Surgical Science, 2011, 2, 369-375
doi:10.4236/ss.2011.27081 Published Online September 2011 (http://www.SciRP.org/journal/ss)
Tissue Engineered Osteogenesis in Bone Defects by
Homologous Osteoblasts Loaded on Sterile
Bioresorbable Coral Scaffold in Rabbits
Arvind Tripathi1, Pandruvada Subramanya Narayana Murthy2, Govind Keshri3, Man Mohan Singh1
1Saraswati Dental College and Hospital, Faizabad Road, Lucknow, India
2Institut des Recherches Cliniques de Montreal, Montreal, Canada
3C-406, Indira Nagar, Lucknow, India
Received January 11, 2011; revised June 28, 2011; accepted August 18, 2011
Abstract
Objectives: This study explores feasibility of tissue-engineered osteogenesis using sterile coral implants
loaded with homologous osteoblasts to repair bone defects. Study Design: A unilateral 4 mm transverse dis-
continuity defect was produced approximately mid-way along left radius of young female rabbits using ro-
tary diamond disc under continuous saline irrigation and stabilised with autoclaved steel miniplate and
screws. The defect was then fitted with sterile bioresorbable coral implant loaded with homologous neonatal
calvarial osteoblasts or control implants without osteoblasts. All animals underwent radiography immedi-
ately post-operative, at weekly intervals for four weeks and at fortnightly intervals thereafter. Operated bones
were histologically evaluated for osteogenesis at 12 weeks. Results: Findings demonstrate osteogenesis and
complete repair of bioresorbable coral implant by homologous osteoblasts loaded on coral scaffold. Conclu-
sions: Single stage surgery using this technique to induce osteogenesis and closure of discontinuity bone de-
fects including palatal clefts and peripheral reduction of large craniofacial defects might prove better thera-
peutic modality than autologous bone grafting or tissue distraction osteogenesis.
Keywords: Osteogenesis, Homologous Neonatal Calvarial Osteoblasts, Bioresorbable Coral Scaffold,
Discontinuity Bone Defects, Rabbit
1. Introduction
Until the beginning of 20th century, amputation was the
only practical solution to treat large bony defects consid-
ered incurable leading to devastating trauma and side
effects for the patient [1]. Later advances led to the de-
velopment of techniques like autologous bone and free
vascularised fibular grafting, still considered as gold
standard with most favorable outcome, to facilitate
treatment of bone defects [2]. While allogenic grafts as
well as synthetic bone substitutes [3] have been used to
fill voids, this approach appeared successful only for
small bone defects.
Osteogenesis is a complex physiological process. It
involves coordinated participation of haemopoietic and
immune cells within the bone marrow in conjunction
with vascular and skeletal cell precursors [4]. It is de-
pendant on adequate number of osteoblasts, which are
recruited predominan tly from the local bo ne marrow and
periosteal cambial layer. Tissue engineered bone genera-
tion has been considered as an effective tool in osteo-
genesis and closure of bone defects including palatal
clefts and peripheral reduction of large craniofacial de-
fects [5]. Use of scaffolds of synthetic as well as natural
biomaterials has also been tried to promote migration,
proliferation and differentiation of bone cells [6]. Previ-
ous studies have proved that natural coral exoskeleton,
because of its interconnected porous structure, like the
spongy bone, has superior mechanical properties of cal-
cium-based ceramic [7]. Transcortical bony defects im-
planted with coral have been reported to become vascu-
larised and display osteogenesis with concurrent resorp-
tion of the coral implant [8]. Such scaffolds, however,
lack osteogenic and osteoinductive potential of bone
grafts [7]. Recent tissue engineering with the use of em-
bryonic stem cells has demonstrated their capability to

370 A. TRIPATHI ET AL.
adopt various cell fates and facilitate development and
regeneration of various tissues [9]. Stromal compartment
of bone marrow contains many multi-potential mesen-
chymal stem cells (MSCs) [10], which can be induced to
differentiate into osteoblasts to form new bone. Avail-
able evidence shows that MSC-loaded implants can elicit
true bone regeneration with complete disappearance of
the biomaterial and formation of cortical bone in a bone
defect of clinically relevant volume [11]. However, since
the use of stem cells for treatment might involve ethical
considerations in clinical setting s [12] together with lim-
ited availability of osteogenic fresh autologous grafts
[13], use of proliferating homologous ultimate bone
forming cells or osteoblasts delivered on a resorbable
natural biomaterial scaffold to induce osteogenesis in
critical bone defects was hypothesized and forms the
basis of this study.
2. Materials and Methods
2.1. Animals and Chemicals
Fourteen neonatal (1 - 3 days old) and fourteen young
(60 days old) female New Zealand white rabbits (body
weight: 600  100 g) maintained under standard condi-
tions (22˚C  1˚C) with alternate 12 h light/dark periods
and free access to regular pellet diet (Lipton India Ltd.,
Bangalore) and tap water were used in this study. This
study was conducted at the Central Drug Research Insti-
tute, Lucknow, India. The Animal Ethical Committee of
the Institute approved the protocol with respect to the
humane care and treatment of animals used in this study.
Minimum essential medium (α-MEM), collagenase,
dispase, fetal calf serum (FCS), glutamine, penicillin-
streptomycin, nonessential amino acid solution, sodium
pyruvate, sodium -glycerophosphate, ascorbic acid, nap h -
thol AS-MX-phosphate, ethylene glycol monomethyl ether,
Fast-Red TR salt, were purchased fro m S i g ma-A l d ri c h , S t .
Louis, MO, trypsin-EDTA (ethylene diamine tetra acetic
acid) from Gibco Life Sciences, Grand Island, NY. All
other chemicals were of analytical grade. Coral exo-
skeleton (Porites spp,) generously provided by the Cen-
tral Marine Fisheries Research Institute, Kochi, India
was identified by the Botany Department of the Insti-
tute.
2.2. Coral Implant Preparation
Cylindrical pieces (Figure 1) of the coral exoskeleton
were cut on a Rotary Diamond Disc mounted on a Dental
Micromotor Handpiece (NSK, Japan) with continuous
physiological saline (0.85% sodium chloride in distilled
water) spray. The coral pieces were sterilized by auto-
claving and used as implants.
Figure 1. Cylindrical pieces of the coral exoskeleton (Porites
spp.) cut on a Rotary Diamond Disc mounted on a Dental
Micromotor Handpiece with continuous physiological saline
spray.
2.3. Primary Osteoblast Cell Culture
Neonatal rabbit calvarial cell cultures (Figure 2) were
established as described previously [14,15] with slight
modifications. Briefly, frontal and parietal bones from
rabbit neonates (1 - 3 day old) were digested in 0.2%
collagenase/0.2% dispase in MEM to obtain 5 sequen-
tial digests. The second through fifth digests were com-
(a)
(b)
Figure 2. Neonatal rabbit calvaria derived primary os-
teoblast cells (a) and showing alkaline phosphatase expres-
sion (b) 7 days after culture in defined medium.

A. TRIPATHI ET AL.
371
bined, resuspended and grown to confluence at 37˚C and
5% CO2 in air in  MEM supplemented with 10% fetal
bovine serum (FBS), 20 mM glutamine, 100 U/ml peni-
cillin-streptomycin, 0.1 mM non-essential amino acid
solution, 1 mM sodium pyruvate, 10 mM sodium
 -
glycerophosphate and 50 g/ml ascorbic acid. Culture
medium was changed every 48 h. Cells were then
trypsinised and approximately 2 × 106 cells were loaded
onto each autoclaved coral implant and cultured for 12 h
for the cells to adhere to the coral implants. The prolif-
eration, differentiation and viability of cells in culture
was confirmed by alkaline phosphatase staining, an early
stage osteoblast differentiation marker. Pertinently, cal-
varia derived primary osteoblast cultures are considered
>95% pure cell population [16]. For alkaline phosphatase
(ALP) expression, the cells were plated at 104 cells per
cover slip (6 mm diameter, Thermanox, Nunc, USA) and
cultured for 7 days in 96-well plates, fixed in 4% for-
maldehyde in phosphate buffered saline (PBS) and incu-
bated with the substrate (5 mg naphthol AS-MX phos-
phate, 0.25 ml ethylene glycol monomethyl ether and 10
mg Fast red TR in 24 mL 0.1 M TBS, pH 9.5) for 1 h at
room temperature. Representative images were captured
using Leica DC 300 camera and Leica IM50 Image Ac-
quisition software fitted to a Leica DMLB microscope.
2.4. Osteotomy
A unilateral discontinuity defect was produced in one
radius bone of each young female rabbit by standardized
surgical procedure under Ketamine (125 mg, i.m.) short
acting anaesthesia. For this purpose, forearm of each
rabbit was shaved and swabbed with Povidone-Iodine
solution (Betadine Lotion, Win Medicare India Ltd.,
New Delhi). About 5 cm longitudinal incision was made
to expose the radius bone. Approximately mid -way alon g
its length, a 4 mm transverse discontinuity defect was
produced using a Rotary Diamond Disc (Horico-Hopf
Ringleb, GmbH, Germany) under continuous sterile PBS
irrigation at room temperature to prevent thermal necro-
sis of the margins and stabilised with autoclaved steel
miniplate and screws. The debris was removed by flush-
ing with the physiological saline. Th e dissected limb was
immediately stabilized with the help of an autoclaved
steel bone miniplate and screws (6 mm length, 1.5 mm
diameter; 2 screws on each side of the defect; Figures
3-5) and tightened with an implant screwdriver. Animals
were then randomized into two groups of seven each.
Animals of Group I received sterile coral implants
loaded with homologous osteoblasts, whereas animals of
Group II received sterile coral implants without os-
teoblasts in a similar manner. Care was taken to ensure a
snug fit of the implant into the discontinuity defect
without side projections. The incision was then sutured
Figure 3. Radiographs of left forearm of a rabbit (#7;
Group I) with unilater al discontinuity de fect in radius bone
fitted with sterile coral implant loaded with homologous
osteoblasts taken immediately post-operative (a) and at 4
(b), 6 (c), 8 (d), 10 (e) and 12 (f) weeks thereafter. Steel bone
miniplate and screws used to stabilize the dissected limb are
visible in the radiographs. Note progressive osteogenesis
with increasing intervals post-surgery and concurrent re-
duction in size of the coral implant. While the coral implant
was almost completely resorbed at 10 ± 1 weeks, complete
repair (arrow) of the discontinuity bone defect was ob-
served at 12 weeks (f,g). This status was observed in 5 of the
7 rabbits of this group. Histological picture (g) also shows
stabilizing screw (*)
with 3.0 silk (Ethicon, Johnson and Johnson, USA) fol-
lowed by dressing with surgical gauge and Micropore
adhesive tape (3M India-Bangalore) after application of
Mupirocin cream (T-Bact, Smith-Kline & French, India,
Mumbai).
2.5. Radiography, Tissue Retrieval and
Histology
Radiographs of each bone were taken immediately
post-operative, at weekly intervals for 4 weeks after sur-
gery and at fortnightly intervals thereafter. Paralleling
cone technique (70 Kvp, 8 mA Machine-Explor-X,
VILLA, Italy) with a 0.7 sec exposure time was used.
Animals were autopsied at 12 weeks post-surgery by
excessive ether inhalation. Operated radial bones from
each animal were carefully dissected free of adhering

372 A. TRIPATHI ET AL.
Figure 4. Radiographs of left forearm of a rabbit (#3;
Group I) with unilateral discontinuity defect in the radius
bone fitted with sterile coral implants loaded with homolo-
gous osteoblasts taken immediately post-operative (a) and
at 4 (b), 6 (c), 8 (d), 10 (e) and 12 (f) weeks after surgery.
Steel bone miniplate and screws used to stabilize the dis-
sected limb are visible in the radiographs. Note only partial
bone formation (arrow) with gradual resorption and ejec-
tion of the implant (**) at 12 weeks post-surgery (f) (g).
This status was observed in only 2 of the 7 rabbits of this
group.
tissue and fixed in 70% ethanol for histology. Undecalci-
fied isolated radial bones were dehydrated using ascend-
ing gradations of isopropanol, cleared in acetone and
embedded in clear self-cure Polymethyl-Methacrylate
following standard procedure [17]. Resin blocks were cut
into 50 m thick longitudinal sections using a dia-
mond-wafering blade fitted to Isomet low speed saw
(Buehler, USA). Images of unstained sections were cap-
tured using Leica DC 300 camera and Leica IM50 Image
Acquisition Software fitted to Leica S6D microscope to
monitor the extent of healing in each animal.
3. Results
3.1. Osteoblast Cell Cultures and ALP
Expression
The cells (Figure 2(a) and (b)) attained >70% conflu-
ency after 7 days of culture and exhibited intense ALP
staining.
Figure 5. Radiographs of left forearm of a rabbit (#10;
Group II) with unilateral discontinuity defect in the radius
bone fitted with empty coral implant (i.e., without os-
teoblasts) taken immediately post-operative (a) and at 4 (b),
6 (c), 8 (d), 10 (e) and 12 (f) weeks after surgery. Steel bone
miniplate and screws used to stabilize the dissected limb are
visible in the radiographs. Note minimal bone formation
and absence of complete bone union. The coral implants (**)
did not resorb fully even 12 weeks after surgery (f,g). Par-
tial bone formation (arrow) with implant loosening (be-
cause of partial resorption) is, however, evident (g). Coral
implant is surrounded by fibrous connective tissue aggrega-
tion. This status was observed in all the 7 rabbits of this
group. Histological picture (g) also shows stabilizing screw
(*).
3.2. Osteogenesis
Marked differences in the extent of osteogenesis were
observed between the two groups of animals. In animals
with discontinuity defects fitted with coral implants
loaded with homologous osteoblasts (Group I), osteo-
genesis was observed at around 4  1 weeks post-surgery
(Figure 3(b)) and bone formation was near complete
with defects appearing radiolucent at 6  1 weeks (Fig-
ure 3(c)). Concurrent with osteogenesis, coral implants
displayed resorption and reduction in size and were al-
most completely resorbed at 10  1 weeks (Figure 3(e)).
In five of the animals of this group, the bone defect was

A. TRIPATHI ET AL.
373
completely filled with new bone (Figure 3(f) and (g)),
while in two animals, partial bone formation with grad-
ual resorption and ejection of the implant was observed
(Figure 4(a)-(g)).
In all the 7 animals of Group II fitted with sterile coral
implants without osteoblasts (Figure 5(a)-(g)), osteo-
genesis was generally minimal and complete bone union,
as observed in animals of Group I, did not occur in any
animal. The coral implants, too, did not get fully re-
sorbed in any animal ev en 12 weeks post-su rgery. Partial
bone formation with loosening of implants due to partial
resorption was, however, evident. Coral implants in ani-
mals of this group were generally surrounded by fibrous
connective tissue ag gregation.
4. Discussion
Results of the present study provide evidence of tissue
engineered osteogenesis and complete repair of small
discontinuity defect in radial bone of rabbits by confluent
homologous calvarial osteoblasts loaded on sterile bio-
resorbable coral scaffold. Pertinently, bone tissue engi-
neering, combining the application of principles of or-
thopedic surgery with basic science and engineering has
been heralded as an alternative to bone regeneration to
replace or restore the function of traumatized, damaged
or lost bone [18].
Critical bone defects fail to heal because of perturba-
tions at the site of fracture [19]. This led to the use of a
variety of materials such as hydroxyapatite, tricalcium
phosphate, hyaluronic acid based polymer, decalcified
bone and chondroid bone for osteoinduction in animal
models [20-22]. However, the main drawback of use of
such implants was their slow resorption, producing bony
ingrowth onto a porous surface rather than true bone
generation [23]. Recent tissue engineering approaches
have attempted to repair cartilag e and bone defects based
on mesenchymal stem cells (MSCs) seeded onto porous
ceramic scaffolds [24]. Compared to harvesting bone
grafts, needle aspiration of bone marrow offers a simple,
minimally traumatic and rich source of MSCs in the in-
fant patient minimizing morbidity. These cells have the
potential to differentiate into bone, cartilage, marrow
element, fat and connective tissue. A series of studies [25]
have strongly indicated the feasibility of porous natural
coral as scaffold material transplanted with marrow de-
rived osteoblasts for osteogenesis. Al-Salihi and Sam-
suddin [26] have demonstrated that the natural coral im-
plant provided excellent and favorable situation for bone
marrow cells to differentiate into osteoblasts that could
lead to formation of a large amount of mineralized tissue
on the coral surface. In an attempt to regenerate bone,
Petite et al. [11] used sea coral, a natural calcium car-
bonate based ceramic, in combination with autologous
MSCs to produce orthopedic implants that facilitated
healing of bony defects in sheep . Over the course of fou r
months of implantation, these coral-stem cell composites
were remodeled into radiographically mature bone and,
in some cases, demonstrated complete unification with
native bone on either side of the composite. Moreover,
the results were superior to those observed when coral
was implanted alone, or in combination with an aspirate
of fresh bone marrow rather than with stem cells. These
investigators speculated that formation of mature bone
from coral-stem cell composite could be attributed to
degradable nature of the natural ceramic matrix, as well
as to interconnectedness of the matrix pores that allowed
infiltration of the bone precursor cells.
According to Guillemin [27], Porites coral scaffold, as
used in the present study, with an average pore size of
250 m and an interconnected structure with no dead end
pockets should facilitate vascu lar invasion an d new bone
formation. As osteogenesis progressed, the coral implant
displayed resorption and reduction in size. This sug-
gested conduction of bone marrow cells through the
coral implant and their participation in coral resorption.
Pertinently, while use of MSCs has the advantage over
osteoblasts, for giving rise to cells of multilineage con-
tributing to fracture healing, homologous/autologous os-
teoblasts provide a potential source for skeletal tissue
engineering. Wang et al. [28] have reported better new
bone growth using perfusion culture of marrow derived
osteoblasts on por ous ceramic substrates.
Bone generation by autologous cell transplantation in
combination with biodegradable scaffold is also one of
the most promising techniques being developed in cra-
niofacial surgery [29]. Despite continued efforts at im-
proving various surgical techniques to achieve superior
functional outcome and aesthetic results in palatal clefts
defects, complications such as postoperative bleeding,
graft rejection and wound dehiscence leading to fistulae
have continued to challenge the surgeons. The therapeu-
tic hypothesis in our study was to put a bioresorbable
organic implant, conforming to the size of the palatal
cleft as estimated from a preoperative occlusal radio-
graph loaded with homologous osteoblasts such that as
the implant resorbed, tissue engineered osteogenesis
would close the defect. Advantages of such an approach
over autologous bone grafting or tissue distraction os-
teogenesis include single stage surgery, unhampered su-
tural and midfacial growth and development of proper
velopharyngeal sphincter vital to normal speech.
Healing time is a major concern in tissue repa ir. In the
present strategy, it was presumed that confluent os-
teoblasts might initiate osteogenesis faster than their
immature pluripotent precursors. This is despite the ini-

374 A. TRIPATHI ET AL.
tial cell death due to lack of vascularisation within the
implant resulting in impeded action of osteoblasts. Dur-
ing the repair process, the pathway of normal embryonic
development is recapitulated with coordinated participa-
tion of several cell types [30]. Currently, as the molecu-
lar and cellular events du ring the cascade of osteogenesis
are being better understood, newer three-component
strategies [31] are being devised and tested in order to
promote bone formation/healing. In essence, tissue engi-
neering aims to combine progenitor cells such as MSCs
or mature osteoblasts for osteogenesis with biocompati-
ble materials or scaffolds for osteoinduction and appro-
priate growth factors and/or certain osteogenic agents [21]
for osteoconduction to be able to generate and maintain
bone [29]. This new strategy though offers great poten-
tial for treatment of conditions requiring bone repair,
needs detailed investigation so that tissue engineered
osteogenesis becomes a viable and simple therapy option
for bone defects including cleft palate and other cranio-
facial defects minus the need of repetitive attempts at
repair and consequent adverse effects of scarring, im-
peded bone growth and bone fusion failure.
5. Acknowledgements
The authors gratefully acknowledge the help offered by
the Botany Department of CDRI, Lucknow, India for
identification and Director, Central Marine Fisheries
Research Institute, Kochi, India for providing the coral
exoskeleton (Porites spp.) and the Ministry of Health and
Family Welfare, Government of India, New Delhi for
financial support. PSNM thanks the Council of Scientific
& Industrial Research, New Delhi for award of Senior
Research Fellowship and MMS thanks the Indian Coun-
cil of Medical Research, New Delhi for appointment as
Emeritus Medical Scientist. Approval of the Institutional
Review Board for its publication has been obtained and
the manuscript has been assigned communication num-
ber 7634.
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