combined with synthetic bone substitute and the use of synthetic bone substitute alone. Key Words: Stem Cell, Oral and maxillofacial surgery, Bone healing.
European Review for Medical and Pharmacological Sciences
2012; 16: 679-686
Effects of mesenchymal stem cells in critical size bone defect S. AGACAYAK, B. GULSUN, M.C. UCAN, E. KARAOZ*, Y. NERGIZ** Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Dicle University, Diyarbakir (Turkey) *Department of Stem Cell, Faculty of Medicine, Kocaeli University, Kocaeli (Turkey) **Department of Histology and Embryology, School of Medicine, Dicle Universty, Diyarbakir (Turkey)
Abstract. – Background and Objectives: The aim of this study was to compare culture-expanded, bone marrow-derived mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) loaded to biphasic calcium phosphate (BCP) bone ceramic in the repair of rat calvarial bone. Materials and Methods: Critical-size (7 mm dia.) calvarial defects were prepared in the frontal-parietal bones of 90 adult female Sprague-Dawley rats. Rats were randomly divided into 5 groups, according to defect filling, as follows: Group I (n=21), BCP; Group II (n=21), BCP+PRP; Group III (n=21), BCP+MSC; Group IV (n=21), BCP+PRP+MSC; Group V (n=6) (control), no treatment. Animals were sacrificed at 2, 8 and 12 weeks postsurgery and bone regeneration was evaluated both histologically and immunohistochemically. Results: Statistically significant differences were observed in bone osteoblastic activity in calvarial defects among the groups (p 30%
5 9 5 6 9 6 5 9 6 19 10 19 2 10 19 2 1 19 2. weeks 8. weeks 2. weeks 12. weeks 8. weeks 12. weeks 2. weeks 8. weeks 12. weeks
63.3% 33.3% 63.3% 6.7% 33.3% 6.7% 63.3% 33.3% 6.7%
Number % Number
Osteocyte density
> 20% > 10%
Weeks
Cell Culture of rBM-MSC MSCs attached to the culture flasks sparsely and majority of the cells displayed a fibroblastlike, spindle-shaped morphology during the early days of incubation. These cells began to proliferate after 3-4 days, and gradually grew to form small colonies that were named as colony forming units (CFU) (Figure 3A). By days 6-8, the number of cellular colonies with different sizes had increased. As growth continued, colonies gradually expanded in size and the adjacent ones interconnected with each other. These primary cells reached monolayer confluence, after plating for 12-15 days in their first passages. In later passages, most of these MSCs exhibited large, flattened or fibroblast-like morphology (Figure 3B-D).
Any osteocyte
Results
Table I. Osteocyte density distribution of the total number of weeks (p < 0,05).
Statistical Analysis Statistical analysis was performed using SPSS 11.5 (SPSS Inc., Chicago, IL, USA). Analysis of variance (Chi-Square) was used to identify differences between treatment groups, with a level of p < 0.05 considered to be statistically significant (Table I, Figure 2).
%
Immunohistochemistry Sections with histologically observable bone formation were subjected to immunohistochemical examination. Samples were cut to 5 µm and dyed using antibodies for osteonectin (OCN) (Novacastra Laboratories Ltd., Newcastle, England) and osteopontin (OPN) (Novacastra Laboratories Ltd.). Primary OCN and OPN antibodies were added at concentrations of 1:500 and 1:100, respectively. A secondary antibody (DAKO, Glostrup, Denmark) was added to enable a colored presentation. Each sample was counterstained with hematoxylin and eosin, paired with a negative control, analyzed using an optical microscope and photographed.
16.7% 30.0% 16.7% 20.0% 30.0% 20.0% 16.7% 30.0% 20.0%
Chi-Square
p
croscope (Olympus, SZX 9, Tokyo, Japan). In addition, defect width and distance between the margins of the original bone defect were recorded for the most central section of each specimen using a microscope digital camera system (3077 CCD, JVC, Tokyo, Japan) and a PC-based image analyzing system (Image-Pro Plust, Media Cybernetic, Silver Spring, MD, USA).
6.7
S. Agacayak, B. Gulsun, M.C. Ucan, E. Karaoz, Y. Nergiz
Effects of mesenchymal stem cells in critical size bone defect
tent throughout the defect (Figure 4). Together with histological observation, these findings suggest that bone formation and mineralization proceeds from the margin to the center of the defect.
Discussion
Figure 2. Bone healing for weeks.
Flow Cytometry-Identification of rBM-MSCs The data indicated that rBM-MSCs expressed CD29 and CD90, but not CD45 (Figure 3E). rBM-MSCs maintained their phenotype throughout passages. Therefore, there was no evidence of hematopoietic precursors in the cultures. In contrast, the expression of other surface antigens agreed with the previous reports regarding murine MSCs, and indicated that the cells used in this study had the characteristics of MSCs reported in other studies19-21. Among the 90 animals examined, calvarial defect sites varied within and between treatments and ranged from limited bone formation to nearly complete defect closure. Histological examination at 2, 8 and 12 weeks showed implant material within fibrous connective tissue in the main portion of the calvarial defect filled with a scaffold containing PRP and MSC. No adverse reaction was observed. Newly formed osteocyte-containing bone was observed mainly at the periphery of the defect. A dense arrangement of osteoblast-like cells adjacent to the newly formed bone suggestive of continuous bone formation through the center of the defect, although less bone-formation activity was evident at the center. The newly formed bone consisted of lamellar and trabecular bone, with no evidence of fatty marrow or cartilage formation. Large amounts of OPN and OCN were observed in and near the newly formed cells, with immunoreaction consis-
Bone regeneration has long been a subject of interest among researchers. Many Authors have examined the osteoinductivity of biological grafts and bio-materials loaded with cells or osteogenic factors22-24, and while some successful results have been reported, their effects on clinical practice have been below expectations25. Biocompatibility of the scaffold is important, as an appropriate ex-vivo matrix is required for cell production. Because of consisting two layers of cortical bone, mandible and cranium have physiological similarities26. Rat calvaria defects have been used to evaluate the biological potential of various devices as well as the osteoinductivity and/or osteoconductivity of biomaterials and biologics in promoting bone regeneration27. However, there is no clear consensus regarding critical size, which has been defined as 8 mm, 6 mm, or sometimes even as small as 4 mm28-31. In the present study, 7 mm full-thickness bone defects were created in rat cranial bone. New bone has been shown to form in a centripedal manner around a center point32. The dura and its interaction with the periosteum have also been reported to play an important role in bone generation33. In this study first periost was elevated, then, cranial bone defect was performed, and periosteum was sutured on the defect without any integrity loss. Biphasic calcium phosphate ceramics may be obtained from a mixture of hydroxyapatite and β-tricalcium phosphate. The micro-structure matrix of BCP permits the establishment of a network between cellular and vascular penetration in which bone formation can easily occur among the pores34. The biphasic calcium phosphate ceramic graft material used in this study had a pore size of 100-500 mM, which supports optimal cellular penetration and vascularization. PRP has been suggested to enhance wound healing and increase the rate of bone healing following oral and maxillofacial surgery35. In a report of their clinical trial, Camargo et al36 concluded suggested two possible reasons for the positive clinical results observed with PRP bone 683
S. Agacayak, B. Gulsun, M.C. Ucan, E. Karaoz, Y. Nergiz
Figure 3. Cell culture and identification.
grafts: (1) the use of autologous fibrin gel may improve adhesion of graft particles and reduce the risk of scattering; and (2) PRP’s strong mitogenic effect on the environment encourages quicker soft-tissue wound healing, thus preventing infection of the defect area36. Platelet alpha granules have been shown to contain potent growth factors needed to initiate tissue repair and regeneration at a wound site 3,37 . In this study,statistically significance was found be684
tween PRP and Graft together used group and other groups, so in PRP combined graft practices it was seen that osteogenesis was influenced positively. Recently studies in tissue engineering about stem cells and growth factors produces positive results for also maxillofacial region38,39. MSCs have been successfully engrafted within criticalsized defects in athymic rat and canine long bones. The implanted cells must be in contact
Effects of mesenchymal stem cells in critical size bone defect
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Acknowledgements
This study was presented orally in 5th ACBID International Conference.
References
Figure 4. After 12 weeks preview of BCP, PRP and MSCs implanted defect. (ONC, × 200).
with the scaffold in order to secure tissue formation in vivo18,40. MSC sources include periosteum, synovial membrane, muscle, fat, vascular pericytes, periodontal ligament, deciduous teeth and dermis. Recently, Tuli et al41 demonstrated that MSCs can also be isolated from explant cultures of collagenase-treated trabecular bone fragments. Inreasing of osteoinductive effect in combination of Graft and PRP with mesenchymal stem cells produced in this study, will provide positive results in bone reconstructions of maxillofacial region.
Conclusions This study compared the osteogenicity of synthetic biphasic calcium phosphate, combined with platelet rich plasma and mesenchymal stem cell fillers in critical size (7 mm dia.) calvarial bone defect sites in rats and found that these combinations may have clinical applications in maxillofacial surgery. The role of osteogenesis agents in bone repair has become increasingly recognized in the treatment of osteoporosis, chemotherapy patients, in fracture repair and maxillofacial reconstruction. However, the mechanisms of these anabolic effects are poorly understood at the cellular level in vivo, particularly in humans. Given the wealth of data on cultured MSCs, it is likely that these intriguing cells play a pivotal role in bone regeneration and repair in vivo, but due to the lack of commonly adopted phenotypic criteria for MSCs in vivo in both human and animal models, this remains unproven. The ability to purify and study non-manipulated MSCs in humans will have important implications in terms of understanding the mechanism(s) of MSC-mediated bone repair of the maxillofacial region.
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