Osteopromotion of mandibular distraction osteogenesis using stem cells from human deciduous teeth and in biphasic calcium phosphate scaffold in rabbit model
| dc.contributor.author | Khalil Mohammed .A, Amera | |
| dc.date.accessioned | 2017-09-07T09:04:49Z | |
| dc.date.available | 2017-09-07T09:04:49Z | |
| dc.date.issued | 2013-05 | |
| dc.description.abstract | Distraction osteogenesis (DO) is described as endogenous bone tissue engineering has become increasingly popular in recent years and the application of distraction technique to the craniofacial skeleton has expanded the number of treatment alternatives for patients with maxillofacial abnormalities and deficiencies. It is applied first in orthopedic surgery for correction of limb length discrepancies, and subsequently has been utilized in the treatment of craniofacial microsomia and bony defect. In DO, new bone formation is induced by gradual separation of bony segments after an osteotomy or corticotomy during which the normal process of fracture healing is interrupted by the application of gradual traction to the soft callus. DO has some distinct advantages over traditional surgical methods which involve the elongation of both hard and soft tissue at the same time. The major disadvantage is the long treatment time which is some time not well tolerated by patients and the chance of associated bony and soft-tissue complications with possible fibrous union or nonunion remains as major limitations impeding its further clinical application. Many studies have focused on the promotion of new bone formation to shorten the course of DO. Physical means have included pulsed electromagnetic fields, low- intensity ultrasound and electrical stimulation, interventional methods such as transplantation of osteoblast-like cells or bone marrow to the distraction site. Bone formation in vivo has become the main cell source for bone tissue engineering, it has been used in critical size defect reconstruction. The application of stem cells from exfoliative deciduous teeth (SHED) during distraction, injection of growth factors or platelet rich plasma and gene therapy have also been applied to accelerate the maturation of the regenerated bone. Some non interventional methods, such as administration of calcitonin, alendronate and zoledronic acid have also shown promising results. Tissue engineering (TE) is a new highly promising field of reconstruction that is drawing attention in recent advances in medicine and surgery. The main 3 element of TE are stem cells, scaffold and growth factor. SHED has been proven to be capable of differentiating into osteoblasts and chondrocytes in vitro. The usage of stem cells from human deciduous teeth (SHD) to promote new bone formation in DO has yet not been reported. Macroporous biphasic calcium phosphate ceramic (MBCP) is biomaterial used as bone filler and as a scaffold in bone tissue engineering. The main aim of this study was to test whether the addition of SHD and a composite consisting of SHD seeded in MBCP granules in osteotomy as a tissue engineering construct increases osteogenic potential in DO. MBCP was synthesized with desirable properties, Calicum / Phosphate ratio, micro and macro porosities and particle size. The material was characterized using x ray diffraction, scanning electron microscope and particle size analyzer. In vitro cytotoxicity was performed to test the biocompatibility of the synthesized macroporous biphasic calcium phosphate. Stem cells were isolated from human pulp of deciduous teeth, expanded in vitro and characterized using 2 antibodies CD 166 and 105. The in vivo study was performed using rabbit’s model, Eighteen New Zealand white rabbits were divided into 3 groups. Group A underwent DO without addition of materials as control, group B had 6 million cells (SHD) transplanted in osteotomy gap and group C had SHD/BCP construct consisted of 6 million cells in 50 mg MBCP transplantation. DO protocol was 4 days latency period, 6 days distraction period 1mm/day and assessed in 3 consolidation periods 3, 18 and 32 days. The regenerate was evaluated in 3 intervals of 2, 4 and 6 weeks postoperative periods, clinically, radiographically using conventional X ray, significance of callus formation, formation of bone cortex and bone marrow cavity were evaluated to compare between groups. Histological sections for new bone formation, blood vessels, cartilage and fibrous tissue were assessed. Quantitative histomorphometric measurements were carried out using Zeiss image analysis system to quantify the amount of bone formed, cartilage and fibrous tissue ruminants. The samples were also analyzed histomorphometrically for the grade of osseous regeneration using an established numerical scoring system for the assessment of bone healing. Serial sections were scored for 2 independent bone-forming indices stage of bone union and grade of bone maturity. The result demonstrated that MBCP synthesized with Ca/P ratio of 1.52 confirmed by XRD, micro and macro porosity of 200-400 μm was confirmed by SEM. In vitro cytotoxicty showed that MBCP is free of toxicity, Mann-Whitney test detected that the optical absorbance (OD) median of SHD for all MBCP concentrations was statically higher than control using 7 days extraction, P value < 0.001 for each. SHD were successfully isolated by enzyme digestion method. Early cell cultures revealed typical fibroblast-like spindle shaped cells arranged in colonies. Results of flow cytometry showed expression of CD 105 and 166, >42 and >95% respectively. The in vivo study showed that both SHD and SHD/MBCP construct enhance bone formation in all time points 2, 4 and 6 weeks postoperatively. Radiographic examination revealed presence of radiological evidence of DO healing in all treatment groups with more bone formation in the transplanted one. Histologically, intramembrance ossification was evidence, the highest bony formation was seen in SHD/MBCP group followed by SHD group and the least amount was in control group. Histomorphometric measurement detected that the percentage of newly formed bone in 2 weeks control, SHD and SHD/BCP were 18.41, 35.97 and 57.28% respectively, in week 4 were 31.68, 59.78 and 66.49% and in week 6 were 52.34, 60.24 and 72.98% respectively. Non parametric ANOVA (Kruskal Whalis Test) showed significant difference between groups P value = 0.003. Bone-forming indices such as stage of bone union and grade of bone maturity were highest in SHD/BCP group and lowest in control group, Kruskal Whalis Test showed significant difference between groups P value = 0.001 and 0.002 respectively. In conclusion, this study may provide additional information and evidence of the various mechanisms of action of SHD in bone formation which may be useful in selecting effective bone promoting construct for bone formation. As we have shown both SHD and SHD/BCP have osteopromoting activities and potential bone forming construct. The osteopromoting effect is better with combination of SHD and BCP scaffold. Further studies on the effect of both SHD and SHD/BCP remain important as it may lead to developing an osteopromotic construct for human distraction osteogenesis and to know whether SHD differentiate directly to form osteoblast or indirectly promote the bone formation in DO. | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/4528 | |
| dc.language.iso | en | en_US |
| dc.publisher | Universiti Sains Malaysia | en_US |
| dc.subject | Distraction osteogenesis (DO) | en_US |
| dc.title | Osteopromotion of mandibular distraction osteogenesis using stem cells from human deciduous teeth and in biphasic calcium phosphate scaffold in rabbit model | en_US |
| dc.type | Thesis | en_US |
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