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org/journal/abseba Review

Microalgae Applications to Bone Repairing Processes: A Review

Lily Margareth Arrieta Payares,* Lizeth Del Carmen Gutiérrez Púa, Leonardo Antonio Di Mare Pareja,
Soliani Carolina Paredes Méndez, and Virginia Nathaly Paredes Méndez*

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ABSTRACT: Research on regeneration and accelerated recovery

processes of bone tissue has driven a growing interest in the
Downloaded via UNIV OF TEXAS AT DALLAS on May 25, 2023 at [Link] (UTC).

scientific community. Implementing natural materials to reduce

rejections due to biocompatibility issues is an important trend.
Biofunctionalization processes have been proposed to promote
osseointegration in implant materials, and those substances able to
generate an adequate environment for cell proliferation are the
object of several studies. Because of their high protein content and
their anti-inflammatory, antibacterial, antimicrobial, and healing
properties, microalgae represent a natural source of bioactive
compounds, and are proposed as candidates for tissue regeneration
applications. In this paper microalgae are reviewed as a source of
biofunctionalized materials focused on orthopedic applications.
KEYWORDS: Microalgae, biofunctionalization, metallic implants, biomedical applications, orthopedic applications, osteoinduction

1. INTRODUCTION However, bones have the capacity to carry out their own
Bones are support for locomotion and attachment points for remodeling, which means that they constantly reabsorb and
ligaments and muscles.1 They consist of a mineralized matrix, generate new tissue to maintain mineral homeostasis and their
made up of organic and inorganic components in which the structure,6 achieving that the amount of reabsorbed bone be
bone cells are housed (Figure 1). Like all hard tissue, bones are identical with that of newly formed bone.6 Thanks to this
frequently damaged due to genetic, metabolic, hormonal, property bones can repair by themselves most of the fractures
infectious, and traumatic disorders, as well as accidents and that do not exceed a critical deformation, less than
aging. Fracture is a common consequence of all these causes.2−5 approximately 10% of the bone to be remodeled. When this
value is exceeded, osteosynthesis materials (implants or fixation
elements) are required for adequate treatment.7,8
Bone implants must have sufficient mechanical strength to
ensure structural and stable support during use, avoid rejection
by the body, and, as far as possible, an anatomical structure that
matches the defect, so that the implant and the bone have very
similar biomechanical properties and the implant integrates with
the original tissue, maintaining its integrity for the required
duration.2,12 Hence, the manufacturing material of the implants
must have physical, mechanical, and biological properties similar
to those of the original bone in order to promote bone
remodeling when it comes into contact with living tissues and/

Received: November 19, 2022

Accepted: April 27, 2023

Figure 1. Bone composition.9−11

© XXXX American Chemical Society [Link]

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or biological fluids;13,14 implants should also be corrosion- proliferation and healing process acceleration.22,23,25,38,39,42−44
resistant in the case of permanent applications.15−17 Therefore, this research focuses on their protein production
Biomaterials intended to come into contact with living tissues capacity to explore the feasibility of using the proteins they may
and/or biological fluids have been developed to aid in the repair produce as a substrate for the biofunctionalization of bone
or replacement of bone tissue.13,14 However, the first moments implant materials, considering that their use as osteoinductive
after implantation, blood serum proteins are adsorbed biomolecules in biofunctionalization processes on metallic
spontaneously to the material; interactions between proteins substrates has not been reported.
and the implant substrate are primarily governed by First, this review will consider some different biomaterials
physicochemical properties, surface properties, surface energy, used for orthopedic implants, and the surface modification
topography, and surface charge. Although this is a complex process focused on the biofunctionalization technique. Sub-
phenomenon which not yet well understood, it is believed that sequently, the characteristics of some common microalgae,
surface fine-tuning properties to trigger specific protein properties of their biomass important for biomedical applica-
adsorption or even the deliberate introduction of biomolecules tions, and their present uses in orthopedic applications are
to the surface prior to implantation can determine a desired shown in order to study the potential use of their biomass in
biological outcome.18 Therefore, one of the strategies used is biofunctionalization of current osteosynthesis materials.
biofunctionalization, a surface modification technique charac-
terized by improving the compatibility of the implant with the 2. BIOMATERIALS IN ORTHOPEDIC APPLICATIONS
bone and altering the surface layer of the material with The remodeling process, whether natural or assisted, begins
immobilized molecules to favor cell adhesion and prolifer- each time a fracture occurs. Its characteristics are determined by
ation.19−25 factors such as bone loss, fracture location, fracture immobiliza-
Synthesized molecules are generally used for biofunctional- tion, blood supply, age, nutritional status, and psychobiological
ization. Usual biomolecules include short peptide sequences habits, among others.7,48 Depending on the complexity of the
(oligopeptides) such as RGD,26−28 FHRRIKA,29 PSHRN,22,29 injury, assistance might be required. Medical criteria are used to
YGFGG,23 and long peptide (protein) chains such as BMP- choose among different technologies developed for the
2,30−32 collagen,33 chitosan,34 and hydroxyapatite.34,35 How- treatment of bone fractures. The high degree of complexity
ever, recent research has used materials extracted from nature as involved in many of them demands the use of surgical devices
a source for alternative biofunctionalization materials.36,37 Their such as screw implants, steel plates, rods, nails, pins, or wires,
advantages include cost reduction, ease of manufacturing, which are often removed after bone healing.49 Materials for
reduction of intoxicants, and increased cell biocompatibility.38,39 fixation elements must be biocompatible. This means that
Microalgae are one of the simplest groups of eukaryotic implantation should not induce any adverse effect on the
beings. Because of this simplicity, microalgae have managed to patient’s health or the implant area. Additionally, implant
thrive in almost any environment imaginable.40 Therefore, they materials must be blood compatible, nontoxic, non-inflamma-
have become the focus of attention as a source of solutions for tory, nonpyrogenic, nonallergenic, and non-carcinogenic.50
different human disciplines. Their ability to synthesize glucose The selection of a biomaterial for a given implant must
from carbon dioxide and water, capital to the evolution of life, is consider the structure and function of the surrounding tissues
proposed as an alternative for the control of CO2 emissions.41 and organs, in order to reduce the risk of postsurgical
Their high CO2 conversion efficiency creates an important complications. Likewise, depending on the implantation
nutrient source for several heterotrophic organisms (including location, materials must have sufficient mechanical resistance
humans), and they have become a viable alternative to many and rigidity to withstand the different conditions to which the
traditional crops.22,23,25,38,39,42−44 bone is exposed, including tension, compression, bending, or
Also, microalgae could be considered the most elegant O2- cyclic loads, since the fracture site will be exposed to constant
producing biomaterial developed by nature. Photosynthetic stress.2,12,51 High-strength metals are the most common
biomaterial strategies, based on the recognition that photosyn- biomaterials (titanium alloys, stainless steels, and chrome-cobalt
thesis is the source of most of the molecular oxygen available for alloys), since their mechanical properties meet the requirements
other living beings, offer a revolutionary and promising solution of fixation elements for the control and stabilization of fractured
to pathologies related to tissue hypoxia, since without the elements.52 However, the search for biodegradable materials,
sustained supply of adequate levels of oxygen by the including nontraditional metals such as magnesium and zinc, has
cardiovascular disease system, the tissues are unable to maintain increased in recent decades, since their eventual degradation in
normal metabolic activity and therefore cannot survive. the human body avoids the need for implant removal and the
Furthermore, hypoxia can aggravate the conditions of many risks associated with a second surgery.52
diseases such as cancer, ischemic heart disease, and chronic The osseointegration capacity of the material must be
wounds. Photosynthetic microalgae can alleviate hepatotoxicity considered for the development of implants. This process is
of the local microenvironment, and several studies have been essential during bone remodeling and depends on the
oriented to clinical applications including areas of engineering biomechanical integrity of the bone−implant interface.53 The
and tissue regeneration, organ preservation, wound healing, surface characteristics of implant materials (surface energy,
diabetic microvascular disease, and cardiovascular, cerebrovas- mechanical properties, topography, chemical composition) are
cular, and vascular diseases.45−47 important in the formation of a stable interface between the
Opportunities above are enhanced by the fact that they may implant surface and bone-generating cells (osteoblasts). The
grow in polluted water and purify them. Their biomass has the success or failure of bone repair will depend on the tissue−
potential to be used as a natural source of biomolecules, due to implant response and the correct osseointegration of the
its high percentage of proteins,22,23,25,38,39,42−44 as well as its implant.54 There are 4 possible responses: 1. Toxic response
anti-inflammatory, antibacterial, and antimicrobial properties, that can cause cell apoptosis around the implant and cause
which have been shown to provide an environment for cell systematic damage to the patient. 2. Biologically almost inert
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response where a nonadherent fibrous membrane develops that chemical bonds between the inorganic material and the
isolates the tissue from the implant and prevents fracture biomolecule.
healing. This is a consequence of low or no interaction between
the implant surface and the tissue (Figure 2). 3. Bioactive • Biological: design of bioactive or bioinspired coatings.
Bioactive material coatings, whether artificial or natural, lead
to a better interaction between the implant material and body
tissues, in any functionalization approach. The most applied
approach in orthopedic implants is the chemical modification of
the surface because it stimulates stable and strong bonds
between the surface and the immobilized molecule through
ionic or covalent chemical bonds resistant to the extreme
conditions of the human body during and after implanta-
tion.18,20,25,60−62
Biofunctionalization is a surface chemical modification
technique that allows the immobilization of biofunctional
molecules (lipids, proteins, nucleic acid, and carbohydrates)
on the material surface to achieve control of the tissue−implant
Figure 2. Biologically almost inert tissue−implant response. interface by biomimicking it, thus improving adhesion of cells,
platelets, bacteria, and the absorption of proteins, peptides,
antibodies and DNA, and the response cell (Figure
response that allows superficial tissue−implant union and 3).22,23,25,63,64 Therefore, in recent years different biofunction-
promotes the initiation of regeneration of damaged tissue. alization techniques have been used for improving osteogenic
This type of response is characterized by nontoxic reaction with activity and mechanical compatibility.21,24,58
body fluids and tissues and correct fracture healing. And finally, Surface biofunctionalization using artificially created peptides
4. Dissolution of the implant and replacement of the material by has been shown to promote cell adhesion and proliferation,20−25
the regenerated tissue, indicating successful bone healing.55,56 In osteoblastic and osteogenic differentiation,23,60−62 mineraliza-
biomaterial implantation processes for fracture healing, it is tion and expression of collagen for the formation of new
desirable to obtain a bioactive response that allows for tissue bone,60,65 healing times reduction,25,60 and, in some experi-
regeneration and subsequent dissolution of the implant. The ments, inhibition of TGF-β1 (an alkaline phosphatase inhibitor)
physicochemical properties of bone-implant surfaces also and osteoblast mineralization.60 On the other hand, the use of
influence adhesion, proliferation, differentiation, new bone short peptides, such as RGD, has been implemented for helping
formation, and reduction of healing times.25,57,58 Therefore, in cell adhesion; results show a higher statistical number of cell
biocompatibility, in terms of implant surface characteristics, is attachment, cell propagation, and proliferation compared to
key to ensure interaction with biological systems in the initial samples coated with other short peptide sequences.20,22,60,61
phase of cell adhesion to the implant. At the same time, natural materials have been implemented to
biofunctionalize metal surfaces in bone implant applications.
3. MATERIAL BIOFUNCTIONALIZATION
Osteogenic growth peptide (ALKRQGRTLYGFGG), obtained
Since the biological response to biomaterials and devices is from bone marrow, and phosphatidylethanolamine, derived
mostly controlled by the surface chemical arrangement, from perivascular cells of the human umbilical cord, are two
modifications can be made to improve the biointeraction of examples that have shown their regulation capabilities on the
the implant without affecting the mechanical properties of the osteoblast differentiation, early mineralization process promo-
material and to increase bioresponse on the tissue−implant tion, bone cell metabolism enhancement, and new bone
interface.20,24,58 formation.66,67 Materials such as collagen, chitosan, cytokine,
A surface can be functionalized to improve its physicochem- albumin, and hydroxyapatite have also been used to biofunction-
ical properties using the following approaches:20,24,58,59 alize metallic surfaces since they have shown high compatibility
• Physical: increasing the roughness that induces the with the human body, participation in bone healing around the
physisorption of biomolecules. implant, and improvement of cell adhesion and proliferation,
• Chemical: through the immobilization of proteins, thus increasing regeneration rates in bone tissue. Additionally,
peptides, calcium phosphates, drugs, etc., generating they improve some chemical and physical properties such as

Figure 3. Biofuctionalization process.

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corrosion resistance, surface roughness, and porosity. All of be helpful in determining biomedical application potential of
these properties preserve morphological stability and chemical microalgae.
composition of the implants thus increasing their useful time
span24,60,61,68−74 (Figure 4). 4. MICROALGAE
Microalgae are unicellular photosynthetic eukaryotic organisms
that use water, solar energy, carbon dioxide, and other nutrients
to grow.85,86 It is estimated that there are approximately 250,000
species of different plants and 800,000 species of microalgae, in
nature. Only some 30,000 microalgal species have been
studied.87−93 Considering the presence of chlorophyl, micro-
organisms from different dominions have been considered as
microalgae: the cyanobacteria (prokaryotic) and eukaryotic
algae.94 Cyanobacteria are usually known as blue-green algae,
while eukaryotic algae include green algae and diatoms.95
Cyanobacteria are unicellular microalgae that have photo-
synthetic pigments called phycobilins that give them their blue
color. They are characterized by having several bioactive
Figure 4. Bioactive tissue−implant response. compounds such as pigments (carotenoids and phycobilipro-
teins) that are studied for their biological properties and
Collagen, hydroxyapatite, and liposome covered surfaces have biomedical potential due to their antioxidant and anti-
been functionalized with PRF, BMP-2 peptides, and even inflammatory activities.96 Additionally, it has been shown that
powders extracted from eggshells modified with MgO nano- they have antimicrobial activity, making them attractive
particles. These materials have shown osteoinductive properties, candidates for the creation of new antibiotics.96,97
which improve angiogenesis, osteogenic differentiation, and Referring to eukaryotic microalgae, there are green microalgae
vascularization.62,65,75−77 This result indicates that biofunction- containing chlorophyll A and B, which give them their
alization helps not only metal surfaces but also natural material characteristic color. They are important in oxygen production
ones.38,39 and are rich sources of proteins, essential amino acids, fatty
New natural materials, like microalgae, have been used in acids, pigments, vitamins, and other bioactive compounds.98,99
different fields such as the food industry; medical, chemical, and The red microalgae are also part of this classification, and they
pharmacological applications (like biofuel production); and are characterized by having polysaccharides and pigments with
water treatment.42,78−84 Also, microalgae have been proposed antioxidant, antitumor, and anti-inflammatory properties and
for functionalization processes in recent years. Diatoms are have been used in traditional medicine for the treatment of skin
among the new proposed species. They have been functionzal- diseases and cancer, among others.100
ized using tyrosine and drugs. Release of therapeutic agents have Finally, there are the diatoms, which are photosynthetic
been controlled through nanopores or silica embedding;36,37 unicellular microalgae with a silica shell (called a frustule) on the
however, no studies of the biofunctionalization of metallic surface that has pores nanometers or micrometers sizes with
surfaces using microalgae as osteoinductive biomolecules are different shapes which respond to various light climates;101,102
presented. The following section shows a bibliographical study they are rich in lipids, sterols, and isoprenoids.102 They have
of properties and compounds obtained from microalgae; it shall received great interest in nanotechnology applications, such as

Figure 5. Average protein content in the biomass of various microalgae.115−133

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Figure 6. Average lipid content in the biomass of various Microalgae.115−133

Figure 7. Average carbohydrate content in the biomass of various Microalgae.115−133

drug delivery, photocatalysis, nanofabrication, and biophoton- etonema are among the most commercially important microalgal
ics.101,103,104 Moreover, diatoms present reactive Si−OH groups species.84
that coat the frustules facilitating the functionalization process 4.1. Chemical Composition of Microalgae Biomass.
on them.101 The correct selection of microalgae, according to the field of
All the microalgae have been characterized as a promising application, is analyzed based on the composition of its biomass.
source of biomass.40,105 Biomass from microalgae is a Therefore, microalgae have the potential to be used as a
considerable protein source and has good amino acid, biomolecule in the biofunctionalization process of implant
polypeptide, lipid, sterol, isoprenoid, carotenoid, fatty acid, materials, since their biomass is mainly composed of proteins,
amide, amine, and ketone body profiles.106−109 However, few carbohydrates, and lipids.112 Average microalgae contain 8−
strains have been studied in detail and have achieved 69.7% carbohydrates, 5−74% proteins, and 7−65% lipids.82
commercial-scale production. For several species of microalgae, Microalgae are considered unconventional protein sources
there are challenges of access, identification, strain isolation, and and have been used for thousands of years for human
extraction, as well as recreating a culture medium that can consumption.113 Many microalgal species have been shown to
provide a sustainable source of supply.40,110,111 Spirulina, contain amounts of protein close to traditional protein sources
Chlorella, Haematococcus, Dunaliella, botryococcus, Phaeodacty- such as soy, milk, beef, and egg.114 However, the use of proteins
lum, Porphyridium, Chaetoceros, Crypthecodinium, Isochrysis, from microalgae is limited in the food industry due to highly
Nannochloris, Nitzschia, Schizochytrium, Tetraselmis, and Skel- concentrated nonprotein compounds such as chlorophyll.113
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Figure 8. Bibliometric analysis using VOSviewer.144

Figure 9. Biomedical properties of microalgae.42,105,143,155−164

Figure 5 shows the average content of protein of some standards for essential amino acids for human nutri-
microalgae. tion.113,135−139
Several species have been identified as major protein Regarding algal lipids, they are present in intracellular droplets
producers, including phycobiliproteins and glycoproteins: containing mainly triacylglycerides (TAGs). Droplets are
Spirulina varieties, filamentous cyanobacterium, have protein encapsulated by a membrane consisting of phospholipids,
contents ranging between 50% and 70% (dry weight basis); glycolipids, and proteins. Studies have been conducted to
Porphyridium cruentum, red microalga, has a steady state protein determine the optimal growth conditions to maximize lipid
range between 30% and 50% of dry weight;134 Synechococcus sp., production; it was determined that the carbon source is essential
unicellular cyanobacteria, has a protein content of 63% (dry for lipid accumulation. Different sources of organic carbon in
weight basis); Aphanizomenon f los-aquae, filamentous cyano- different microalgal cultures lead to different metabolic
bacterium, 62% (dry weight basis); while Chlorella varieties, mechanisms for the synthesis and accumulation of lipids.
green microalga, exhibit protein contents ranging between 54% Different light/dark cycles and combinations of heterotrophic
and 58% + (dry weight basis). Varieties of both Spirulina and and phototrophic culture conditions are also considered
Chlorella may provide high-grade protein with well-propor- effective strategies to improve their efficiency in the usage of
tioned amino acid profiles that meet WHO/FAO/UNU organic carbon sources and in their lipid production. However,
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 Table 1. Patents on Microalgae in Biomedical Applications
Title IPC Field Description Summary of the invention ref
Method for inducing os- A61P Biological/Medi- The objective of the invention is the design a methodology to induce The peptide has a molecular weight of less than or equal to 10 000 Da, and the biological activity 180
teoblast differentiation 19/08 cal osteoblast differentiation through mitogen-activated protein kinase of the polypeptide can be easily maintained. It shows high biological activity, is easily absorbed
through MAPK and (MAPK) and NF-kB by using peptides separated from microalgae for and can be safely used in the human body.
NF-kB by utilizing A61K osteoporosis treatments. The resulting synthetic peptide promotes bone formation and can be used as an effective
peptides separated 38/01 ingredient in the treatment of osteoporosis, osteoarthritis, or cranial defects.
from microalgae
A61P
19/10
A61P
19/02
Pharmaceutical composi- A61P Pharmaceutical/ Development of a drug and functional food for the prevention or The pharmaceutical composition and the functional food developed in the invention proved to be 181
tion containing peptide 19/00 Medical treatment of bone diseases through a peptide isolated by enzymatic effective in the prevention or treatment of bone diseases such as osteoporosis by promoting
separated from biodie- A61K hydrolysis of biodiesel byproducts of marine microalgae of the species osteoblast differentiation. The pharmaceutical composition can be formulated orally as
sel byproduct of marine 38/17 Nannochloropsis oculata as an active ingredient. powders, granules, capsules, suspensions, emulsions, syrups, or as injections. It can be given
microalgae as an active intravenously, intraperitoneally, subcutaneously, rectally, and topically.
ingredient for prevent- A61K
38/16
ACS Biomaterials Science & Engineering

ing or treating bone

diseases
Extract from microalgae A61K Cosmetic/Food/ Design and description of microalgae extract comprising fucoxanthin, The microalgae extract obtained integrates fucoxanthin and fucoxanthinol, with other 182
comprising fucoxan- 36/03 Pharmaceutical/ fucoxanthinol, and some fatty acids for use as an anti-inflammatory, components of the algae, especially fatty acids. This can be used as an anti-inflammatory,
thin, fucoxanthinol, (2006.1) Nutraceutical/ antiobesity, or antiangiogenic agents. antiobesity agent, due to its fat-burning effect, antiangiogenic, antibacterial, antifungal,
and fatty acids, the A61K Medical anticancer, antineoplastic, antihypertensive, antiallergic agents, as a neovascularization
process for its produc- 36/04 inhibitor, in skin pigmentation treatments, for the prevention of bone diseases such as
tion, and applications (2006.1) osteoporosis and arthritis, depression, diabetes, among others.
thereof
A61P

G
29/00
(2006.1)
A61K
8/00
(2006.1)
A potent immunostimu- C07K Pharmaceutical/ Identification and extraction of immunostimulatory lipoproteins from The obtained lipoproteins prove to be useful as agents for immunotherapy and can be used in the 183
latory component in 14/405 Medical microalgae of the Spirulina species, Chlorella species, Haematococcus treatment of immunodeficiency disorder, cancer, wound healing, and infectious diseases. The
microalgae extract A61P Pluvialis, and Aphanizomenon f los-aquae through different procedures, lipoprotein preparations designed in the patent can be combined with other pharmaceutical
37/04 for their use in pharmaceutical and botanical applications due to their vehicles, active ingredients, or excipients. Likewise, various manufacturing methods can be
A61P great potential as an immunostimulatory agent and monocyte activator. implemented to obtain the desired presentation, for example, conventional mixing, dissolving,
38/16 Additionally, the chemical methods and bioactivity-based stand- granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing
ardization used to extract lipoproteins from microalgae are described. processes.
Potent immunostimula- A61K Biological/Phar- Design of a methodology for the extraction of immunostimulant The polysaccharide preparations obtained in the invention can be used for treatments of 184
tory polysaccharides 31/715 maceutical polysaccharides from food-grade microalgae (Aphanizomenon f los- immunodeficiency, cancer, wound healing, and infectious diseases and as an active ingredient in
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extracted from micro- A61K aquae (AFA), Chlorella pyrenoidosa, and Spirulina platensis). A dietary supplements. They can be mixed with pharmaceutical vehicles, active ingredients, and
algae 31/726 transcription factor-based assay for NF-kappa B in human monocytes excipients. The polysaccharides obtained are soluble in water and aqueous ethanol solutions.
A61K was implemented for extraction, isolation, characterization, and The most efficient conditions for the extraction of immunostimulant polysaccharides were
31/736 preparation development. identified: 50% ethanol concentration at temperatures between 60 and 70 °C. Instant
C07H polysaccharide preparations are a thousand times more active for monocyte activation than
1/08 polysaccharide preparations currently used clinically for immunotherapy in cancer patients.
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 Table 2. Use of Microalgae for Bone Applications
Microalgae type Application Manufacturing Method Results ref
Green Microalgae Porous bone scaffolds based on hydroxyapatite with Solvent Casting Scaffolds are composed of rod-shaped hydroxyapatite nanocrystals, without the formation of toxic compounds. 39
microalgae biomass as a fugitive additive and pore- For composite scaffolds with the same proportion in weight of microalgae and HA, it obtained an improved
forming agent. compressive strength (2.89 ± 0.50 MPa) and yield strength of 2.36 ± 0.30 MPa
Scaffolds with the same proportion of binder and HA showed an interconnected morphology with a porosity of
65.29 ± 2.63% and a maximum pore diameter of 258 μm.
The thermal stability of the scaffold increases with higher HA content in the composite suspension.
The synthesized scaffolds have no harmful effect on the human body
Spirulina LEB 18 Bone scaffolds based on nanofibers composed of poly Electrospinning For weight contents of up to 67% biomass into nanofibers and for PEO concentrations well below 4% wt.
(Arthrospira) (ethylene oxide) (PEO)/Spirulina In the Spinning solution, bead-free nanofibers with a diameter of about 110 nm can be produced for different
biomass.
The porosity of the composite nanofibers indicates that the system possesses favorable properties that support
their use as bone scaffolds.
Poly(D,L-lactic acid) synthetic bone scaffolds (PDLLA) Electrospinning The developed scaffolds have a fibrous and porous structure similar to the natural extracellular matrix (ECM) of 185
/Spirulina the cells, which provides a suitable environment for the culture of these.
ACS Biomaterials Science & Engineering

The addition of Spirulina biomass produced improvements in the adherence and viability of the stem cells used.
The designed scaffold is nontoxic to the patient.
Bone scaffolds based on biodegradable polymer nano- Electrospinning The addition of biomass did not alter the thermal properties of the material. 186
fibers with the addition of Spirulina biomass as a Spirulina improved the tensile strength and elongation of the scaffolds.
bioactive component.
The designed scaffold showed enzymatic action, simulating the behavior of natural tissue.
The presence of microalgae in the scaffolds reduces the healing time and stimulates cell growth.
Thalassiosira Biosilica from diatoms Thalassiosira weissflogii function- In vivo functionalization The biosilica obtained showed good osteoconductive capacity and was effective in activating the tissue 187
weissflogii alized with Alendronate Sodium. regeneration model (assessed in SaOS-2 cells and bone marrow stem cells (BMSC)).

H
Inhibition of the metabolic activity of J774 of functionalized biosilica.
Nanostructured biosilica from Thalassiosira weissflogii Covalent functionalization The TEMPO biosilica obtained allows the controlled release of the drug ciprofloxacin and exhibits antioxidant 188
covalently functionalized with 2,6,6-tetramethylpiper- properties.
idine-N-oxyl cyclic nitroxide (TEMPO) The material evidenced the function of eliminating reactive oxygen species capable of preventing inflammatory-
type side effects.
An improvement in the adhesion and proliferation of bone cells was observed.
Nanostructured biosilica from Thalassiosira weissflogii Covalent functionalization with The MPTMS-functionalized surface showed higher vitality and better cell adhesion than the APTES- 189
functionalized organosilanes (APTES and MPTMS) organosilanes functionalized substrates.
Assays with Saos-2 cells showed greater sensitivity to bare biosilica compared to NHDF cells.
NHDF presented higher sensitivity, in terms of viability, on the functionalized surface.
Pavlova lutheri Fermentation of P. lutheri to produce small molecule Fermentation with Hansenula Fermented P. lutheri showed an increase in alkaline phosphatase activity in MG-63 osteoblast cells. 190
peptides that enhance bone differentiation polymorpha Fermentation peptide increases alkaline phosphatase release in MG-63 osteoblast cells and mineralization in a
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dose-dependent manner.
Intracellular levels of protein alkaline phosphatase and osteocalcin are increased after fermentation treatment.
Microalgae fermentation produced an increase in differentiation in MG-63 at concentrations of 50 and 100 μg/
mL
Nannochloropsis Osteoblast differentiating peptide from N. oculata Enzymatic hydrolysis The peptide-induced osteoblastic cells differentiation (MG-63) both in the early and the terminal stage due to the 191
oculata biodiesel byproducts increase in the levels of alkaline phosphatase, collagen type I, osteocalcin, BMP-2, BMP2/4, and bone
mineralization both in MG-63 and murine mesenchymal stem cells (D1)
The produced peptide increased the phosphorylation of the expression of mitogen-activated protein kinase and
Smads.
Cyclotella mene- Cyclotella meneghiniana diatom biosilica for implant Surface functionalization with Diatom biosilica does not generate an inflammatory response and does not represent a toxic risk. 192
ghiniana material silane groups (Si−OH) The material has a wide variety of surface morphologies that can be systematically mapped to quantify the role of
silica in bone regeneration or drug-delivery system.
The two silanes used for functionalization have a significant effect on the cellular response in vitro
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as happens to the other studied components, their concentration

193
ref depends on the species, culture conditions, and extraction
Oral treatment with biomass and polar fractions increased bone mineral density and serum phosphorus level. It method.140 Lipids obtained from biomass are used in the form of

The fractions ameliorated bone loss in osteoporosis through downregulation of serum OPG in conjunction with
fatty acids in nutraceuticals and cosmetics, but the principal
market for lipids is biodiesel.113 Figure 6 shows the average

H. Pluvialis fraction with high astaxanthin content proved to be beneficial in the control of age-related
content of lipid of some microalgae. Spirogyra sp., a green
filamentous microalga, have lipid contents ranging between 33%
and 66% (dry weight basis); Chlamydomonas sp., green
biflagellate microalga, has a lipid content of 14% to 65.8%
(dry weight basis); while Oscillatoria amphibia, filamentous
cyanobacterium, exhibit protein contents ranging between 23%
and 41% (dry weight basis) depending on their culture
decreased serum levels of calcium, BALP, interleukin 6, OPG, and RANKL.
The carotenoid treatment restored these parameters to their nominal values.

conditions.
Carbohydrates are complex organic compounds formed
within the chloroplast as a product of photosynthesis. Within
microalgal cells, their main function is energy storage and
protective activities, as well as functions for the structural
Results

features of the cell wall. Figure 7 shows the average content of

carbohydrates of some microalgae. Compared to lipids and
proteins, these compounds are found in better content in the
microalgae biomass. Chlorella varieties have approximately 10%
to 52% carbohydrate, Spirulina varieties exhibit carbohydrate
contents ranging between 8% and 63% (dry weight basis), 10−
15%, and Chlamydomonas reinhardtii is 15% to 17%.135,136,141
upregulation of serum RANKL.

The carbohydrates extracted from microalgae are used for the

production of biofuels through anaerobic digestion, anaerobic
fermentation, and biological production of biohydrogen; in the
food industry as a raw material in thermochemical conversion
technologies; they have also been used in the cosmetic industry
osteoporosis

as antioxidants and creams.113,142,143

Considering the general characteristics of microalgae biomass,
the potential of these species in biomedical applications was
studied. For this purpose, a descriptive bibliometric analysis of
In vivo study in osteoporotic rats -

the research trend on this topic was carried out, then the
Oral treatment. Microcom-
Manufacturing Method

biomedical properties exhibited by the biomass of microalgae

puted tomography scan.

were investigated, and some patents on microalgae in the

medical field and studies of their use in orthopedic applications
were collected.
4.2. Use of Microalgae in Biomedical Applications.
4.2.1. Descriptive Bibliometric Analysis of the Use of
Microalgae for Biomedical Applications. In order to better
understand research background of microalgae usage in medical
applications, a bibliometric analysis was made using VOS
viewer.144 Bibliographic records were obtained from the Web of
Biomass, carotenoids, and the polar fractions of H.

Science database. Keywords used for the search were microalgae

medical (1762), microalgae biomedical (251), microalgae
biomedical applications (145), microalgae drug delivery
pluvialis for the control of osteoporosis

(128), microalgae pharmacological (94), bone microalgae

(84), osteoporosis microalgae (10), osteoblastic microalgae
Application

(4), and osteoactive microalgae (2). A total of 2480 articles were

extracted as of June 2022, and keywords with at least 20
concurrences were filtered. Figure 8 shows the bibliometric map
obtained. Five clusters were identified:
• Industrial uses (30.43%): It groups a total of 49 items.
Keywords with the highest frequency were Microalgae;
Biomass; Biodiesel production; Wastewater.
Table 2. continued

• Biomedical applications (29.81%): It includes a total of

48 items. Keywords with the highest frequency were
Microalgae type

Oxidative stress; Antioxidant; Biosynthesis; Carotenoids.

Heamatococcus

• Biotechnology (19.88%): It groups a total of 32 items.

pluvialis

Keywords with the highest frequency were Photosynthesis;

Expression; Cyanobacteria; Gene expression.

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Figure 10. Biofunctionalization with microalgae.

• Soil treatment (10.56%): It covers a total of 17 items. studies in the orthopedic field or biofunctionalization of implant
Keywords with the highest frequency were Algae; surface.
Bioremediation; Removal; Toxicity. 4.2.2. Biomedical Properties of Microalgae Biomass. There
• Aquaculture feed (9.32%): It includes a total of 15 items. is a wide field of applications for microalgae in medicine, ranging
Keywords with the highest frequency were Fatt-acid from drug delivery uses of the frustules from diatoms145 to the
composition; Chlorella vulgaris; Aquaculture; Fish. production of hormones, antibodies, vaccines, and immune
regulators.146 Fatemeh Khavari et al.147 proposed 4 pharmaco-
The subject of study was positioned in the center of the map:
logical applications of microalgae, as antimicrobial agents in
microalgae (cluster 1). According to the keywords of this group,
antibacterial and antiviral activities, as anti-inflammatory and
use of microalgae in biofuel production processes and
wastewater treatment is related to traditional applications of anticancer due to their antiproliferative and apoptotic effects, as
microalgae in industry. These uses have been widely investigated recombinant proteins for the creation of vaccines and
and reported by scientific literature. Likewise, uses of microalgae monoclonal antibodies, and in drug delivery processes where
for soil treatments (cluster 4) and aquaculture crops (cluster 5) diatom-based nanocarriers have been implemented in cancer
have been reported. In these fields, microalgae are linked to therapy.
bioremediation processes, removal of toxic compounds and Photosynthetic materials have been developed from micro-
heavy metals, biodegradation, aquaculture, feeding of marine algae that promise unlimited possibilities for regenerative
species, and studies on the composition of fatty acids and medicine. Microalgae can improve oxygen delivery to tissues
essential elements in animal feed. Cluster 3 group research is and poor vascularization in vivo leading to hypoxia.148 Some
related to biotechnology and genetic modification techniques. strategies to increase tissue oxygenation include the combined
These keywords are related to cultivation conditions such as use of scaffolds with synthetic oxygen carriers, and although this
light and temperature, which may indicate that the effect of review focuses on applications to bone repairing process, it is
variation in cultivation methodology has been studied to considered appropriate to dedicate a few paragraphs to this type
generate changes in the microalgae biomass. of applications, due to their high impact, given that they
Biosynthesis is correlated with the antioxidant, antimicrobial, represent an emerging field with enormous potential to address
and antibacterial activity of microalgae (cluster 2), and keywords diseases aggravated by oxygen deficiency, such as such as cancer,
such as cancer, inflammation, in vivo and in vitro studies, and ischemic heart disease, clinical applications of medicine that
bioactive compounds are found but with less concurrence, include the areas of tissue engineering and regeneration, organ
indicating a slight presence of research on the use of microalgae preservation, and wound healing, among others.45,148−150
in biomedical applications. However, it was not possible to find The first use of a photosynthetic microorganism to repair
papers whose keywords related to osteoinduction processes or tissue hypoxia in vivo was reported in 2012. Chlorella vulgaris was
bone regeneration, which indicates the low or null existence of used in the peritoneal cavity to improve the pancreatic capacity
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of transplanted rats, most of which were diabetic rats that and hepatoprotective agent during malnutrition and ethionine
acquired pancreas stored in a solution with Chlorella have intoxication.84
normal blood glucose levels and survived longer after surgery Specifically, Chlorella sp. contains peptides that could prevent
than those who worked with traditional solutions.151 Likewise, cell damage and tissue generation. This microalga provides high-
the development of photosynthetic biomaterials with microalgae grade proteins with amino acid profiles that meet WHO/FAO/
such as Chlamydomonas reinhardtii, Chlorella vulgaris, Synecho- UNU standards for essential amino acids for human
coccus elongates, and Chlorococcum Littorale has been studied. nutrition.113,135,136,138,139,174 On the other hand, Chlorella
The results indicate that respiratory support through photosyn- vulgaris has been used to accelerate the healing of cutaneous
thesis helps to improve not only the blood gas status in the case wounds through a gel with a high percentage of biomass that
of respiratory failure but also graft recovery after transplantation, exhibited anti-inflammatory, antioxidant, antibacterial, and
as has been reported in organ preservation studies such as antimicrobial activity against Klebsiella pneumoniae, Enterococcus
porcine kidneys or pancreas.149,151 It has also been suggested faecalis, and Escherichia coli and showed no atopic or allergic
that implantation of photosynthetic cells can provide oxygen to reactions in vivo in mice.42 In addition, the microalgae extract
tissues in the absence of vascular supply, and for this purpose, showed excellent hemagglutination results, an increase in
wound healing scaffolds or dressings, photosynthetic sutures for collagen deposition, and a decrease in fibroblasts.42 This
oxygenation, and growth factor delivery have been developed microalgae species showed a slow degradation that benefits
that promote tissue regeneration and treat a broad spectrum of the growth and survival of cells, especially those that require a
hypoxic conditions.45,148,150 higher property of regeneration and proliferation. For this
The aforementioned studies do not contemplate the potential reason, Chlorella vulgaris has been used as a support material for
use of microalgae in bone regeneration and biofunctionalization the cardiovascular system.153
of implants. However, microalgae exhibit a unique combination On the other hand, for the cell to be restored in a three-
of characteristics typical of higher plants (efficient oxygenic dimensional tissue, artificial or natural scaffolds or supports were
photosynthesis and simple nutritional requirements) combined created to guide the optimal growth of tissues,38,154,168 because
with biotechnological attributes of microbial cells (rapid growth in addition to providing mechanical support, it allows the human
and ability to accumulate or secrete metabolites under specific cell to effectively attach, grow, and migrate as in its original
culture conditions), which contributes to a more environ- environment;153 Chlorella extracts have been studied as
mentally friendly approach in the production of biomass and candidates for regenerative tissue, because they can provide an
bioactive compounds with a controlled and standardized atmosphere for cell proliferation.38
composition.152 Similarly, Chlamydomonas reinhardtii, a species composed of
In the biomass of microalgae has been reported the presence
48−51% protein, 15−17% carbohydrate, and 19−22%
of anti-inflammatory, antimicrobial, and antibacterial activity,
lipid,21,141 has been used to improve skin blemishes. This
and the ability to stimulate the regeneration of tissues is a key
microalga has been seeded on a collagen base to create
indicator that demonstrates the potential of microalgae to be
photosynthetic scaffolds to generate tissues that contain a
used in biomedical applications, especially in the orthopedic
local source of oxygen.40,154,168 In vitro studies have shown that
field where the display of these characteristics allows for
increasing success of the fixation elements or im- this microalga not only survives on the scaffold but also
plants.38,42,153,154 Figure 9 shows the compilation of the proliferates.40,154,168 Also, the scaffold was installed in mice,
biomedical properties of some species of microalgae. where it showed that microalgae live for at least 5 days after
Previous research showed that microalgae biomass stimulates transplantation, discovering chimeric tissues of algae and
growth,165 cell adhesion, and proliferation.38,166 Studies mammals. Likewise, no macroscopic sign of infection or foreign
reported generation of scaffolds for tissue regeneration in the body reaction was demonstrated in the wound area; on the
human body using Chlorella,39,42,153,158,167 Chlamydomonas contrary, anti-inflammatory properties were observed as a key
reinhardtii, and Spirulina,38,154,168 among other. mediator in tissue repair and regeneration.26,154
Chlorella has been one of the most studied strains with a Chlamydomonas reinhardtii is used as a propulsion and
significant increase in research on it.169 This species has steering agent for micro robots in medical applications for the
antibacterial,42,170 antioxidant,42,135,170 anti-inflammatory,42,170 transport of medicines, since it can move through places that are
antimicrobial,42 immunomodulatory,170 hemagglutination,42 difficult to access in the human body thanks to its flagella.
and curative properties,42,153 high biocompatibility in the Additionally, it allows the modification of the cell wall, is
human body,153 and exhibits the ability to rapidly absorb biocompatible with the animal cell, and does not release toxins
nutrients and assimilate them for growth. Currently, it is one of during its decomposition.50,175−177 By having a negatively
the most cultivated strains in the world, with large-scale charged cell wall on the surface, due to the presence of pectin
cultivation in the United States, Germany, and Japan.171,172 and glycoproteins with anionic carboxylate groups, a non-
Hildebrand et al. and Vadiveloo et al.135,136 have reported that covalent electrostatic interaction takes place between the film
this microalga is composed of approximately 40−58% protein in that will distribute the positively charged drug and the
dry weight, 18−23% carbohydrate, and 28−32% lipid. It has microorganism.51,177,178 Finally, when comparing Chlamydomo-
vitamin E, B, and C, minerals such as iron, magnesium, nas reinhardtii to bacteria such as Serratia marcescens, Salmonella
potassium, and calcium.135,136 Therefore, it has been used in typhimurium, or Escherichia coli, which have been used for
pharmaceutical applications and tissue engineer- microrobotic applications, C. reinhardtii was shown to have a
ing.39,42,153,158,167 Chlorella exhibited various health benefits, better drug-carrying capacity.51,175,177,178
for example, due to its content of β-1,3-glucan, an active Same electrostatic charging mechanism has been used with
immunostimulant, it can scavenge free radicals and lower blood the microalgae Spirulina platensis for the sustained release of
lipids.84,173 Likewise, it can increase hemoglobin concentration, drugs52,179. This microalga has a biochemical composition of
decrease blood sugar levels, and act as a hypocholesterolemic around 60−65% protein, 10−15% carbohydrates, 5−10% lipids,
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and 10−15% phycocyanin, so it has great potential in the The new biofunctionalized surface will promote the growth and
pharmaceutical and medical industries.107 proliferation of bone cells, encouraging bone regeneration.
In recent years, Spirulina species have been investigated for Likewise, it could offer anti-inflammatory, antimicrobial, and
biomedical applications in scaffold preparation due to its ability antibacterial properties that could reduce healing times and the
to heal and repair damaged tissues, as well as its biocompati- risk of infections.
bility. These species are the microalgae with the highest Additionally, biofunctionalization not only allows generating
percentages of protein content (60−70% dry weight) and a more bioactive surface but also improves the surface properties
have been used to reduce inflammation and allergies. of the material such as roughness and chemical stability.194,195 A
Furthermore, it was discovered that spirulina extracts have corrosion inhibitor like this usually contains nitrogen, sulfur,
anticoagulant activity in low concentration ranges and help cell oxygen, phosphorus, and a multiple bond or aromatic ring
adhesion and proliferation on the scaffold. Even better, they do organic structure. In this sense, some compounds such as plant
not have significant cytotoxic effects in the fibroblast cell line.38 extracts, chitosan, and cellulose have structures or functional
Given the above-mentioned properties, the use of microalgae groups that can act as green corrosion inhibitors through
in various biomedical applications has been studied. Some biofunctionalization processes. Likewise, amino acids, the basic
patents have been published on microalgae ranging from the unit of proteins, have excellent biocompatibility, high purity, and
induction of osteoblast differentiation to the design of low cost, making them excellent candidates for inhibitors.196
immunostimulatory polysaccharides extracted from microalgae. There have been reports on using protein in biofunctionalization
Table 1 presents the most important published patents on the to control degradation speed in metallic implants;196−205 results
subject and the description and summary of the invention. indicate that the presence of the biomolecules improves the
Application for these patents confirms the potential of adhesion and proliferation of osteoblasts and provides greater
microalgae as biomedical materials for several applications. resistance to corrosion, acting as green corrosion inhibitors and
Study of the microalgae biomass properties could open new improving the biocompatibility of the materials. Therefore,
perspectives in the medical and materials field since, thanks to considering the high percentage of proteins in the biomass of
their ease of cultivation, industrialization capacity, and low cost, microalgae, it is expected that their use in biofunctionalization
they could be attractive options for designing alternative processes will not only promote tissue regeneration but also
treatments in tissue engineering and bone regeneration. In the increase the resistance to corrosion of materials in order to
orthopedic field, two patents related to bone disease treatments decrease the risks associated with the release and toxicity of
were identified. Therefore, in the following section, the potential metal ions, and increase the useful life of the implant.55 The
of microalgae in the processes of regeneration and healing of microalgae are a highly versatile species since its biomass can be
bone tissue will be studied. used directly, but it is also possible to work with peptides created
4.3. Use of Microalgae for Orthopedic Applications. from it, which have been shown to promote osteoblastic
Despite the healing and medical properties that microalgae differentiation, improve cell adhesion and bone regeneration,
possess, extensive research on their use in orthopedic and can be absorbed and safely used in the human
applications has not been reported. Table 2 summarizes the body.180,181,190,191 Finally, in recent years interest in the
studies found related to the subject. bioinduced production of nanostructured materials for bio-
Microalgae promote cell growth, as well as proliferation and mineralization has increased,206 the process where organisms
regeneration of bone tissues (osteoconductive), encourage the produce inorganic minerals in the form of bones, shells, and
growth of fibroblasts and cells similar to osteoblasts, and inhibit teeth. This takes place in environments composed of
the osteoclast-like cell growth. They are nontoxic biomaterials biomolecules that build scaffolds for mineralization, while
that do not produce inflammatory responses. They improve controlling the growth, morphology, and orientation of the
mechanical and thermal properties and increase porosity of bone compounds formed.207,208 The use of microorganisms such as
scaffolds. The presence of microalgae in these structures reduces microalgae, marine plants, and bacteria has been proposed as
healing time and stimulates cell growth. Peptides created from these species can adapt physiologically and biochemically faster
microalgae promote osteoblastic differentiation and enhance the in a wide variety of environmental conditions, genetic
performance of bone remodeling and bone repair functions. modifications can be used to control the morphology and size
Finally, they have shown potential in drug delivery systems. of nanomaterials created, and the production and maintenance
4.4. Microalgae for Biofunctionalization Processes. of these micro-organisms requires simple and easily controllable
Considering the properties of the microalgae biomass that have culture conditions. These species could eliminate the problems
been exposed in this review, it is possible to confirm the potential associated with the traditional synthesis of nanoscale materials
of these species as sources for biomolecules in biofunctionaliza- such as the requirement of strict synthesis conditions, low yield
tion processes of metal surfaces for orthopedic implants. of the materials, and energy inefficiency.206 This application
Biomass of microalgae has the four types of biomolecules opens a new possibility to use microalgae in orthopedic
currently used to biofunctionalize implants (lipids, proteins, applications as a biomolecule to encourage bone tissue
nucleic acid, and carbohydrates), which makes them promising regeneration.179,180,189,190,205−207
sources of bioactive products to improve the biocompatibility of
biomaterials. A scheme of how biofunctionalization with
microalgae would work is presented in Figure 10. A decision is
■ CONCLUSIONS
Microalgae are versatile microorganisms that have been used in a
made considering the area and severity of the fracture and the wide range of applications, from wastewater and soil treatment,
estimated time of bone regeneration. Next, the microalgae as a source of protein in food and the production of biocrude.
species is selected based on biomass composition, species However, their involvement in the medical field has been
availability, and culture requirements. Subsequently, the minuscule. Therefore, there are great opportunities to
biofunctionalization process is carried out and, as a result, it is investigate the applications of microalgae in bone repair
expected to obtain a bioactive surface with microalgal biomass. processes. For that reason, this paper compiled evidence on
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the potential of microalgae to be used in biomedical National master’s modality. CALL No. 809 of 2018;
applications, especially in orthopedic implants. No literature Biotechnology laboratory of Universidad del Norte; Alliance
review on this topic has been reported, focused on studying the 4U contract number 2022−55951.
medical potential of these microorganisms. Properties exhibited Notes
by their biomass, such as anti-inflammatory, antimicrobial, and
The authors declare no competing financial interest.
antibacterial activity, as well of their high protein contents are
key indicators that highlight the advantages of using these
species in clinical devices. In the orthopedic field, few studies
have been reported on the use of microalgae, in which their
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