yoffe, M. Ronen, Garlic burns, Burns 27 (7) (2001)
781–782.
[88] K.C. Srivastava, Aqueous extracts of onion, garlic and ginger inhibit platelet
aggregation and alter arachidonic acid metabolism, Biomed. Biochim. Acta 43
(8-9) (1984) S335–S346.
[89] V. Tyler, L. Brady, J. Robbers, Pharmacognosy (1981) 156-15.
[90] SP. Ginger, Common Spice and Wonder Drug, 3rd ed., Herbal Free Press Ltd,
Brattleboro (VT), 1996.
[91] A.R. Swain, S.P. Duton, AS. Truswell, Salicylates in foods, J. Am. Diet Assoc. 85
(1985) 950–960.
[92] F. Kiuchi, S. Iwakami, M. Shibuya, F. Hanaoka, U. Sandawa, Inhibition of
prostaglandin and leukotriene biosynthesis by gingeroles and diarylhep-
tanoids, Chem. Pharm. Bull. (Tokyo) 40 (1992) 187–191.
[93] U. Jana, R.N. Chattopadhayay, BP. Shaw, Preliminary studies on anti-in-
flammatory activity of Zingiber officinale Rosc., Vitex negundo Linn. and
Tinospora cordifolia (Willid) miers in albino rats, Indian J. Pharmacol. 31
(1999) 232–233.
[94] R.D. Altman, K.C. Marcussen, Effects of a ginger extract on knee pain in pa-
tients with osteoarthritis, Arthritis Rheumatol. 44 (11) (2001) 2531–2538.
[95] H. Bliddal, A. Rosetzsky, P. Schlichting, M.S. Weidner, L.A. Andersen,
H-H. Ibfelt, K. Christensen, O.N. Jensen, J. Barslev, A randomized, placebo–
controlled, cross-over study of ginger extracts and ibuprofen in osteoarthritis,
Osteoarthritis Cartilage 8 (1) (20 0 0) 9–12.
[96] J.L. Funk, J.B. Frye, L.E. Wright, B.N. Timmermann, Effects of Ginger (Zingiber
officialis L) on Inflammation-Induced Bone Loss, FASEB J. 26 (1 Supplement)
(2012) 263–265.
[97] J.Z. Fan, X. Yang, Z.G. Bi, The effects of 6-gingerol on proliferation, differenti-
ation, and maturation of osteoblast-like MG-63 cells, Braz. J. Med. Biol. Res.
48 (7) (2015) 637–643.
[98] K.C. Srivastava, MT. Ginger, (Zingiber officinale) and rheumatic disorders,
Med. Hypothesis 29 (1989) 25–28.
[99] K.C. Srivastava, MT. Ginger, Zingiber officinale) in rheumatism and muscu-
loskeletal disorders, Med. Hypotheses 39 (1992) 342–348.
[100] A. Leung, Encyclopedia of Common Natural Ingredients Used in Food, Drugs,
and Cosmetics, John Wiley, New York, 1980.
[101] J.K. Kundu, H.K. Na, YJ. Surh, Ginger-derived phenolic substances with cancer
preventive and therapeutic potential, Forum Nutr. 61 (2009) 182–192.
[102] A.K. Pillai, KK. Sharma, YK. Gupta, S. Bakhshi, Anti-emetic effect of ginger
powder versus placebo as an add-on therapy in children and young adults
receiving high emetogenic chemotherapy, Pediatr. Blood Cancer 56 (2) (2011)
234–238.
[103] N. Mascolo, R. Jain, S.C. Jain, F. Capasso, Ethnopharmacologic investigation of
ginger (Zingiber officinale), J. Ethnopharmacol. 27 (1-2) (1989) 129–140.
S. Bose, N. Sarkar and D. Banerjee
[104] J.E. Pizzorno, Textbook of Natural Medicine, Elsevier Health Sciences, 2013.
[105] A.E. Hak, K.H. Polderman, I.C. Westendorp, C. Jakobs, A. Hofman, J.C. Wit-
teman, C.D. Stehouwer, Increased plasma homocysteine after menopause,
Atherosclerosis 149 (1) (20 0 0) 163–168.
[106] J.B. van Meurs, R.A. Dhonukshe-Rutten, S.M. Pluijm, M. van der Klift, R. de
Jonge, J. Lindemans, M.M. Breteler, Homocysteine levels and the risk of os-
teoporotic fracture, N. Engl. J. Med. 350 (20) (2004) 2033–2041.
[107] LE. Brattström, BL. Hultberg, J.E. Hardebo, Folic acid responsive post-
menopausal homocysteinemia, Metabolism 34 (11) (1985) 1073–
1077.
[108] Y. Sato, Y. Honda, J. Iwamoto, T. Kanoko, K. Satoh, Effect of folate and
mecobalamin on hip fractures in patients with stroke: a randomized con-
trolled trial, JAMA 293 (9) (2005) 1082–1088.
[109] A. Cagnacci, F. Baldassari, G. Rivolta, S. Arangino, A. Volpe, Relation of homo-
cysteine, folate, and vitamin B12 to bone mineral density of postmenopausal
women, Bone 33 (6) (2003) 956–959.
[110] R.A.M. Dhonukshe-Rutten, S.M.F. Pluijm, L.C. Groot, P. Lips, JH. Smit,
WA. Staveren, Homocysteine and vitamin B12 status relate to bone turnover
markers, broadband ultrasound attenuation, and fractures in healthy elderly
people, J. Bone Miner. Res. 20 (6) (2005) 921–929.
[111] J.B. Goerss, C.H. Kim, E.J. Atkinson, R. Eastell, W.M. O’Fallon, L.J. Melton, Risk
of fractures in patients with pernicious anemia, J. Bone Miner. Res. 7 (5)
(1992) 573–579.
[112] G.S. Kim, C.H. Kim, J.Y. Park, K.U. Lee, C.S. Park, Effects of vitamin B12 on cell
proliferation and cellular alkaline phosphatase activity in human bone mar-
row stromal osteoprogenitor cells and UMR106 osteoblastic cells, Metabolism
45 (12) (1996) 1443–1446.
[113] J.M. Koh, Y.S. Lee, Y.S. Kim, D.J. Kim, H.H. Kim, J.Y. Park, G.S. Kim, Homocys-
teine enhances bone resorption by stimulation of osteoclast formation and
activity through increased intracellular ROS generation, J. Bone Miner. Res. 21
(7) (2006) 1003–1011.
[114] G. Barber, G. Spaeth, Pyridoxine therapy in homocystinuria, Lancet 1 (1967)
337.
[115] Abnormalities in fracture healing induced by vitamin B6 deficiency in rats,
Bone 7 (1986) 489–495.
[116] S. Muraki, S. Yamamoto, H. Ishibashi, H. Oka, N. Yoshimura, H. Kawaguchi,
K. Nakamura, Diet and lifestyle associated with increased bone mineral den-
sity: cross-sectional study of Japanese elderly women at an osteoporosis out-
patient clinic, J. Orthop. Sci. 12 (4) (2007) 317–320.
[117] C-Li Shen, JJ. Cao, RY. Dagda, TE. Tenner, M.-.C. Chyu, JK. Yeh, Supplementa-
tion with green tea polyphenols improves bone microstructure and quality in
aged, orchidectomized rats, Calcif. Tissue Int. 88 (6) (2011) 455–463.
[118] C-Li Shen, JK. Yeh, JJ. Cao, J.-.S. Wang, Green tea and bone metabolism, Nutr.
Res. 29 (7) (2009) 437–456.
[119] C.L. Shen, P. Wang, J. Guerrieri, J.K. Yeh, J.S. Wang, Protective effect of green
tea polyphenols on bone loss in middle-aged female rats, Osteoporos. Int. 19
(7) (2008) 979–990.
[120] C.L. Shen, M.C. Chyu, J.K. Yeh, Y. Zhang, B.C. Pence, C.K. Felton, J.S. Wang, Ef-
fect of green tea and Tai Chi on bone health in postmenopausal osteopenic
women: a 6-month randomized placebo-controlled trial, Osteoporos. Int. 23
(5) (2012) 1541–1552.
[121] C.H. Chen, M.L. Ho, J.K. Chang, S.H. Hung, G.J. Wang, Green tea catechin en-
hances osteogenesis in a bone marrow mesenchymal stem cell line, Osteo-
poros. Int. 16 (12) (2005) 2039–2045.
[122] R.C. Mühlbauer, A. Lozano, A. Reinli, Onion and a mixture of vegetables, sal-
ads, and herbs affect bone resorption in the rat by a mechanism independent
of their base excess, J. Bone Miner. Res. 17 (7) (2002) 1230–1236.
[123] A. Murakami, H. Ashida, J. Terao, Multitargeted cancer prevention by
quercetin, Cancer Lett. 269 (2) (2008) 315–325.
[124] M.N. Horcajada-Molteni, V. Crespy, V. Coxam, M.J. Davicco, C. Rémésy, J.P. Bar-
let, Rutin inhibits ovariectomy-induced osteopenia in rats, J. Bone Miner. Res.
15 (11) (20 0 0) 2251–2258.
[125] Y.J. Yang, Z.L. Yang, D.C. Wang, X.C. Xiao, P. Li, Comparative study on effects
of rutin and quercetin on metabolism in osteoblast cells, Zhong yao cai=
Zhongyaocai= J. Chin. Med. Mater. 29 (5) (2006) 467–470.
[126] H.A. Wetli, R. Brenneisen, I. Tschudi, M. Langos, P. Bigler, T. Sprang,
R.C. Mühlbauer, A γ -glutamyl peptide isolated from onion (Allium cepa L.)
by bioassay-guided fractionation inhibits resorption activity of osteoclasts, J.
Agric. Food Chem. 53 (9) (2005) 3408–3414.
[127] C.M. Rassi, M. Lieberherr, G. Chaumaz, A. Pointillart, G. Cournot, Modulation
of osteoclastogenesis in porcine bone marrow cultures by quercetin and rutin,
Cell Tissue Res. 319 (3) (2005) 383–393.
[128] CR. Cederroth, S. Nef, Soy, phytoestrogens and metabolism: a review, Mol.
Cell. Endocrinol. 304 (1-2) (2009) 30–42.
[129] F. Branca, Dietary phyto-oestrogens and bone health, Proc. Nutr. Soc. 62 (4)
(2003) 877–887.
[130] B. Li, S. Yu, Genistein prevents bone resorption diseases by inhibiting bone
resorption and stimulating bone formation, Biol. Pharm. Bull. 26 (6) (2003)
780–786.
[131] N. Sarkar, S. Bose, Controlled release of soy isoflavones from multifunc-
tional 3D printed bone tissue engineering scaffolds, Acta Biomater. 114 (2020)
407–420 2020.
[132] SL. Silverman, RE. Madison, Decreased incidence of hip fracture in Hispanics,
Asians, and blacks: California Hospital Discharge Data, Am. J. Public Health
78 (11) (1988) 1482–1483.
89
Acta Biomaterialia 126 (2021) 63–91
[133] SE. Putnam, AM. Scutt, K. Bicknell, CM. Priestley, EM. Williamson, Natu-
ral products as alternative treatments for metabolic bone disorders and for
maintenance of bone health, Phytotherapy Res.: Int. J. Devoted Pharmacol.
Toxicol. Eval. Nat. Prod. Deriv. 21 (2) (2007) 99–112.
[134] A. Cotter, KD. Cashman, Genistein appears to prevent early postmenopausal
bone loss as effectively as hormone replacement therapy, Nutr. Rev. 61 (10)
(2003) 346–351.
[135] BH. Arjmandi, EA. Lucas, DA. Khalil, L. Devareddy, BJ. Smith, J. McDonald,
AB. Arquitt, ME. Payton, C. Mason, One year soy protein supplementation
has positive effects on bone formation markers but not bone density in post-
menopausal women, Nutr. J. 4 (1) (2005) 8.
[136] A.H. Roudsari, F. Tahbaz, A. Hossein-Nezhad, B. Arjmandi, B. Larijani,
S.M. Kimiagar, Assessment of soy phytoestrogens’ effects on bone turnover
indicators in menopausal women with osteopenia in Iran: a before and after
clinical trial, Nutr. J. 4 (1) (2005) 30.
[137] D.A. Khalil, E.A. Lucas, B.J. Smith, D.Y. Soung, L. Devareddy, S. Juma,
MP. Akhter, R. Recker, B.H. Arjmandi, Soy isoflavones may protect against
orchidectomy-induced bone loss in aged male rats, Calcif. Tissue Int. 76 (1)
(2005) 56–62.
[138] W.-.P. Koh, AH. Wu, R. Wang, Li-W Ang, D. Heng, J.-.M. Yuan, MC. Yu, Gen-
der-specific associations between soy and risk of hip fracture in the Singa-
pore Chinese Health Study, Am. J. Epidemiol. 170 (7) (2009) 901–909.
[139] JD. Adachi, R. Rizzoli, S. Boonen, Z. Li, MP. Meredith, CH. Chesnut, Vertebral
fracture risk reduction with risedronate in post-menopausal women with os-
teoporosis: a meta-analysis of individual patient data, Aging Clin. Exp. Res. 17
(2) (2005) 150–156.
[140] U. Gröber, J. Reichrath, M.F. Holick, K. Kisters, Vitamin K: an old vitamin in a
new perspective, Dermato-Endocrinol. 6 (1) (2014) e968490.
[141] N. Sarkar, S. Bose, Controlled delivery of curcumin and vitamin K2 from hy-
droxyapatite-coated titanium implant for enhanced in vitro chemoprevention,
osteogenesis, and in vivo osseointegration, ACS Appl. Mater. Interfaces 12 (12)
(2020) 13644–13656.
[142] N. Sakamoto, T. Nishiike, H. Iguchi, K. Sakamoto, The effect of diet on blood
vitamin K status and urinary mineral excretion assessed by a food question-
naire, Nutr. Health 13 (1) (1999) 1–10.
[143] Y. Koshihara, K. Hoshi, H. Ishibashi, M. Shiraki, Vitamin K 2 promotes 1α , 25
(OH) 2 vitamin D 3-induced mineralization in human periosteal osteoblasts,
Calcif. Tissue Int. 59 (6) (1996) 466–473.
[144] J.P. Hart, M.J. Shearer, L. Klenerman, A. Catterall, J. Reeve, P.N. Sambrook,
R.A. Dodos, L. Bitensky, J. Chayen, Electrochemical detection of depressed cir-
culating levels of vitamin K1 in osteoporosis, J. Clin. Endocrinol. Metabolism
60 (6) (1985) 1268–1269.
[145] T. Kanai, T. Takagi, K. Masuhiro, M. Nakamura, M. Iwata, F. Saji, Serum vitamin
K level and bone mineral density in post-menopausal women, Int. J. Gynecol.
Obstetr. 56 (1) (1997) 25–30.
[146] S.J. Hodges, K. Akesson, P. Vergnaud, K. Obrant, P.D. Delmas, Circulating levels
of vitamins K1 and K2 decreased in elderly women with hip fracture, J. Bone
Miner. Res. 8 (10) (1993) 1241–1245.
[147] M. Kaneki, S.J. Hedges, T. Hosoi, S. Fujiwara, A. Lyons, N. Ishida, M. Nakagawa,
M. Takechi, Y. Sano, Y. Mizuno, S. Hoshino, Japanese fermented soybean food
as the major determinant of the large geographic difference in circulating lev-
els of vitamin K2: possible implications for hip-fracture risk, Nutrition 17 (4)
(2001) 315–321.
[148] SA. Jamal, WS. Browner, DC. Bauer, SR. Cummings, Warfarin use and
o risk for osteoporosis in elderly women, Ann. Intern. Med. 128 (10) (1998)
829–832.
[149] L.J. Sokoll, S.L. Booth, M.E. O’Brien, K.W. Davidson, K.I. Tsaioun, J.A. Sadowski,
Changes in serum osteocalcin, plasma phylloquinone, and urinary gamma–
carboxyglutamic acid in response to altered intakes of dietary phylloquinone
in human subjects, Am. J. Clin. Nutr. 65 (3) (1997) 779–784.
[150] A.M. Craciun, J. Wolf, M.H.J. Knapen, F.J.P.H. Brouns, C. Vermeer, Improved
bone metabolism in female elite athletes after vitamin K supplementation,
Int. J. Sports Med. 19 (7) (1998) 479–484.
[151] M.H.J. Knapen, K. Hamulyák, C. Vermeer, The effect of vitamin K supplemen-
tation on circulating osteocalcin (bone Gla protein) and urinary calcium ex-
cretion, Ann. Intern. Med. 111 (12) (1989) 1001–1005.
[152] T. Akjba, S. Kurihara, K. Tachibana, Vitamin K increased bone mass in hemo–
dialysis patients with low-turnover bone disease, J. Am. Soc. Nephrol. 608
(1991) 42P.
[153] H. Orimo, Clinical evaluation of menatetrenone in the treatment of in-
volutional osteoporosis-A double-blind multicenter comparative study with
1α -hydroxyvitamin D_3, J. Bone Miner Res. 7 (1992) S122.
[154] J. Iwamoto, T. Takeda, S. Ichimura, Effect of combined administration of vi-
tamin D 3 and vitamin K 2 on bone mineral density of the lumbar spine
in postmenopausal women with osteoporosis, J. Orthop. Sci. 5 (6) (20 0 0)
546–551.
[155] C. Bolton-Smith, P.A. Mole, M.E.T. McMurdo, M.J. Shearer, Two-year interven-
tion with phylloquinone (vitamin K1), vitamin D and calcium: effect on bone
mineral content of older women, Ann. Nutr. Metabolism 45 (Suppl. 1) (2001)
246.
[156] Y. Ishida, H. Soh, S. Ogawa, S. Kawahara, H. Murata, A one-year randomized
controlled trial of hormone replacement therapy, bisphosphonate, calcitonin,
vitamin D, and vitamin K, in women with postmenopausal osteoporosis, J.
Bone Miner. Res. 15 (1) (20 0 0) SA409-SA409.
[157] I.O.M Copper, Dietary Reference Intakes for Vitamin A Vitamin K, Arsenic,
Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Sil-
S. Bose, N. Sarkar and D. Banerjee
icon, Vanadium, and Zinc, The National Academies Press, Washington, DC,
2001.
[158] H. Orimo, M. Shiraki, A. Tomita, H. Morii, T. Fujita, M. Ohata, Effects of
menatetrenone on the bone and calcium metabolism in osteoporosis: a
double-blind placebo-controlled study, J. Bone Miner. Metab. 16 (2) (1998)
106–112.
[159] H.H Conaway, P. Henning, UH. Lerner, Vitamin A metabolism, action, and role
in skeletal homeostasis, Endocr. Rev. 34 (6) (2013) 766–797.
[160] EH. Harrison, Mechanisms involved in the intestinal absorption of dietary
vitamin A and provitamin A carotenoids, Biochimica et Biophysica Acta
(BBA)-Mol. Cell Biol.Lipids 1821 (1) (2012) 70–77.
[161] S.B Wolbach, Vitamin-A deficiency and excess in relation to skeletal growth,
JBJS 29 (1) (1947) 171–192.
[162] H. Melhus, K. Michaelsson, A. Kindmark, R. Bergstrom, L. Holmberg,
H. Mallmin, A. Wolk, S. Ljunghall, Excessive dietary intake of vitamin A is
associated with reduced bone mineral density and increased risk for hip frac-
ture, Ann. Intern. Med. 129 (10) (1998) 770–778.
[163] H.H Conaway, A. Pirhayati, E. Persson, U. Pettersson, O. Svensson, C. Lind-
holm, P. Henning, J. Tuckermann, UH. Lerner, Retinoids stimulate pe-
riosteal bone resorption by enhancing the protein RANKL, a response inhib-
ited by monomeric glucocorticoid receptor, J. Biol. Chem. 286 (36) (2011)
31425–31436.
[164] E. Mellanby, Vitamin A and bone growth: the reversibility of vitamin A-defi-
ciency changes, J. Physiol. 105 (4) (1947) 382–399.
[165] I. Dickson, J. Walls, Vitamin A and bone formation. Effect of an excess
of retinol on bone collagen synthesis in vitro, Biochem. J. 226 (3) (1985)
789–795.
[166] HB. Fell, E. Mellanby, Effects of hypervitaminosis A on foetal mouse bones
cultivated in vitro, Br. Med. J. 2 (4678) (1950) 535.
[167] SQ. Cohlan, Congenital anomalies in the rat produced by excessive intake of
vitamin A during pregnancy, Pediatrics 13 6 (1954) 556–567.
[168] D. Feskanich, V. Singh, WC. Willett, GA. Colditz, Vitamin A intake and hip
fractures among postmenopausal women, JAMA 287 (1) (2002) 47–54.
[169] J.J.B. Anderson, Oversupplementation of vitamin A and osteoporotic fractures
in the elderly: to supplement or not to supplement with vitamin A, J. Bone
Miner. Res. 17 (8) (2002) 1359–1362.
[170] S. Saneshige, H. Mano, K. Tezuka, S. Kakudo, Y. Mori, Y. Honda, A. Itabashi,
T. Yamada, K. Miyata, Y. Hakeda, J. Ishii, Retinoic acid directly stimulates os-
teoclastic bone resorption and gene expression of cathepsin K/OC-2, Biochem.
J. 309 (3) (1995) 721–724.
[171] T. Rokkaku, R. Kimura, C. Ishikawa, T. Yasumoto, M. Senba, F. Kanaya, N. Mori,
Anticancer effects of marine carotenoids, fucoxanthin and its deacety-
lated product, fucoxanthinol, on osteosarcoma, Int. J. Oncol. 43 (4) (2013)
1176–1186.
[172] J. McGuire, JP. Lawson, Skeletal changes associated with chronic isotretinoin
and etretinate administration, Dermatology 175 (Suppl. 1) (1987) 169–181.
[173] S. Chambial, S. Dwivedi, K.K. Shukla, PJ. John, P. Sharma, Vitamin C in dis-
ease prevention and cure: an overview, Indian J. Clin. Biochem. 28 (4) (2013)
314–328.
[174] VH. Guaiquil, J.C. Vera, DW. Golde, Mechanism of vitamin C inhibition of cell
death induced by oxidative stress in glutathione-depleted HL-60 cells, J. Biol.
Chem. 276 (44) (2001) 40955–40961.
[175] P. Aghajanian, S. Hall, MD. Wongworawat, S. Mohan, The roles and mecha-
nisms of actions of vitamin C in bone: new developments, J. Bone Miner. Res.
30 (11) (2015) 1945–1955.
[176] H. Melhus, K. Michaëlsson, L. Holmberg, A. Wolk, S. Ljunghall, Smoking, an-
tioxidant vitamins, and the risk of hip fracture, J. Bone Miner. Res. 14 (1)
(1999) 129–135.
[177] S. Bose, N. Sarkar, S. Vahabzadeh, Sustained release of vitamin C from PCL
coated TCP induces proliferation and differentiation of osteoblast cells and
suppresses osteosarcoma cell growth, Mater. Sci. Eng.: C 105 (2019) 110096.
[178] N. Sarkar, H. Morton, S. Bose, Effects of vitamin C on osteoblast proliferation
and osteosarcoma inhibition using plasma coated hydroxyapatite on titanium
implants, Surf. Coat. Technol. (2020) 125793.
[179] PE. Boyle, OA. Bessey, PR. Howe, Rate of dentin formation in incisor teeth of
guineapigs on normal and on ascorbic acid-deficient diets, Arch. Pathol. 30
(1940) 90–107.
[180] T. Alcantara-Martos, A.D. Delgado-Martinez, M.V. Vega, M.T. Carrascal,
L. Munuera-Martinez, Effect of vitamin C on fracture healing in elderly Os-
teogenic Disorder Shionogi rats, J. Bone Joint Surg. Br. 89 (3) (2007) 402–
407.
[181] MT. Hannan, DT. Felson, JJ. Anderson, Bone mineral density in elderly men
and women: results from the Framingham osteoporosis study, J. Bone Miner.
Res. 7 (5) (1992) 547–553.
[182] S. Sahni, MT. Hannan, D. Gagnon, J. Blumberg, L. Adrienne Cupples, DP. Kiel,
KL. Tucker, High vitamin C intake is associated with lower 4-year bone loss
in elderly men, J. Nutr. 138 (10) (2008) 1931–1938.
[183] RT. Franceschi, BS. Iyer, Relationship between collagen synthesis and expres-
sion of the osteoblast phenotype in MC3T3-E1 cells, J. Bone Miner. Res. 7 (2)
(1992) 235–246.
[184] A. Falchetti, R. Cosso, The interaction between vitamin C and bone health: a
narrative review, Expert Rev. Precis. Med. Drug Dev. (2018) just-accepted.
[185] DJ. Morton, EL. Barrett-Connor, DL. Schneider, Vitamin C supplement use and
bone mineral density in postmenopausal women, J. Bone Miner. Res. 16 (1)
(2001) 135–140.
90
Acta Biomaterialia 126 (2021) 63–91
[186] SG. Leveille, AZ. LaCroix, TD. Koepsell, SA. Beresford, G. Van Belle, DM. Buch-
ner, Dietary vitamin C and bone mineral density in postmenopausal women
in Washington State, USA, J. Epidemiol. Commun. Health 51 (5) (1997)
479–485.
[187] J. Zhang, RG. Munger, NA. West, D.R Cutler, HJ. Wengreen, CD. Corcoran, An-
tioxidant intake and risk of osteoporotic hip fracture in Utah: an effect mod-
ified by smoking status, Am. J. Epidemiol. 163 (1) (2006) 9–17.
[188] JA. Simon, ES. Hudes, Relation of ascorbic acid to bone mineral density and
self-reported fractures among US adults, Am. J. Epidemiol. 154 (5) (2001)
427–433.
[189] J. Lykkesfeldt, A.J. Michels, B. Frei, C. Vitamin, Adv. Nutr. 5 (1) (2014) 16–18
Published 2014 Jan 1, doi:10.3945/an.113.005157.
[190] S.W.F. Kletzien, V.M. Templin, H. Steenbock, B.H. Thomas, Vitamin D and the
conservation of calcium in the adult, J. Biol. Chem. 97 (1932) 265–280.
[191] L.G. Raisz, C.L. Trummel, M.F. Holick, H.F. DeLuca, 1,25-Dihydroxycholecalcif-
erol: a potent stimulator of bone resorption in tissue culture, Science 175
(1972) 768–769.
[192] HA. Bischoff-Ferrari, E. Giovannucci, WC. Willett, T. Dietrich, B. Daw-
son-Hughes, Estimation of optimal serum concentrations of 25-hydroxyvita-
min D for multiple health outcomes, Am. J. Clin. Nutr. 84 (1) (2006) 18–28.
[193] J. Porthouse, S. Cockayne, C. King, L. Saxon, E. Steele, T. Aspray, M. Baverstock,
Y. Birks, J. Dumville, R. Francis, C. Iglesias, Randomised controlled trial of cal-
cium and supplementation with cholecalciferol (vitamin D3) for prevention
of fractures in primary care, BMJ 330 (7498) (2005) 1003.
[194] RG. Cumming, Calcium intake and bone mass: a quantitative review of the
evidence, Calcif. Tissue Int. 47 (1990) 194–201.
[195] RECORD Trial Group, Oral vitamin D3 and calcium for secondary prevention
of low-trauma fractures in elderly people (Randomised Evaluation of Calcium
Or vitamin D, RECORD): a randomised placebo-controlled trial, Lancet North
Am. Ed. 365 (9471) (2005) 1621–1628.
[196] HA. Bischoff-Ferrari, WC. Willett, JB. Wong, E. Giovannucci, T. Dietrich,
B. Dawson-Hughes, Fracture prevention with vitamin D supplementation:
a meta-analysis of randomized controlled trials, JAMA 293 (18) (2005)
2257–2264.
[197] H.A. Bischoff, H.B. Stähelin, W. Dick, R. Akos, M. Knecht, C. Salis, M. Nebiker,
R. Theiler, M. Pfeifer, B. Begerow, R.A. Lew, Effects of vitamin D and calcium
supplementation on falls: a randomized controlled trial, J. Bone Miner. Res.
18 (2) (2003) 343–351.
[198] M. Pfeifer, B. Begerow, HW. Minne, C. Abrams, D. Nachtigall, C. Hansen, Ef-
fects of a short-term vitamin D and calcium supplementation on body sway
and secondary hyperparathyroidism in elderly women, J. Bone Miner. Res. 15
(6) (20 0 0) 1113–1118.
[199] A.A. Vu, S. Bose, Effects of vitamin D 3 release from 3D printed calcium phos-
phate scaffolds on osteoblast and osteoclast cell proliferation for bone tissue
engineering, RSC Adv. 9 (60) (2019) 34847–34853.
[200] A.A. Vu, S. Bose, Vitamin D 3 release from traditionally and additively
manufactured tricalcium phosphate bone tissue engineering scaffolds, Ann.
Biomed. Eng. 48 (3) (2020) 1025–1033.
[201] N. Binkley, R. Ramamurthy, D. Krueger, Low vitamin D status: definition,
prevalence, consequences, and correction, Endocrinol. Metab. Clin. North Am.
39 (2) (2010) 287–301.
[202] CF. Garland, CB. French, LL. Baggerly, RP. Heaney, Vitamin D supplement doses
and serum 25-hydroxyvitamin D in the range associated with cancer preven-
tion, Anticancer Res. 31 (2) (2011) 607–611.
[203] S.P. Wasser, AL. Weis, Therapeutic effects of substances occurring in higher
Basidiomycetes mushrooms: a modern perspective, Crit. Rev. Immunol. 19
(1999) 65–96.
[204] S. Genaro Pde, L.A. Martini, Vitamin A supplementation and risk of skeletal
fracture, Nutr. Rev. 62 (2004) 65–67.
[205] MF. Holick, Sunlight and vitamin D for bone health and prevention of au-
toimmune diseases, cancers, and cardiovascular diseases, Am. J. Clin. Nutr. 80
(Suppl 6) (2004) 1678S-188S.
[206] M.J. Lee, S.E. Kim, J. Park, G.Y. Ahn, T.H. Yun, I. Choi, H.-.J. Kim, S.-.W. Choi,
Curcumin-loaded biodegradable polyurethane scaffolds modified with gelatin
using 3D printing technology for cartilage tissue engineering, Polym. Adv.
Technol. 30 (12) (2019) 3083–3090.
[207] R. Sedghi, N. Sayyari, A. Shaabani, H. Niknejad, T. Tayebi, Novel biocompatible
zinc-curcumin loaded coaxial nanofibers for bone tissue engineering applica-
tion, Polymer 142 (2018) 244–255.
[208] A. Merolli, L. Nicolais, L. Ambrosio, M. Santin, A degradable soybean-based
biomaterial used effectively as a bone filler in vivo in a rabbit, Biomed. Mater.
5 (1) (2010) 015008.
[209] M. Santin, C. Morris, G. Standen, L. Nicolais, L. Ambrosio, A new class of
bioactive and biodegradable soybean-based bone fillers, Biomacromolecules
8 (9) (2007) 2706–2711.
[210] I.M.V. Soares, G.V.O. Fernandes, L.C. Cavalcante, Y.K.P.C. Leite, D.O. Bezerra,
M.A.M. Carvalho, C.M.R.S. Carvalho, The influence of Aloe vera with mes-
enchymal stem cells from dental pulp on bone regeneration: characterization
and treatment of non-critical defects of the tibia in rats, J. Appl. Oral Sci.
(2019) 27.
[211] S. Jain, S.R.K. Meka, K. Chatterjee, Curcumin eluting nanofibers augment os-
teogenesis toward phytochemical based bone tissue engineering, Biomed.
Mater. 11 (5) (2016) 055007.
[212] S. Sebastiammal, A.S.L. Fathima, S. Devanesan, M.S. AlSalhi, H. Johnson,
M. Govindarajan, B. Vaseeharan, Curcumin-encased hydroxyapatite nanopar-
S. Bose, N. Sarkar and D. Banerjee
ticles as novel biomaterials for antimicrobial, antioxidant and anticancer ap-
plications: a perspective of nano-based drug delivery, J. Drug Delivery Sci.
Technol. (2020) 101752.
[213] Y. Li, Z.Z. Zhang, Sustained curcumin release from PLGA microspheres im-
proves bone formation under diabetic conditions by inhibiting the reactive
oxygen species production, Drug Des. Dev. Therapy 12 (2018) 1453.
[214] Y. Wang, W. He, H. Hao, J. Wu, N. Qin, Eggshell derived Se-doped HA
nanorods for enhanced antitumor effect and curcumin delivery, J. Sol-Gel Sci.
Technol. 87 (3) (2018) 600–607.
[215] D. Godoy, D. Jeanne, J. Chokboribal, R. Pauwels, W. Banlunara, P. Sangvanich,
S. Jaroenporn, P. Thunyakitpisal, Acemannan increased bone surface, bone
volume, and bone density in a calvarial defect model in skeletally-mature
rats, J. Dent. Sci. 13 (4) (2018) 334–341.
[216] V. Mouriño, AR. Boccaccini, Bone tissue engineering therapeutics: controlled
drug delivery in three-dimensional scaffolds, J. R. Soc., Interface 7 (43) (2010)
209–227.
[217] S. Bose, S. Tarafder, Calcium phosphate ceramic systems in growth factor and
drug delivery for bone tissue engineering: a review, Acta Biomater. 8 (4)
(2012) 1401–1421.
[218] T.A. Holland, A.G. Mikos, Biodegradable polymeric scaffolds. Improvements in
bone tissue engineering through controlled drug delivery, in: Tissue Engineer-
ing I, Springer, Berlin, Heidelberg, 2005, pp. 161–185.
[219] S. Maher, A. Mazinani, M.R. Barati, D. Losic, Engineered titanium im-
plants for localized drug delivery: recent advances and perspectives of Ti-
tania nanotubes arrays, Expert Opin. Drug Deliv. 15 (10) (2018) 1021–
1037.
[220] J.R. Porter, T.T. Ruckh, K.C. Popat, Bone tissue engineering: a review in bone
biomimetics and drug delivery strategies, Biotechnol. Prog. 25 (6) (2009)
1539–1560.
[221] J. Panyam, V. Labhasetwar, Biodegradable nanoparticles for drug and gene de-
livery to cells and tissue, Adv. Drug. Deliv. Rev. 55 (3) (2003) 329–347.
[222] A.K. Ekenseair, F.K. Kasper, A.G. Mikos, Perspectives on the interface of drug
delivery and tissue engineering, Adv. Drug. Deliv. Rev. 65 (1) (2013) 89–92.
91
Acta Biomaterialia 126 (2021) 63–91
[223] X. Bao, L. Zhu, X. Huang, D. Tang, D. He, J. Shi, G. Xu, 3D biomimetic ar-
tificial bone scaffolds with dual-cytokines spatiotemporal delivery for large
weight-bearing bone defect repair, Sci. Rep. 7 (1) (2017) 1–13.
[224] H. Nasri, H. Shirzad, Toxicity and safety of medicinal plants, J. Herbmed Phar-
macol. 2 (2) (2013) 21–22.
[225] SS. Deshpande, in: Handbook of Food Toxicology. Marcel Dekker, Marcel
Dekker. Inc., New York, 2002, pp. 1–5.
[226] S. Desai, P. Babaria, M. Nakarani, K. Shah, A. Paranjape, Antiosteoporotic effect
of hemidesmus indicus linn. On ovariectomised rats, J. Ethnopharmacol. 199
(2017) 1–8, doi:10.1016/j.jep.2017.01.031.
[227] WHO, in: WHO Guidelines on Safety Monitoring of Herbal Medicines in Phar-
macovigilance Systems, World Health Organization, Geneva, 2004, p. 82.
[228] G. Balammal, M.S. Babu, PJ. Reddy, Analysis of herbal medicines by mod-
ern chromatographic techniques, Int. J. Preclin. Pharmaceut. Res. 3 (1) (2012)
50–63.
[229] L. Lv, W. Huang, X. Yu, H. Ren, R. Sun, Comparative research of different
Bupleurum chinense composition to influence of hepatotoxicity of rats and
oxidative damage mechanism, China J. Chin. Materia Medica 34 (18) (2009)
2364–2368.
[230] P.K. Mukherjee, Quality evaluation of herbal medicines: challenges and op-
portunities, Qual. Control Eval. Herb. Drugs (2019) 53–77.
[231] S.B. Goodman, J. Pajarinen, Z. Yao, T.D. Lin, Inflammation and bone repair:
from particle disease to tissue regeneration, Front. Bioeng. Biotechnol. 7
(2019) 230.
[232] R. Mazzei, M. Leonti, S. Spadafora, A. Patitucci, G. Tagarelli, A review of
the antimicrobial potential of herbal drugs used in popular Italian medicine
(1850s–1950s) to treat bacterial skin diseases, J. Ethnopharmacol. 250 (2020)
112443.
[233] R.A. Carano, E.H. Filvaroff, Angiogenesis and bone repair, Drug Discov. Today
8 (21) (2003) 980–989.
[234] T. Hoffman, A. Khademhosseini, R. Langer, Chasing the paradigm: clinical
translation of 25 years of tissue engineering, Tissue Eng. Part A 25 (9-10)
(2019) 679–687.