ORIGINAL ARTICLE
Eggshell-Derived Hydroxyapatite: A New Era in Bone
Regeneration
Vivekanand Kattimani, MDS, Krishna Prasad Lingamaneni, MDS,
Pandi Srinivas Chakravarthi, MDS, T.S. Sampath Kumar, PhD,y and Arjunan Siddharthan, PhDz
Background: Defects of maxillofacial skeleton lead to personal
(functional and aesthetic), social and behavioral problems; which
make the person to isolate from the main stream of society. So,
bone regeneration is the need for proper structure, function, and
aes-thetics following cyst enucleation, trauma, and tumor ablative
surgery; which helps for overall health of the individual.
Aim and Objectives: The preliminary study is planned to evaluate and
compare the efficacy of eggshell-derived hydroxyapatite (EHA) and
synthetic hydroxyapatite (SHA) following cystectomy. Material and
Methods: Microwave-processed calcium deficient EHA and
commercially available SHA are used for grafting. Total 20 patients
enrolled in this study, consisting 10 in each group between 20 and 45
years of age. All the patients were evaluated for bone regeneration at
first, second, third, and sixth month’s interval, postsurgically, using
radiovisiograph and clinical parameters.
Results: The bone formation characteristics vary at second month
when compared to SHA. This difference may be because of the
kinetics involved in the regeneration pattern. The pattern of bone
healing was trabecular after third month, indicating complete bone
formation. The study showed constant raise of density and
remained same at the end of study period.
Conclusions: Both EHA and SHA graft materials are equally
efficient in early bone regeneration. Within the limitations of this
study the EHA showed promising results. Which indicates the
eggshell waste-bio mineral is worthwhile raw material for the
production of HA and is a Go Green procedure. Eggshell-derived
hydroxyapatite is economic, compared with SHA.
Key Words: Apicoectomy, bone substitute, cystectomy, hen’s
eggshell, graft, osteoconductivity, osteoinductivity, synthetic
hydroxyapatite
(J Craniofac Surg 2016;27: 112–117)
To promote healing of large bone defects using graft material is well known.1–3
Missing bone is replaced traditionally with
From the Department of Oral and Maxillofacial Surgery, Sibar Institute of
y
Dental Sciences, Guntur; Medical Materials Laboratory, Department of
Metallurgical and Materials Engineering, Indian Institute of Technology
z The SHA was obtained from synthetic calcium HA in low-
Madras; and Department of Production Technology, MIT Campus, Anna
University, Chennai, India.
Received May 26, 2015.
Accepted for publication September 27, 2015.
Address correspondence and reprint requests to Vivekanand Kattimani,
Department of Oral and Maxillofacial Surgery, Sibar Institute of Dental
Sciences, Guntur 522509, Andhra Pradesh, India;
E-mail: drvivekanandsk@gmail.com
The authors report no conflicts of interest.
Copyright # 2015 by Mutaz B. Habal, MD
ISSN: 1049-2275
DOI: 10.1097/SCS.0000000000002288
112 The Journal of Craniofacial Surgery Volume 27, Number 1, January 2016
Copyright © 2015 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
material either from patient or donor.1–3 Clinicians and scientists are
in search of alternative bone graft substitutes because of factors like
donor site morbidity, time, and skill required for second surgery to
harvest graft, inadequate quantity available with respect to auto grafts,
and so.4–9 Multiple sources and different forms of bone grafts have
been used for bone augmentation and reconstruction in oral
maxillofacial and orthopedic surgery to stimulate bone healing since
long time.3,7–10 In recent years, use of synthetic or processed bone
graft substitute has gained popularity over traditional grafting
3,7–9
methods.
Hydroxyapatite (HA) is apatite calcium phosphate
Ca10(PO4)6(OH)2. In many disciplines of surgery HA is being used as
graft material successfully.11 In particular, HA shows highest level of
bioactivity and forms a quick bond with bone.10–12 A recent study
showed bioactive effect of the HA ceramic is because of the ability to
concentrate active fibronectin on its surface.13 Hydro-
xyapatite is available in porous/dense block or granular form for
2,3,7–9
clinical use. It is an inorganic ceramic substance which does
not cause any foreign body reaction; in porous grafts the hard
11–15
tissue grows into it and mingles with natural tissues.
The eggshell formulations are being used since beginning as trace
element and mineral supplying agents.16,17 The eggshell powder has
been examined in rats for bone healing.16–20 Few studies show
surface modified eggshell powder as osteoconductive
bone filling material with variable benefit in bone regener-
16,17,21
ation. The literature showed material properties of egg-
shell-derived hydroxyapatite (EHA) are superior when compared
22,23
with the commercially available graft materials. So, the EHA
has been introduced as bone graft substitute recently. 23 Histomor-
phometric evaluation showed that the EHA has excellent new bone
formation ability.18–21 This has led to the curiosity to prepare the
EHA from eggshell waste in a very economical way.22–24
The aim of this study is to evaluate and compare the efficacy of
indigenously prepared EHA with commercially available synthetic
hydroxyapatite (SHA) for enhancement of the bone regeneration.
In addition, the effect of grafting was evaluated using direct digital
radiographic imaging technique and computer densitometry in
bone regeneration of human cystic bone defects.
MATERIALS AND METHODS
Synthetic Hydroxyapatite
crystalline form. It is a mixture of HA, tri-calcium phosphate,
calcium phosphate, and calcium carbonate. Polycrystalline struc-
ture of HA is responsible for its strength. In our study, SHA used
is acquired from market; commercially available as G-synthetic
gran-ules (G Surgiwear, Shahjahanpur, India).
Eggshell-Derived Hydroxyapatite
Eggshell-derived hydroxyapatite sample preparation and
characterization22,23 is well described in our previous study. Many
preparatory methods were proposed for preparation of nano-HA in
The Journal of Craniofacial Surgery Volume 27, Number 1, January 2016 Eggshell-Derived Hydroxyapatite

TABLE 1. Showing Distribution of Subjects According to Groups and Sex TABLE 3. Showing the Size of the Lesion Preoperatively in Both the Groups

Sex SHA Group % EHA Group % Total % Size of the Lesion SHA EHA
Preoperatively Group % Group % Total %
Male 5 50.00 5 50.00 10 50.00
Female 5 50.00 5 50.00 10 50.00 <2 cm 5 50.00 2 20.00 7 35.00
Total 10 100.00 10 100.00 20 100.00 >2 cm 5 50.00 8 80.00 13 65.00
Total 10 100.00 10 100.00 20 100.00
EHA, eggshell-derived hydroxyapatite; SHA, synthetic hydroxyapatite.
EHA, eggshell-derived hydroxyapatite SHA, synthetic hydroxyapatite.

25–35
the literature, but in this study EHA is indigenously synthes-
ized by microwave processing. The EHA produced was RESULTS
chemically pure form of nanocrystalline calcium deficient
22– 24
hydroxyapatite with eggshell origin alike any other SHA. Clinical Observations
The sizes of cysts were ranged from 2 to 4 cm in diameter. All
Clinical and Radiologic Evaluation the patients were healed well without significant adverse clinical
The study was conducted in the Department of Oral and Max- observations irrespective of the size of the lesion in both the
illofacial Surgery in our institute after ethical committee approval. groups (Table 3). First day postsurgical clinical examination
The study registered in Clinical Trial Registry of India (CTRI) showed similar edema among patients in both the groups with
retrospectively with CTRI numbers CTRI/2014/09/004947 and reddish overlying mucosa in all the patients. At the time of suture
CTRI/2014/12/005340. A total number of 20 patients aged removal, the mucosal color and contour was normal in all the
between 20 and 45 years with periapical cyst/residual cyst of patients. Moreover, no flap dehiscence was observed.
anterior maxilla, requiring cystectomy/apicoectomy, and grafting
were enrolled (Tables 1-2). All the patients were divided randomly Digital Radiographic Observations
into 2 groups with 10 patients in each group. In Group-1 the cystic
defects were filled with SHA, whereas in Group-2 with EHA. Radiologic evaluation of surgical site outline and bone formation
characteristics in both the groups were significant with time interval
All the patients were followed up during first and second week
(Table 4; Fig. 1), but on comparison between the groups both SHA
postsurgically to evaluate the signs and symptoms of infection or any
and EHA showed similar pattern of bone formation and bone healing
other complications related with the surgical procedure. During
characteristics along with the margin blend with material margin at
clinical evaluation mucosal color, postoperative pain or swelling was
first, third, and second month’s interval (Table 5). The mean radio-
noted. Pain measurements were carried out using visual analog scale.
The wound healed uneventfully in all the patients. Seventh day of graphic density of grafted cystic bone defects were continued to
postsurgery, suture removal was done. Infected patients were excluded increase until it reached its maximum at 24 weeks (Table 6). The
from the study. Bone regeneration was assessed postopera-tively at mean density of SHA and EHA group were similar at third and sixth
first, second, third, and sixth month’s intervals using modified month’s interval. The formation of bone and bone margin blending
with material margin were well correlated with density of bone
observer strategy.36,37 The radiographs were examined blindly by 2
formation. The radiographs shows well demarcation of both material
examiners. In case of any gross inconsistency with observations, the and bone margin during first month follow-up (Fig. 2 A-B), and was
third examiner observed the radiographs to prevent bias and the results
invisible with passage of time (Figs. 3-5). At sixth month’s interval on
were tabulated. Surrounding normal bone density was also measured
both the sides it showed trabecular appearance indicating complete
as a control since beginning and correlated till last follow-up for
bone formation (Fig. 6). The observations of bone regeneration
comparison using the variable intensity light.
parameters by both the examiners were in very good agreement with
each other (k: 0.81–0.92).
Statistical Analysis
Radiographic changes in surgical site outline, internal portion
of surgical site after surgery were statistically compared using TABLE 4. Showing the Comparison of Groups (Synthetic Hydroxyapatite, Eggshell-
Mann-Whitney test and Wilcoxon matched-pairs test. The kappa Derived Hydroxyapatite) With Radiologic Evaluation of Surgical Site Outline at First,
(k) correlation was taken into consideration to assess the degree of Second, Third, and Sixth Month by Wilcoxon Matched-Pairs Test
observer agreement for radiologic assessment. The mean values Groups Treatments N t z P
and standard deviations of each parameter were calculated. The
Student t-test used to evaluate the differences between the means; SHA First month versus second month 10 0.00 2.8031 0.0051
P < 0.05 was considered as significant.
First month versus third month 10 0.00 2.0226 0.0431
First month versus sixth month 10 0.00 — —
TABLE 2. Showing Mean and Standard Deviation Age of Men and Women Second month versus third month 10 15.00 1.2741 0.2026
by Groups and Sex
Second month versus sixth month 10 3.00 2.4973 0.0125
SHA Group EHA Group Total Third month versus sixth month 10 0.00 1.8257 0.0679
EHA First month versus second month 10 0.00 2.8031 0.0051
Sex Mean SD Mean SD Mean SD First month versus third month 10 0.00 2.0226 0.0431
First month versus sixth month 10 0.00 — —
Male 28.20 7.66 25.00 4.53 26.60 6.17 Second month versus third month 10 15.00 1.2741 0.2026
Female 34.40 4.98 29.60 14.06 32.00 10.26
Second month versus sixth month 10 3.00 2.4973 0.0125
Total 31.30 6.91 27.30 10.14 29.30 8.69 Third month versus sixth month 10 0.00 1.8257 0.0679

EHA, eggshell-derived hydroxyapatite; SD, standard deviation; SHA, synthetic EHA, eggshell-derived hydroxyapatite; SHA, synthetic hydroxyapatite.
hydroxyapatite.

# 2015 Mutaz B. Habal, MD 113

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Kattimani et al The Journal of Craniofacial Surgery Volume 27, Number 1, January 2016

TABLE 6. Showing the Comparison of Radiologic Evaluation of Bone Density

(SHA, EHA) at First, Second, Third, and Sixth Month’s Interval
Treatment Time Group N Mean SD T P

First month SHA 10 102.10 8.65 1.8365 0.0829

EHA 10 108.00 5.33
Second month SHA 10 121.30 9.79 2.1745 0.0433
EHA 10 129.30 6.29
Third month SHA 10 145.10 9.39 0.4636 0.6485
EHA 10 147.30 11.71
Sixth month SHA 10 165.70 8.58 0.6441 0.5277
EHA 10 160.90 21.95
First–second month SHA 10 19.20 5.79 0.6679 0.5127
EHA 10 21.30 8.08
First month–third month SHA 10 43.00 6.22 0.7549 0.4601
EHA 10 39.30 14.20
FIGURE 1. Graph shows the comparison of eggshell-derived hydroxyapatite First month–sixth month SHA 10 63.60 10.01 1.3079 0.2074
and synthetic hydroxyapatite groups with radiologic evaluation of bone EHA 10 52.90 23.85
formation at first week, first, third and sixth month interval.

EHA, eggshell-derived hydroxyapatite; SHA, synthetic hydroxyapatite; SD, stan-

DISCUSSION dard deviation.
4–6 Significant at (P < 0.05).
The limitations of autografts and allografts have necessitated
2,3,7–9
the pursuit of alternatives. Osteoconduction and
osteoinduction are the 2 basic properties of the grafts used by nanocrystalline HA is osteoinductive in nature,10,38 and stimulates
investigators to develop several alternatives. Such materials are cells for periodontal tissue regeneration.12 The rapid bone regen-
available for clinical use and some of which are still in the eration associated with graft is because of enhancement of selective
2,3,7–9 adsorption of attachment proteins and growth factors by HA, which
developmental stage. These alternative materials include
natural/synthetic polymers, ceramics, and composites; whereas stimulates osteoblast adhesion and bone deposition12,13 or through ion
remaining are incorp-orated with different factors and cell-based release.39 This versatile biomaterial derived from many sources eg,
2–3,7–9
strategies used either alone or in combination. bone, corals, synthetic etc.3 Hydroxyapatite is unique biocom-patible
Hydroxyapatite is a naturally occurring mineral component of versatile biomaterial can be machined to many shapes or
bone and is osteoconductive in nature, which is used to graft bone consistencies.39–41 The porous form of HA stabilizes because of rapid
2,3,7–9
defects to stimulate bone healing. Recent literature showed fibrovascular tissue ingrowths, and resist micromotion. 42

TABLE 5. Comparison of 2 Groups (Synthetic Hydroxyapatite, Eggshell-Derived Hydroxyapatite) With Radiologic Evaluation Surgical Site Outline, Bone
Formation and Bone Healing Patterns at First, Second, Third, and Sixth Month by Mann-Whitney Test
Treatment Time Groups Sum of Ranks U z P

Radiologic evaluation surgical site outline First month SHA 105.00

EHA 105.00 50.00 0.0000 1.0000
Second month SHA 105.00
EHA 105.00 50.00 0.0000 1.0000
Third month SHA 105.00
EHA 105.00 50.00 0.0000 1.0000
Sixth month SHA 105.00
EHA 105.00 50.00 0.0000 1.0000
Radiologic evaluation of bone Formation First month SHA 105.00
EHA 105.00 50.00 0.0000 1.0000
Second month SHA 85.00
EHA 125.00 30.00 1.5119 0.1306
Third month SHA 110.00
EHA 100.00 45.00 0.3780 0.7055
Sixth month SHA 110.00
EHA 100.00 45.00 0.3780 0.7055
Radiologic evaluation of bone Healing First month SHA 105.00
EHA 105.00 50.00 0.0000 1.0000
Second month SHA 85.00
EHA 125.00 30.00 1.5119 0.1306
Third month SHA 110.00
EHA 100.00 45.00 0.3780 0.7055
Sixth month SHA 110.00

EHA 100.00 45.00 0.3780 0.7055

EHA, eggshell-derived hydroxyapatite; SHA, synthetic hydroxyapatite.

114 # 2015 Mutaz B. Habal, MD

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The Journal of Craniofacial Surgery Volume 27, Number 1, January 2016 Eggshell-Derived Hydroxyapatite

FIGURE 2. A, Immediate radiograph after synthetic hydroxyapatite grafting on the

right side showing the well demarcation of margins at periapical area of right
central incisor and right lateral incisor. B, Immediate radiograph after eggshell-
derived hydroxyapatite grafting on the left side showing the well demarcation of
margins at periapical area of left central incisor and left lateral incisor.

Hydroxyapatite is used for sinus grafting, socket preservation,

grafting of bony defects, ridge augmentations, and as a bone graft
expander when combined with autogenous bone and so.43–44 This
mixing could decrease the volume of autogenous bone graft needed,
which in turn potentially reduces the donor site morbidity. 44–46 This
study compared the EHA versus SHA as a novel bone graft material.
In our study, nanocrystalline calcium deficient EHA was used
which is very similar to natural bone apatite. The main inorganic
component of bone is calcium–hydroxyapatite, which contains
carbonated (3–8 w/t %), calcium deficient biologic HA and is
47
nonstoichiometric, noncrystalline, and ionsubstituted. Synthetic
hydroxyapatite is often stoichiometric with a specific atomic Ca/P
molar ratio of 1.67 and chemical formula of Ca10(PO4)6(OH)2.22,23
Synthetic hydroxyapatite is similar in chemical and crystallo-
graphic structure with that of bone material with excellent
FIGURE 4. Radiograph after two month of the grafting showing improved
bone formation at periapical area of right central incisor, right lateral incisor,
left central incisor, and left lateral incisor.
47–50
biocompatibility. One of the main components of HA and all
living hard tissues is calcium. So the source of calcium needed for
the preparation of HA can be obtained from natural precursors like
eggshells, which minimizes impurities like silica in the
material; also the cost of production can be reduced a lot as there is no
need to purify.22,24 Eggshells are one of the richest sources of
calcium, consisting of calcium carbonate (94%), calcium phosphate
(1%), organic matter (4%), and magnesium carbonate (1%). 22,24 A
huge amount of eggshells are produced daily which are basically of no
use causing environmental pollution. So, an EHA was produced from
waste eggshells and was used for grafting clinically. Results of the
current study were associated with a greater rate of bone regeneration
both in SHA- and EHA-grafted sites and the results are in accordance
with similar studies in the literature.20,23
The radiologic evaluation of bone formation is the initiation of
bone formation after grafting of cystic defects marked by radio
opacity. Bone formation pattern was different in both graft materials

FIGURE 3. Radiograph after one month of the grafting showing improved

bone formation at periapical area of right central incisor, right lateral incisor, FIGURE 5. A, Radiograph after 3 months of synthetic hydroxyapatite grafting.
left central incisor, and left lateral incisor. B, Radiograph after 3 months of eggshell-derived hydroxyapatite grafting.

# 2015 Mutaz B. Habal, MD 115

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Kattimani et al The Journal of Craniofacial Surgery Volume 27, Number 1, January 2016
FIGURE 6. Radiograph after 6 months showing uniform radio opacity
indicating well-healed lesion on both sides.
at the second month’s interval which may be attributed to early or late
resorption kinetics of graft particles.20 At third and sixth month’s
interval, the pattern of bone formation in both the SHA and EHA
groups, however, remains the same (P > 0.05). All the bone
regeneration parameters were well correlated with the observations of
the study performed by other investigators in the literature. 18–20,23
Eggshell-derived hydroxyapatite seems to be promising graft
material with excellent properties for grafting18–20,23 with viable
various production techniques.25–35 Lack of disease transfer risks,
biocompatibility, and ease of use makes EHA a viable choice as
regenerative material. Eggshell-derived hydroxyapatite is hydro-philic
in nature, absorbed by body fluids and blood, so that handling
becomes easy for placing it in the surgical site. Because of ethical
concerns collection of tissue for histologic examination has excluded
in this study but histologic examination of EHA has been studied in
animal models16–20 and surface modified eggshell in humans which
showed promising results.21 The preliminary reports of EHA showed
good results in humans by Kattimani et al.23 Other interesting trends
for HA include applications in drug delivery, cell culture, purification
of antibodies on industrial scale, as an artificial
blood vessel or trachea, as well as a catheter made of an HA-
51,52
composite. Many bone grafts are being marketed today for
clinical use. Because of newer innovative technology in the pro-
duction and combinations of existing materials for newer appli-
cations is emerging as new field in the bone regeneration for
successful clinical utility.
CONCLUSIONS
Eggshell-derived hydroxyapatite becomes a viable choice as regen-
erative material because of its biocompatibility, lack of disease
transfer risks, ease of use, and unlimited availability of eggshell raw
material. Eggshell-derived hydroxyapatite is a versatile novel bone
graft substitute that yielded promising results within the limitations
116
Copyright © 2015 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
of this study. Eggshell-derived hydroxyapatite is very cost-
effective which can be prepared in a very economical way. It is a
worthwhile bone substitute because it is safe and easily available
material and process involved in the production is environment
friendly. This study warrants a larger sample size with variable
age group, including hisomorphometry to confirm its nature of
bone regener-ation. Further research is going on to use EHA in
combination with collagen, cell factors, platelet-rich plasma, and
platelet-rich fibrin (PRF) lysate for bone repair. The future of
EHA graft material continues to be an expanding topic.
ACKNOWLEDGMENT
The authors thank S.B. Javali for his continued support in the
research.
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