| Objectives1. To identify the effects ofdifferent scaffoldon hBMSCs in tissue engineered bone construction.2. To identify the dose-effect relationship between the cell seeding amount and in vivo bone yield.3. To perform the individualclinical trial of bone defects repair of alveolar cleft using scaffolds combined withconcentrated autologous bone marrow.Methods1. The hBMSCs were isolated from bone marrow by density gradient centrifugation combined with adherent screening method. The hBMSCs were cultured, expanded and induced into osteoblast and adipocyte in vitro. Themesenchymal stem cells surface markers of hBMSCswere detected by flow cytometry. Theβ-TCP/hBMSCs group, CHA/hBMSCs group, FDBA/hBMSCs group were constructed in vitro respectively and implanted in nude mice subcutaneously. The adhesion, proliferation, viability, distribution, matrix deposition, and osteogenic differentiation of hBMSCs seeded on different scaffolds were evaluated duringculture in vitro,and thein vivoosteogenesis were compared by micro-CT and histological assay.2. TEB were constructedwith (3-TCP using10μL of P3hBMSCs at different seeding densities of5×106/mL,10×106/mL,20×106/mL,30×106/mL, and50×106/mL. After2weeks osteogenic induction in vitro, they were implanted in nude mice subcutaneously. The adhesion, proliferation, viability, distribution, matrix deposition, and osteogenic differentiation of hBMSCs with different seeding densities on P-TCP scaffolds were evaluated duringculture in vitro, and the in vivo bone yield were compared by micro-CT and histological assay.3. TEB were constructedwith CHA using10μL of P3hBMSCs at different seeding densities of5×106/mL,10×106/mL,20×106/mL,30×106/mL, and50×106/mL. The adhesion, proliferation, viability, distribution, matrix deposition, and osteogenic differentiation of hBMSCs with different seeding densities on CHA scaffolds were examined during culture in vitro, and the in vivo bone yield were compared by micro-CT and histological assay.4. A total of7patients (2males and5females), aged8-27years old with alveolar cleft were enrolled and treated with concentrated autologous bone marrow graftcombined withβ-TCP scaffold. To evaluate the bio-safety and clinical efficacy, the exogenous factor such as endotoxin, bacteria, fungi, and mycoplasma were tested, and the patients were followed up for3months,6months,12months, and2years by X-ray and CT scan. The postoperative complications, such as rejection, poor wound healing, and infection were observed at the same time. To date, the clinical data from3of7patients were analyzed retrospectively and evaluated with Bergland’s score system based on X-ray and CT scan.Results1.The adhesion, proliferation, and spatial distribution of hBMSCs seeded on FDBA were significantly lower than (3-TCP and CHA groups as well as the gene expression level of OCN and OPN. The FDBA group was unable to form new bone after3months ectopic implantation. Compared with FDBA group, β-TCP and CHA group showed high positive bone fraction of ectopic bone formation, and in vivo bone yield of β-TCP group were significantly higher than that of CHA group.2. Seeded onβ-TCP, the proliferationof hBMSCs in5×106/mL group was significantly lower than in the other4groups, but the oxygen metabolic activity, cell viability, and the gene expression level of ALP, RUNX2, OCN, and OPNof hBMSCs in these5groups had no significant differences. The5×106/mL group was unable to form new bone after3months ectopic implantation, and the positive bone fraction of ectopic bone formation were higher than50%in the rest4groups. In vivo bone yield represented by bone area, bone volume fraction (BVF), bone mass fraction (BMF) of hBMSCs in groups of30×106/mL and50×106/mL were significantly higher than that in groups of10×106/mL and20×106/mL.3. Seeded on CHA, the proliferationof hBMSCs in5×106/mL group was significantly lower than in the other4groups, at the same time the10×106/mL group showed significantly lower than the20×106/mL,30×106/mL, and50×106/mL groups. The statistic results of comparison of in vivo bone yield among the5groups were consisted with that in vitro comparison in proliferation of hBMSCs.4. At the follow-up of3months,2minor patients achieved bone fusion,1adult patient elevated nasal base by bone formation.Conclusions1.β-TCP and CHA were demonstrated to be the suitable scaffolds for constructionof tissue engineered bone. 2. The dose-effect relationship between cell seeding quantity and in vivo bone yield was demonstrated as follows:when TEB constructed with hBMSCs using the seeding density below the minimum seeding density, TEB was not able to form new bone. Higher than the minimum seeding quantity, the in vivo bone yield of TEB with the cell seeding quantitywas dose-dependent and has a’ceiling effect’ that the bone yield didn’t increase when the seeding density was higher than saturation seeding quantities.3. Different scaffolds have different saturation seeding quantities which wasaffected by the charateristic of scaffold.4. Concentrated autologous bone marrow combined withβ-TCPscaffold could repair alveolar cleft. |
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