| [Background and Objectives]Bone defects are common clinical symptoms, including congenital and acquired defects. The methods of healing bone defects include:autologous bone transplantation, allogeneic bone transplantation and artificial material filling. But they were unable to overcome the limitations:In 1980s, there was a new concept providing new ideas and methods which is proposed to solve this problem:tissue engineering.As for the increasing frequence and urgency of healing bone defects in clinic, bone tissue engineering is becoming the fastest growing field. Using tissue engineering technology is expected to avoid the shortcomings of autogenous bone graft. Bone tissue engineering employs cells which have autologous ossification activity and biodegradable compo.site materials replanted in vivo to heal bone defects. With gradually degradation of materials, osteogenic cells secretes extracellular matrix, eventually forms tissue engineered bone to repair the defects, which avoids the secondary injury of autologous bone donor site.There are three essential components of bone tissue engineering, which are seed cells, scaffolds and growth factorsAdipose-derived stem cells become the superior seed cells because of its wide distribution, adequate sources which are accessible easily in tissue engineering.β-tricalcium phosphate is similar to the main component of the human bone and may be an ideal scaffold for bone tissue engineering. Preparation of platelet-rich plasma is simple and platelet-rich plasma contains high concentrations of growth factors, which is the main source of autologous growth factors. Platelet-rich plasma contains a variety of factors promoting the repair of bone defects, accelerating the formation of new bone.Avidin-biotin binding system is a biology technique developing very rapidly in recent years. The covalent bond between avidin and biotin is the most stable covalent bond so far, which promotes cell adhesion and proliferation in the early stage.In this study, avidin-biotin binding system modified adipose-derived stem cells and β-TCP scaffold were combined with platelet-rich plasma in vivo to construct tissue engineered bone ectopic and in situ. Then the study also investigates the effect of promoting osteogenesis of platelet-rich plasma.[Materials and Methods](A) Adipose-derived stem cells(ADSCs) isolation, identification, culture and induced differentiation1) The fat tissue is obtained from the groin of New Zealand white rabbits. ADSCs are extracted by conventional enzyme digestion, then we observe the cell morphology.2) Use monoclonal antibody to label the ADSCs, then identify stem cells surface markers by immunohistochemistry:CD29、CD34、CD44、CD105.3) Use conditional induction medium to induce ADSCs to differentiate into adipocytes and osteoblasts. We stained oil red 0 to detect adipogenic; we use Alkaline phosphatase stain and Von Kossa stain to detect osteogenesis.(B) The preparation and characterization of β-tricalcium phosphate 1) Use solution of calcium nitrate and diammonium phosphate as raw materials; adopt liquid precipitation technique to prepare β-TCP powder; use ammonium chloride as pore-forming agent; adopt temperature programmed methods to sinter porous β-TCP nanoscale scaffolds.2) Use X-ray diffraction to analyze the phase of material subject, then use fourier transform infrared spectrometer testing to identify the ingredient of material; use the universal testing machine to measure the compressive strength of material; adopt hydrostatic weighing method to determine the porosity and water absorption of the scaffolds.3) Use scanning electron microscopy to test the microstructure of the scaffolds.(C) Avidin-biotin binding system promotes adipose-derived stem cells to adhere on the porous β-TCP nanoscale scaffolds1) Use CCK-8 assay to test the toxicity of the porous β-TCP nanoscale scaffolds.2) Use immunofluorescence and flow cytometry to test the efficiency of biotinylated ADSCs.3) Use CCK-8 assay to test the adhesion efficiency of ADSCs on the porous β-TCP nanoscale scaffolds and the avidin-biotin binding system modified porous β-TCP nanoscale scaffolds;.4.) Use scanning electron microscopy to detect ADSCs biocompatibility with the porous β-TCP nanoscale scaffolds and the avidin-biotin binding system modified porous β-TCP nanoscale scaffolds.(D) Platelet-rich plasma promotes adipose-derived stem cells and avidin-biotin binding system modified porous β-TCP nanoscale scaffolds to build ectopic bone in nude mice1) Obtain platelet-rich plasma by centrifugation twice.2) Divide into four groups:β-TCP group, β-TCP+PRP group, ADSCs+ β-TCP group and ADSCs+β-TCP+PRP group, to test the ectopic bone formation in nude mice.3) Remove the specimens after 8 weeks to do hematoxylin and eosin (HE) staining and osteocalcin (OCN) staining. (F) Platelet-rich plasma promotes adipose-derived stem cells and avidin-biotin binding system modified porous β-TCP nanoscale scaffolds to build bone in rabbit mandibular defects1) In this study, six New Zealand white rabbits were divided into two groups, including Group A:Control Group, ADSCs+β-TCP group; Group B: Experimental Group, ADSCs+β-TCP+PRP group.2) Experimental rabbits were sacrificed after 4 weeks; do parallel head CT scan, and observe the fusion between the scaffolds and the surrounding bone.3) Remove the specimens after 4 weeks to do hematoxylin and eosin (HE) staining and osteocalcin (OCN) staining.4) Measure the expression of osteocalcin (OCN), alkaline phosphatase (ALP) through fluorescence quantitative RT-PCR[Results]1. ADSCs from New Zealand white rabbits groin fat were obtained from conventional enzymatic digestion. ADSCs had stem cell morphology, and Immunohistochemical detection confirmed that the surface marker expression of CD29, CD44, CD105 expression was positive, while the CD34 expression was negative. The differentiation assay revealed ADSCs had adipogenic, osteogenic differentiation potential.2. The composition is β-TCP; the grain size is 31.5nm; the porosity is 71.26±0.28Vol%(n=5); the water absorption-is 38.51±0.21Wt%(n =5); the compressive strength is 7.93±0.06MPa (n=5); the scaffolds have rich and uniform pores.3. CCK-8 assay found that ADSCs cultured with P-TCP extract concentration 100%,50%,10%,1%, the relative growth rates were 107.48±10.32%, 105.63±9.85%,108.51±12.06%,109.04±8.17%. Biotinylated ADSCs staining showed biotin binding sites in the cell cytoplasm, and flow cytometry tips biotinylated positive cells was 95%. At different time points (10min,30min,60min,12hour,24hour) the adhesion of ADSCs with the porous P-TCP vs avidin-biotin binding system modified porous β-TCP scaffolds were 2.31±0.14% vs 21.75±4.69%,11.96±2.53% vs 54.82 ±12.37%,33.48±9.51% vs 78.69±15.65%,78.29±10.63% vs 95.46 ±7.38%,94.79±10.42% vs 98.13±1.45%.4. As for ectopic tissue engineered bone in nude mice, hematoxylin and eosin (HE) staining and osteocalcin (OCN) staining in group A and group B have only a very small amount of osteoblastic cells and extracellular matrix coloring. However, HE staining and OCN staining of group C and group D have large number of cells and the extracellular matrix of osteoblasts coloring, moreover, the D group is more obvious than group C.5. Head CT shows scaffold was fused with the surrounding bone without significant rejection. Hematoxylin and eosin (HE) staining and osteocalcin (OCN) staining showed many cells and extracellular matrix coloring. The experimental group was significantly more than the control group.6. The RT-PCR assay reveals that the OCN expression value of group A was 17.49±3.52, and the OCN expression value of group B was 26.38±7.17; the ALP expression value of group A was 15.12±4.21, and the ALP expression value of group B was 49.62±8.34. Group B was more obvious than group A.[Conclusion]1. Adipose stem cells (ADSCs) were simple to isolate and culture, easy to access, stable when passaged and have multiple differentiation potential.2. The prepared β-TCP powder was complete, free of impurities, fine grains. The nanoscale porous β-TCP scaffolds have good pore structure and pore connectivity, and a certain strength, which can be used as scaffolds for further experiments3. The nanoscale porous β-TCP scaffolds have no cytotoxicity, and avidin-biotin binding system modified β-TCP scaffolds can promote the adhesion of ADSCs in early stage.4. Platelet-rich plasma promotes adipose-derived stem cells and avidin-biotin binding system modified porous β-TCP nanoscale scaffolds to build tissue engineered bone in nude mice and rabbits.5. Platelet-rich plasma promotes the formation of bone in nude mice and rabbit mandibular defects. |
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