Comparison of autogenic and allogenic bone marrow derived mesenchymal stem cells for repair of segmental bone defects in rabbits

Research in Veterinary Science 94 (2013) 743–752

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Comparison of autogenic and allogenic bone marrow derived mesenchymal stem cells for repair of segmental bone defects in rabbits Rahul Kumar Udehiya a, Amarpal a,⇑, H.P. Aithal a, P. Kinjavdekar a, A.M. Pawde a, Rajendra Singh b, G. Taru Sharma c a b c

Division of Surgery, Indian Veterinary Research Institute, Izatnagar 243122, India Centre for Advanced Disease Research and Diagnosis, Indian Veterinary Research Institute, Izatnagar 243122, India Division of Physiology and Climatology, Indian Veterinary Research Institute, Izatnagar 243122, India

a r t i c l e

i n f o

Article history: Received 16 April 2012 Accepted 20 January 2013

Keywords: Allogenic Bone marrow Mesenchymal stem cells Bone defect Healing Rabbits

a b s t r a c t Autogenic and allogenic bone marrow derived mesenchymal stem cells (BM-MSCs) were compared for repair of bone gap defect in rabbits. BM-MSCs were isolated from bone marrow aspirates and cultured in vitro for allogenic and autogenic transplantation. A 5 mm segmental defect was created in mid-diaphysis of the radius bone. The defect was filled with hydroxyapatite alone, hydroxyapatite with autogeneic BM-MSCs and hydroxyapatite with allogenic BM-MSCs in groups A, B and C, respectively. On an average 3.45  106 cells were implanted at each defect site. Complete bridging of bone gap with newly formed bone was faster in both treatment groups as compared to control group. Histologically, increased osteogenesis, early and better reorganization of cancellous bone and more bone marrow formation were discernible in treatment groups as compared to control group. It was concluded that in vitro culture expanded allogenic and autogenic BM-MSCs induce similar, but faster and better healing as compared to control. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Comminuted fractures of long bones involving varying amount of bone loss are frequently encountered in veterinary practice. Management of such fractures requires not only proper fixation but also maintaining the structural integrity at fracture site by preserving the loose bone pieces. In the cases of bone loss the gold standard method of bone replacement for the treatment of bone gap defects or non-union is the autologous bone graft, where a piece of bone is taken from another body site, and transplanted into the defect (Salgado et al., 2004). Though the success rate of this procedure is quite high, the number of cases in which it can be used are small, due to the limited amount of available tissue, and increased risk of donor site morbidity (Rose and Oreffo, 2002; Spitzer et al., 2002). The second most common treatment is allografting, using tissue from another animal of the same species after processing to reduce antigenicity. This treatment, however leads to a lower rate of graft incorporation with the host tissue (Salgado et al., 2004) and involves the risk of immune rejection and pathogen transmission in the recipient (Herberts et al., 2011). Alternative techniques to bone grafting encompass the use of ceramic or metal implants enriched with osteoinductive cells. ⇑ Corresponding author. Tel.: +91 9012339489; fax: +91 5812301327. E-mail address: [email protected] ( Amarpal). 0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2013.01.011

It is estimated that one in 100,000 cells in the bone marrow is a mesenchymal stem cell (MSCs). MSCs have been shown to differentiate along several lineages including bone (Pittenger et al., 1999; Jaiswal et al., 1997; Kadiyala et al., 1997), cartilage (Pittenger et al., 1999; Mackay et al., 1998; Johnstone et al., 1998) and fat (Pittenger et al., 1999). MSCs are the most commonly used seed cells, having the potential for in vitro expansion and osteogenic differentiation (Patel et al., 2008; Barry and Murphy, 2004; Chao et al., 2007; Deans and Moseley, 2000). Autologous MSCs are the optimal type of seed cell; both animal experiments and clinical trials indicate that bone constructed using autologous MSCs has strong osteogenic ability (Lucarelli et al., 2004; Quarto et al., 2001). Ease of availability and capability of allogenic BM-MSCs to avoid immune rejection (Ryan et al., 2005) have made these cells an attractive alternative to autogenic marrow-derived cells (MDCs) for reconstructive surgery. Allogeneic mesenchymal stem cells loaded on hydroxyapatite– tricalcium phosphate implants enhanced the repair of the canine femur without the use of immunosuppressive therapy (Arinzeh et al., 2003). Planka et al. (2008) reported that the transplantation of both autogenous and allogeneic MSCs into a defect of the growth plate appears as an effective method of surgical treatment of physeal cartilage injury. However, comparative evaluation of autogenic and allogenic BM-MSCs with hydroxyapatite granules for diaphyseal bone defect repair in rabbits is not well documented.

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The purpose of this study was to compare the therapeutic efficacy of autogenic and allogenic bone-marrow-derived mesenchymal stem cell transplantation with hydroxyapatite for the repair of bone segmental defect model in rabbits. 2. Materials and methods 2.1. Animals Fifty-four clinically healthy, six to seven months old New Zealand white rabbits of either sex, weighing 1.4–2.2 kg were used. The Institute Animal Ethics Committee of the Indian Veterinary Research Institute, Izatnagar, India approved the study. Animals were housed in rabbit cages individually under uniform feeding and management conditions, water was provided ad libitum. The animals were acclimatized to approaching and handling for a period of 15 days before start of the study. 2.2. Experimental design The rabbits were divided randomly into three groups viz. Group A (Hydroxyapatite with Dulbecco’s Modified Eagle’s Medium – low glucose (DMEM-LG)), Group B (Hydroxyapatite with culture expanded autogenic BM-MSCs) and Group C (Hydroxyapatite with culture expanded allogeneic BM-MSCs). Each treatment was awarded to 18 animals, out of which six animals were euthanized using over dose of thiopental at each interval i.e. on 30th, 60th and 90th days to evaluate extent of bone healing by radiography, angiography and histology. 2.3. Isolation and culture of BM-MSCs The rabbits were anesthetized with 6 mg/kg xylazine administered intramuscularly, followed 10 min later by 60 mg/kg ketamine (Amarpal et al., 2010). The area over the left and right iliac crests was prepared aseptically by shaving, scrubbing with cetrimide and painting with povidone iodine. The bone marrow aspirate was collected from the lateral aspect of the iliac crest using an 18 gage bone marrow biopsy needle. Approximately 2.5 ml of bone marrow aspirate was collected in the syringe containing 2500 IU of heparin. The same procedure was repeated for the contra-lateral iliac crest to collect another 2.5 ml bone marrow aspirate in the same syringe. Thus, a 5 ml of bone aspirate was collected from each animal. The marrow sample was washed with equal volume of Dulbecco’s phosphate buffered saline (DPBS, Thermo Scientific HyClone, Chemicals Co., USA) (5 ml) and disaggregated, by passing it gently through a 21-gage intravenous catheter and a syringe, to create a single cell suspension. Marrow sample with 5 ml of DPBS

Fig. 1. Fully confluent monolayer of BM-MSCs before trypsinization of the cells for transplantation.

was loaded onto 5 ml of Ficoll–Paque plus (GE Healthcare BioSciences, Sweden). The mono-nucleated cells were collected from the interface by centrifugation at 805 g for 30 min, and diluted with two volumes of DPBS. The cells were washed with DPBS and centrifuged at 201 g. After centrifugation, 5 ml of RBC lysis buffer was added to the cells, mixed properly and again centrifuged at 201 g for 10 min. The cells were again washed with DPBS at the same speed of centrifugation. The cells were resuspended in DMEM-LG (Thermo Scientific HyClone, Chemicals Co., USA) containing 10% fetal bovine serum (FBS, Thermo Scientific HyClone, Chemicals Co., USA) and antibiotics (mixture of 100 units/ml of penicillin and 100 lg/ml of streptomycin) (Sigma–Aldrich, India). The cells were counted by Neubar’s counting chamber and plated at an average of 2.2  105 cells/cm2 in T-25 flasks and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air in a CO2 incubator. After 3 days of primary culture, the non-adherent cells were removed with medium and fresh medium was added to the flasks. The medium was then changed every 3rd day until 14th– 18th day. MSCs grew as symmetrical colonies. Upon reaching 80–90% confluency, as assessed by visual inspection under inverted microscope (Fig. 1), the cells were passaged at lower densities into new culture flasks. To obtain the cells from the culture flasks, culture medium was removed, and cells were washed with 0.05% trypsin and 0.53 mM ethylenediaminetetraacetic acid (EDTA) for 5 min. Trypsin–EDTA activity was stopped by adding

Fig. 2. (a) A 5 mm bone defect created in the central diaphysis of radius bone, (b) the defect filled with hydroxyapatite granules, and (c) application of BM-MSCs at the defect site.

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R.K. Udehiya et al. / Research in Veterinary Science 94 (2013) 743–752 Table 1 Modified Lane and Sandhu (1987) and Heiple et al. (1987) radiological and histopathological scoring system. S.no.

Feature

I 1

Radiological scoring Reduction in defect size Less than 25% reduction 25–50% reduction 50–75% reduction More than 75% reduction No gap

1 2 3 4 5

Radiographic density No density Slight Moderate Dense

0 1 2 3

Remodeling of bone No remodeling Less than 25% reduction in size of callus 25–50% reduction in size of callus 50–75% reduction in size of callus More than 75% reduction in size of callus and canalization of marrow cavity

0 1 2 3 4

2

3

Score

4 II

Maximal possible radiological score Histopathological scoring

1

Osteogenesis No osteogenesis Weak osteogenesis Medium osteogenesis Good osteogenesis Perfect osteogenesis

0 1 2 3 4

Union No evidence of union Fibrous union Osteochondral union Bony union Complete organization of shaft

0 1 2 3 4

Marrow None is resected area Beginning to appear Present in more than half of the defect Complete colonization by red marrow Mature fatty marrow

0 1 2 3 4

Cancellous bone/medullary bone No osseous cellular activity Early apposition of new bone Active apposition of new bone Reorganizing cancellous bone Completely reorganization of cancellous bone

0 1 2 3 4

Cortical/compact bone None Early appearance Formation under way Mostly reorganized Completely formed

0 1 2 3 4

Total point possible per category Osteogenesis Union Marrow Cancellous bone/ medullary bone Cortical/ compact bone

4 4 4 4 4

2

3

4

5

6

Total histological score

3 ml of DMEM-LG having 10% FBS, the contents were collected in centrifuge tube and centrifuged at 358 g for 6 min. The supernatant was discarded and the pellet was resuspended in 10 ml of DMEM. The suspension was aspirated through a 20 gage needle three times to obtain a single cell suspension and the cells were replated onto T25 culture flasks at half of their original density. Cultures were maintained at 37 °C in a 95% air 5% CO2 incubator. Supplemented DMEM was changed every 3rd–4th day. After 7–10 days when cells reached to full confluency, they were again trypsinized as described above. After centrifugation, the supernatant was dis-

12

20

carded and the pellet was resuspended in 200 ll of supplemented DMEM. After adjusting the average cell count to 3.45  106/200 ll, the cells were used for transplantation in groups B in autogenic mode and in group C in allogenic mode. 2.4. Surgery and transplantation procedure The surgical procedure for creation of bone gap started immediately after processing of BM-MSCs, so as to apply fresh cells at the osteotomy site. The animals were anesthetized with xylazine and

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ketamine anesthesia as described earlier. The left forelimb (arm and forearm) was prepared for aseptic surgical procedure, and the animals were restrained in left lateral recumbency. Two to three cm long skin incision was made on the medial aspect of the limb approximately equally distant from the elbow and the carpal joint. The muscles were separated with the help of a fine artery forceps and the radius bone was exposed. A length of 5 mm was marked in the central diaphysis of the radius and the marked piece of the bone was cut with the help of a heck saw, and removed to create a segmental defect (Fig. 2a). The segmental defect in each animal was filled with HA granules of 0.1–0.4 mm diameter (Fig. 2b). Autogenic BM-MSCs and allogenic BM-MSCs diluted in 200 ll DMEM were implanted at the defect site in groups B and C, respectively, using a micropipette (Fig. 2c). In the animals of control group A only 200 ll DMEM was implanted at the defect site. 3. Observations 3.1. Clinical signs Clinically, the animals were observed for the status of cutaneous wound healing in terms of swelling, infection or wound dehiscence etc. 3.2. Radiography Two orthogonal, medio-lateral and antero-posterior, radiographs of the defect sites were made3 immediately after surgery and subsequently on 30th, 60th and 90th postoperative days at 14 mAs; 50 kVp and 85 cm FFD. The radiographs were observed for presence of new bone formation, extent and size of callus, bridging of the gap, radiographic density at the defect site and signs of remodeling. Radiological findings were graded according to modified Lane and Sandhu (1987) and Heiple et al. (1987) X-ray scoring systems (Table 1). Mean scores were calculated for each parameter and scores for different parameters were added to find overall radiographic score to quantify the healing in each group. The group attaining higher radiographic score was considered to have better healing. 3.3. Angiography After sacrifice, two animals from each group were subjected to angiographic studies of the test limb at each interval. A 5 cm long cutaneous incision was made on the medial aspect of elbow joint of the test limb. Brachial artery was exteriorized and canulated with polyethylene canula no. 26. The artery was flushed with heparin solution in normal saline. The flushing of the vessel was continued until clear fluid started flowing out from the corresponding vein. A positive contrast medium prepared by homogenization of red lead oxide (20%) in 10% formalin and soap water was then in-

fused into the limb vasculature through the canulated brachial artery (Singh et al., 2009; Nandi et al., 2008). The limb was disarticulated from the elbow joint and mediolateral and anteroposterior radiographs of perfused limb were made at 55 kVp, 6.5 mAs and 85 cm FFD. Angiograms of normal limbs were also made in the same fashion in three randomy selected animals for comparison. Image Pro Plus 6 software (Media cybernetics) was used to obtain reverse image for interpretation of the angiograms. 3.4. Histological study The test bones were collected from the animals sacrificed on 30th, 60th and 90th postoperative days and a 2.5 cm long piece of the radius including the defect site and normal bone on both sides was cut using heck saw. The bone specimens were washed thoroughly with normal saline and fixed in 10% formalin for 48– 72 h. The bone specimens were decalcified in Goodling and Stewart’s fluid containing 15 ml formic acid, 5 ml formalin and 80 ml distilled water (Culling, 1963). The solution was stirred daily and changed once in 3 days. The sections were checked regularly for the status of decalcification by observing flexibility, transparency and pin penetrability of the bone sections. Decalcified specimens were processed in a standard manner and 4 l thick longitudinal sections were cut and stained with Hematoxylin and Eosin (Luna, 1968). The sections were examined under different magnifications and bone healing was assessed in each group with triple blinding according to modified Lane and Sandhu (1987) and Heiple et al. (1987) histopathological scoring system (Table 1). Mean scores for each parameter were calculated and scores for different parameters were added to find overall histologic score to quantify the healing. Healing was considered better in the group attaining higher histologic score. 3.5. Statistical analysis Mean values and standard deviations were calculated. Differences between groups were calculated by factorial analysis of variance using the GLM program and checked in post hoc tests (Tukey’s studentized range (HSD) tests for variables) with SPSS 16.0 for Windows (SPSS Inc., Chicago, IL). A values of P < 0.05 was considered significant. 4. Results 4.1. Propagation of mesenchymal stem cells Most nucleated cells in the buffy coat were non-adherent which were subsequently removed at the first change of medium. MSCs grew in colonies and achieved confluence after 14–18 days. They had a fibroblast-like appearance. After first passage the cells grew uniformly throughout the surface of tissue culture flask.

Table 2 Radiographic Mean ± SD scores of various parameters in three groups (A–C), Time intervals (30, 60 and 90 days). Groups

Days

Reduction in defect size

Radiographic density

Remodeling

Total score

A B C A B C A B C

30 30 30 60 60 60 90 90 90

1.00 ± 0.55A 1.75 ± 0.68B 2.28 ± 0.73B 2.37 ± 1.41A 4.11 ± 1.17B 4.67 ± 0.71B 4.00 ± 0.82a 5.00 ± 0.00b 5.00 ± 0.00b

1.43 ± 0.51A 2.06 ± 0.57B 2.00 ± 0.68B 2.00 ± 0.75a 2.55 ± 0.53ab 2.88 ± 0.44b 2.50 ± 0.58 3.00 ± 0.00 3.00 ± 0.00

0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.87 ± 0.35 1.67 ± 1.00 1.56 ± 0.88 1.75 ± 0.50 3.00 ± 0.82 3.00 ± 0.82

2.43 ± 0.65A 3.81 ± 1.11B 4.28 ± 1.14B 5.25 ± 1.83A 8.33 ± 2.29B 9.11 ± 1.17B 8.25 ± 1.71a 11.00 ± 0.82b 11.00 ± 0.82b

Values bearing different superscripts differ significantly between the groups for a parameter at different intervals (Capital alphabets P < 0.01; small alphabets P < 0.05).

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Fig. 3. Mediolateral radiographs of the animals of group A (control), group B (autogenic BM-MSCs) and group C (allogenic BM-MSCs) on days 0, 30, 60 and 90 showing complete bridging of the defect by day 60 in groups B and C, however, a radiolucent line was still present at the defect site on day 90 in group A.

4.2. Clinical signs The surgical wounds healed by first intention in all the groups. However, surgical wounds in the animals of groups B and C were dry early without apparent signs of inflammation. The suture line

became dry as early as 3–4 days following application of both autogenic and allogenic BM-MSCs in groups B and C, respectively, albeit there was no appreciable difference in lameness between the groups. Weight bearing gradually improved and led to quick return of normal gait after removal of bamboo splints in all the groups.

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4.3. Radiographic observations Mean ± SD values of score for reduction in size of defect, radiographic density and remodeling and total radiographic scores in the animals of different groups are presented in Table 2. On day 0, in mediolateral views of radiographs, a gap of 5.03 ± 0.64 mm was clearly visible almost at the center of the radial diaphysis in all the animals. On day 30, new bone formation was clearly evident on the edges of the defect in the animals of groups B and C, however, in group A only mild periosteal reaction was seen (Fig. 3). Maximal reduction in the defect size was recorded in group C. Scores for reduction in size for groups B and C were significantly (P < 0.01) higher as compared to that for group A. There was an increase in the radiographic density at the defect site in the animals of groups B and C (Fig. 3).The scores for radiographic density in groups B and C were significantly (P < 0.01) higher than that of control group. Remodeling of the callus and canalization of marrow cavity was not evident in any of the groups. Size of the bone defect reduced further on day 60 in groups B and C, but in group A the size of the defect remained almost unchanged. Radiographic density at the defect site was significantly (P < 0.05) higher in groups C and B than that of group A. The remodeling of bone had started at this interval in the animals of group B and group C treated with mesenchymal stem cells. On day 90, the defect was filled completely with new bone in all the animals of groups B and C, however a radiolucent line was still present at the defect site in the animals of group A (Fig. 3). The mean scores for reduction of the defect size were higher in the animals of groups B and C treated by mesenchymal stem cells as compared to control. The radiographic bone density at the defect site in the animals of groups B and C was still higher than that in the animals of group A. The signs of remodeling were

clearly appreciable in the animals of groups B and C, but not in the animals of group A. The scores for remodeling of callus were also higher in groups B and C as compared to group A. The total mean radiographic scores on 30th and 60th days were significantly (P < 0.01) higher in animals of group B and C as compared to group A. The scores on 90th day improved further as compared to the respective scores on day 60 in all the groups, but scores on 90th day were still significantly (P < 0.05) higher in the animals of groups B and C than that in the animals of group A. 4.4. Angiographic observations At the defect site, the vessels were more prominent in the angiograms made on 30th day as compared to normal limb angiogram. The vascular network increased further on 60th day in all the groups, but vessels were more prominent in groups B and C as compared to that in group A (Fig. 4). Day 30, angiograms showed an increased vascularity not only at the healing site but in the whole limb too. This hyper-vascularity was slightly more intense in treatment groups B and C as compared to the control group A (Fig. 4). In groups B and C, treated by mesenchymal stem cells, the blood vessels were more intense at the defect site, however, in control group A, the vascularization was minimal over the defect site. On day 90, vascularity at the healing site was reduced as compared to day 60 interval in all the groups and angiograms appeared near to normal (Fig. 4). 4.5. Histological observations The mean ± SD scores for histological parameters in the animals of different groups at 30th 60th and 90th days intervals are pre-

Fig. 4. Mediolateral angiograms of the animals of a normal limb (0) group A (control), group B (autogenic BM-MSCs) and group C (allogenic BM-MSCs) on days 30, 60 and 90 showing more prominent vasculature particularly on day 60 in groups B and C as compared to group A.

Table 3 Histological Mean ± SD scores of various parameters in three groups (A–C), Time intervals (30, 60 and 90 days). Group A B C A B C A B C

Days 30 30 30 60 60 60 90 90 90

Osteogenesis A

1.00 ± 0.00 2.75 ± 0.50B 3.00 ± 0.00B 1.75 ± 0.50a 3.25 ± 0.50b 3.25 ± 0.96b 2.25 ± 0.50A 3.75 ± 0.50B 3.75 ± 0.50B

Union

Marrow A

1.00 ± 0.00 3.00 ± 0.00B 3.00 ± 0.00B 2.00 ± 1.15 3.25 ± 0.50 3.25 ± 0.50 2.50 ± 0.58a 3.75 ± 0.50b 3.75 ± 0.50b

0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00a 1.25 ± 0.50b 1.25 ± 0.96b 1.00 ± 0.00A 2.50 ± 0.58B 2.50 ± 0.58B

Cancellous bone a

1.00 ± 0.00 2.00 ± 0.82ab 2.50 ± 0.58b 1.75 ± 0.50A 3.00 ± 0.00B 3.00 ± 0.00B 2.50 ± 0.58a 3.50 ± 0.58ab 3.75 ± 0.50b

Cortical bone

Total score

0.00 ± 0.00 0.50 ± 0.58 0.50 ± 0.58 0.00 ± 0.00 2.00 ± 0.00 2.00 ± 0.00 1.25 ± 0.50a 2.50 ± 0.58b 2.50 ± 0.58b

3.00 ± 0.00A 8.25 ± 1.71B 9.00 ± 0.82B 5.50 ± 1.00A 12.75 ± 0.96B 12.75 ± 2.22B 9.50 ± 0.58A 16.00 ± 1.41B 16.25 ± 1.50B

Values bearing different superscripts differ significantly between the groups for a parameter at different intervals (Capital alphabets P < 0.01; small alphabets P < 0.05).

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sented in Table 3. On day 30, the mean scores for osteogenesis were significantly higher (P < 0.01) in the animals of treatment groups B and C as compared to control group A. The mean score for union of bone edges were also significantly higher in groups B and C as compared to group A. Formation of cancellous bone with presence of bone marrow between trabeculae was significantly (P < 0.05) higher in the animals of group C treated by allogenic mesenchymal stem cells as compared to the animals of group A. Formation of cortical bone was not evident in control group while a few specimens from groups B and C showed initiation of cortical new bone formation (Fig. 5b, c). At higher magnifications only few osteoblasts were seen in the sections of healing site in group A, whereas numerous osteoblasts, osteocytes and even osteoclasts were discernible in the sections of healing site in groups B and C (Fig. 6b, c). On day 60, the mean scores for osteogenesis increased over the score of day 30 in all the groups. The scores were significantly higher (P < 0.05) in the animals of mesenchymal stem cells treated groups B and C than the control group A. The mean score for union of bony edges increased in all the groups but did not differ significantly from each other, albeit the score was still higher in treatment groups. The union of the bony edges was classified as bony union in treatment groups and fibrous to osteochondral union in control groups. Significantly (P < 0.05) higher scores for the formation of bone marrow were recorded in the animals of mesenchymal stem cells treated groups B and C as compared to the control group A. The bone marrow formation had started to cover more than half of the defect in groups B and C (Fig. 5e, f). Scores for cancellous bone formation were significantly (P < 0.01) higher

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in treatment groups B and C as compared to control group A. Evidences of cortical bone formation were discernible in animals of groups B and C but not in control group. Newly formed bone was well organized in the animals of groups B and C, but it was highly unorganized with the presence of fibrous tissue in group A (Fig. 6d–f). Signs of development of Haversian system were evident in the animals of groups B and C (Fig. 6 e, f). The scores for histological parameters did not differ significantly (P > 0.05) between the groups B and C. On day 90, mean scores for osteogenesis increased as compared to day 60 scores, and significantly higher scores were recorded in the mesenchymal stem cells treated animals of groups B and C as compared to control group A. The microscopic architecture of the specimen was suggestive of good to perfect osteogenesis in groups B and C (Fig. 5h, i). The mean scores for union of bone edges were significantly (P < 0.05) higher in the animals of mesenchymal stem cells treated groups B and C compared to control group A. The microscopic appearance suggested complete organization of shaft in treatment groups (Fig. 5h, i). The mean score for the formation of bone marrow was significantly (P < 0.01) higher, in the animals of groups B and C as compared to group A. There was only scattered appearance of bone marrow in control groups but complete colonization by red marrow was seen in groups B and C treated with mesenchymal stem cells (Fig. 5h, i). The score for the formation of cancellous bone was still significantly (P < 0.05) higher in allogenic mesenchymal stem cells treated group C as compared to control group A. The mean scores for cortical bone formation were significantly (P < 0.01) higher in the animals of groups B

Fig. 5. Photomicrograph showing fibrous connective tissue (F), periosteal fibrosis (PF), woven bone formation (WB), osteochondral union (Och), bone marrow formation (BM) in between the gaps with grafted hydroxyapatite (HA) and few areas of new bone formation (NB) with host bone (HB) in group A (control), group B (autogenic BM-MSCs) and group C (allogenic BM-MSCs) at 30, 60 and 90 day intervals (H&E stain; x4). Note better new bone formation and more bone marrow formation in groups B and C.

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Fig. 6. Photomicrograph showing fibrous connective tissue (F), osteocytes (Os), osteoblast (Ob), osteoclast (Oc) and haversian systems (H) in between the gaps with grafted hydroxyapatite (HA) and new bone formation in group A (control), group B (autogenic BM-MSCs) and group C (allogenic BM-MSCs) at 30, 60 and 90 day intervals (H&E stain; x40). Note better organization of bone, osteoclastic activity and formation of Haversian system in groups B and C.

and C as compared to control group. Higher magnifications showed better organization, osteoclastic activity and development of Haversian system in treatment groups B and C (Fig. 6h, i) as compared to control group A (Fig. 6g). The total histological mean scores were significantly (P < 0.01) higher in the animals of groups B and C as compared to that in control group A at all the intervals. Overall histological evaluation thus suggested better healing response in the mesenchymal stem cells treated animals of groups B and C than that in the animals of control group A. Both treatment groups showed similar histological appearance of the defect site at all the intervals, and total score did not vary significantly (P > 0.05) between the groups B and C.

5. Discussion In cases of fractures associated with bone loss due to high energy trauma or excision of some pathological lesion, the regeneration of the bone is a major difficulty in veterinary practice because the progression of fracture healing has ceased. Progression of healing requires filling of the gap with bone grafts. It has been extensively evaluated by several workers with promising results (Friedlaender, 1987; Van Heest and Swiontkowski, 1999; Shafiei et al., 2009). However, various setbacks associated with autogenic as well as allogenic or xenogenic grafts have dictated the researchers to find other alternatives to the bone grafting. Application of scaffolds, proteins, growth factors and cells was introduced as an

alternative solution to induce bone regeneration instead of bone graft (Canalis, 1980; Hock et al., 1988; Joyce et al., 1990; Lind, 1998). Despite of lots of advances in the application of various synthetic and natural bone substitutes, problems still exist in the healing of large bony defects associated with bone loss. Bone marrow-derived MSCs facilitate bone repair when implanted locally, usually on an artificial matrix, such as hydroxyapatite/tricalcium phosphate or hydroxyapatite ceramic in the case of craniotomy or long-bone defects (Kon et al., 2000; Quarto et al., 2001). Kim et al. (2007) loaded an acrylated hyaluronic acid scaffold with recombinant human BMP-2 protein and/or human MSCs and used to regenerate a rat calvarial defect. Use of autogenic MSCs may be preferred over the allogenic MSCs for the minimal risk of rejection but their use may be limited for the want of time constraints. Since there is accumulating data suggesting that MSCs have capacity to evade immune rejection, they can be used allogenically. Therefore, the present study was designed to test the hypothesis that both autogenic and allogenic MSCs would induce comparable healing of diaphyseal bone defect in rabbits. Angiography of the limb suggested increased vascularity in treatment groups, which could be due to more angiogenesis attributable to release of factors such as vascular endothelial growth factor at the fracture site by MSCs. The findings of the present study conformed to the observations of Bouletreau et al. (2002), who reported that MSC-derived endothelial cells secreted bone morphogenic protein (BMP) to promote osteogenesis and stimulate

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osteoblasts and their precursor cells to secrete vascular endothelial growth factor (VEGF), which can significantly promote the proliferation of endothelial cells (ECs), vascularization and osteogenesis (Lammert et al., 2001). Increased vascularization at the defect site in the treatment groups might have helped faster healing of the defect as compared to control group. Radiographic observations and histological findings complimented each other and suggested faster osteogenesis and remodeling of the bone in the animals of mesenchymal stem cells treated groups B and C as compared to control group A. This increased osteogenesis could mainly be attributable to conversion of mesenchymal stem cells into osteoblasts in treatment groups B and C as reported by Jiang et al. (2010). Jang et al. (2008) also recorded increased osteogenesis and new bone formation through differentiation of MSCs to osteoblast progenitors in canine when tricalcium phosphate (TCP) was loaded on canine umbilical cord derived mesenchymal stem cells (UCBMSCs). In addition to direct differentiation, there might be several factors that would have contributed to early healing in treatment groups. It has been proposed that MSCs secrete distinctively different cytokines and chemokines such as vascular endothelial growth factor (VEGF), insulin-like growth factor-1, epidermal growth factor, keratinocyte growth factor, angiopoietin-1, stromal derived factor-1, macrophage inflammatory protein-1alpha, protein-1beta and erythropoietin (Chen et al., 2009), which might have accelerated healing in treatment groups. The mesenchymal stem cells treated groups showed bony union which could be due to early lying down and mineralization of the healing tissue due to conversion of osteoblast into osteocytes and release of Ca ion in the matrix. Breitbart et al. (2010) also observed more bony union in mesenchymal stem cells treated group as compared to the groups treated by demineralized bone matrix (DBM) in diabetic animals at the 4 week time point. The newly formed bone showed early initiation of remodeling in treatment groups. Bruder et al. (1997) and Arinzeh et al. (2003) recorded similar increase in radiopacity and new bone formation after treating the defects in dogs with autogenic and allogeneic mesenchymal stem cells, respectively. It was inferred that mesenchymal stem cells have little or no immunogenicity and could be well incorporated in healing bone. The results of the present study supported the hypothesis that mesenchymal stem cells can be used allogenically in animals. Previously reported studies suggested that mesenchymal stem cells lack in MHC II on their cell surface, responsible for the antigenicity of the cells (Planka et al., 2008; Guo et al., 2009; Jung et al., 2009). Guo et al. (2009) also reported that pig MSCs did not stimulate lymphocyte proliferation and activation in vitro, indicating that these MSCs also had little or no immunogenicity. The mesenchymal stem cells avoid allogeneic rejection by three broad mechanisms that contribute to this effect, firstly, mesenchymal stem cells are hypoimmunogenic, often lacking MHC-II and costimulatory molecule expression. Secondly, these stem cells prevent T cell responses indirectly through modulation of dendritic cells and directly by disrupting NK as well as CD8 + and CD4 + T cell function (Di Nicola et al., 2002; Uccelli et al., 2006; Nauta and Fibbe, 2007). Thirdly, mesenchymal stem cells induce a suppressive local microenvironment through the production of prostaglandins and interleukin-10 as well as by the expression of indoleamine 2,3,-dioxygenase, which depletes the local milieu of tryptophan (Ryan et al., 2005). Our findings conformed to the observations of Guo et al. (2009), who reported that MSCS have very little or no immunogenicity in mini-pigs.

6. Conclusions Present study suggested that in vitro cultured bone marrow derived mesenchymal stem cells can enhance osteosynthesis in gap

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