| Backgrounds Bone loss due to trauma or disease is an increasingly serious health problem. Bone grafting is frequently used to augment bone healing with the numerous approaches to reconstructing or replacing bone defects. Grafting substitutes currently available include autogenous bone, allograft bone, ceramics, demineralized bone matrix, bone marrow, and composite grafts. But all have their own shortcomings and complications, including limited quantities of bone for harvest and donor-site morbidity, immunologic rejection, and so on. To overcome the drawbacks of the current bone graft materials, bone tissue engineering using bone mesenchymal stem cells (BMSCs) has been suggested as a promising technique for reconstructing bone defects. However, until recently, no convincing successes have been achieved in humans. One of the problems is inadequate diffusion and insufficient oxygen and nutrient supply for the cell-based approach. With regard to the former, only a sufficient number of new blood vessels within a short period of time guarantee an optimal survival rate of implanted cells. In the near future, several approaches to improve the oxygen and nutrient supply will be further investigated. One approach is to stimulate vessel growth by adding angiogenetic growth factors or endothelial cells to the tissue engineered construct.Alginate hydrogels have been proving to have a wide applicability as biomaterials. They have been used as scaffolds for tissue engineering, as delivery vehicles for drugs, and as model extracellular matrices for basic biological studies. BMSCs can be expanded and induced to terminally differentiate into osteoblasts, chondrocytes, adipocytes, tenocytes, myotubes, neural cells, and hematopoietic-supporting stroma. Their multipotential, their ease to isolate and culture, as well as their high expansive potential make them be selected as a seeding cell in bone tissue engineering.It is difficult to construct tissue engineered bone by using osteoblast and EC directly because it is so hard to harvest the two kind cells. We are interested in whether BMSCs can differentiate into the two kinds of cells in the same microenvironment when precisely induced. If they can, the construction will be simplified accordingly and formation of complex organ will be promoted. So we chose BMSCs as seeding cell and alginate as scaffold, analysed the applicability of alginate as eligible scaffold in bone tissue engineering and investigated preliminarily the potency of constructing vascularized tissue engineered bone by constructs of BMSCs/ alginate hydrogels. Objectives1 To detect the biological effects (biocompatibility and osteoinductive) of 2D alginate gels on BMSCs and investigate the possibility of alginate gels to be eligible scaffolds in bone tissue engineering.2 To induce BMSCs to differentiate into osteoblasts and observe whether BMSCs can be promoted by cultured with osteoinductive medium on 2D alginate gels.3 To induce BMSCs to differentiate into EC on the alginate gels.4 To explore the possibility of inducing BMSCs to differentiate into bone and EC in same microenvironment simultaneously.5 To search the construct methods of 3D alginate gels and to investigate the possibility of the 3D scaffold used in bone tissue engineering.6 To form the constructs of BMSCs/ 3D alginate gels and explore the possibility of forming vascularized tissue engineered bone.Methods1 Rat BMSCs were isolated from rat bone marrow by density gradient centrifugation, and 3% alginate scaffolds were prepared in 6-well plates. BMSCs were cultured on 2D alginate gels with SαMEM. There are 2 groups: A. cell cultured on alginate gels (the experimental group); B. cell cultured on tissue culture plastic (TCP) (as control). The quantity of alginate gels after dewatering weighed in 0, 3, 6, 9, 12d; The surface was observed by scaning electron microscope (SEM); Cell proliferation was detected by MTT; The mRNAs of alkaline phosphatase, collogen I and osteocalcin, which all are bone related genes were determined by RT-PCR.2 BMSCs were cultured on 2D alginate gels with Dex-SαMEM which contains dexamethasone, ascorbic acid,β-glycerophosphate for 12 days. There were 3 groups: A. Dex-SαMEM with TCP; B. SαMEM with alginate gels; C. Dex-SαMEM with alginate gels. The quantity of alginate gels after dewatering in group B and C weighed in 0, 3, 6, 9, 12d; Cell proliferation was detected by MTT; Morphology was observed by microscope and toluidine blue stain in group B and C. von Kossa stain in group A and immunocytochemistry of Osteocalcin of cells on the coverslip in groups B and C and RT-PCR for the mRNAs of alkaline phosphatase, collogen I and osteocalcin, which all are bone related genes were used to identify the calcification.3 BMSCs were cultured on 2D alginate gels with the VEGF- SαMEM which contains VEGF and L-glutamine for 12 days. Cells were observed under light microscope. Subsequent cells on the coverslip were observed by immunocytochemistry and immunofluorescence with anti-von Willebrand factor polyclonal antibody (vWF); The mRNA of endothelin-1(ET-1), which was EC related gene was determined by RT-PCR.4 BMSCs were cultured on 2D alginate gels with the mixing medium which contains dexamethasone, ascorbic acid,β-glycerophosphate, VEGF and L-glutamine for 12 days. There were 4 groups: A. the mixing medium with alginate gels; B. the mixing medium with coverslip; C. Dex-SαMEM with alginate gels; D: VEGF-SαMEM with alginate gels. Cells were observed by microscope and HE stain. Subsequent cells on the coverslip were incubated with anti-vWF and anti-Osteocalcin, and analysed the differentiation rates from BMSCs to EC by image analysis.Immunofluorescence with anti-vWF and anti-Osteocalcin were observed by 1aser scanning confocal microscopy (LSCM). Cell ultrastructure was observed through scanning electron microscope.5 Prepared 3D alginate gels by freezing and detected factor of porosity of scaffolds by liquid substitution; the pore formation was observed by SEM and section HE staning and aperture size was analysed by image analysis.6 Prepared the constructs of BMSCs/ 3D alginate and transplanted the 4 group constructs into Wistar rat dorsal subcutaneous tissue. There were 4 groups: A. the constructs cultured with mixing medium; B. the constructs cultured with Dex-SαMEM; C. alginate gels only; D. nothing transplanted. To identify the calcification in 0, 4, 6, 8w by X-ray; Tissue sections observed by von Kossa and HE staning. The density of blood vessel was detected by image analysis.Results:1 The surface of alginate gels are rough;Alginate gels degradated along the time when cultured in medium. There was no significant difference in the cell proliferation between the experimental group and the control group. BMSCs in the experimental group formed colony proliferation obviously. mRNA expressions of the alkaline phosphatase and collagen I were positive in the two groups, but the levels of their expressions in the experimental group were higher; at the same time, osteonectin mRNA was positive in the experimental group only.2 The alginate gels both in group B and C degradated along the time, but there was no significant difference in the 2 groups. BMSCs of the 3 groups grew well and formed colony proliferation obviously; the calcification was proved on the group A by von Kossa stain and immunocytochemistry of Osteocalcin. The mRNAs of alkaline phosphatase, collogen I and osteocalcin were positive in the three groups and all levels of the 3 gene expression was highest in bone inductive medium on the alginate gels among the three groups.3 The immunofluorescence and immunocytochemistry of vWF were both positive. The mRNA of ET-1 showed positive.4 The different morphous can be observed by light microscope and HE stain: some cells became round, small, aggregated and laminated; calcification was found in these cells; some cells formed a tube similarity. Osteocalcin and vWF were positive in the two kinds of cells on group B; the density of EC in the slide was 14.1%. Under SEM, cells were maturity, and some cells with the features of EC (such as tight junctions, pseudopodiums) formed a tube similarity. After fluorescence immunocytochemistry staining, two colors can be detected in cells simultaneously with LSCM. The mix of two colors was yellow, but the colors of some cells were much red, some were much green.5 The factor of porosity was 91.14%,Section HE staning and SEM showed that the scaffolds were porosity, round, not uniformity and disposition uniform. It is connected among pores. Average pore diameter was 205.26±78.98μm.6 Detected by X-ray and tissue paraffin section: The calcification was found at 6 w, and more at 8 w in group A. The calcification was not found at 6 w, and found in group B at 8 w only. There was no calcification in group C and D. With the HE staning, there were abundant blue calcification, proliferative blood vessels, chondroblast and chondrocyte precursor around which is matrix and cava at 8 w in group A; there are less calcification and blood vessels, chondroblasts were found but chondrocyte precursor was not at 8 w in group B.Conclusions:1 BMSCs have potentiality to differentiate into osteoblasts spontaneously.2 Alginate gels have osteoinductive; it is applicable materials for scaffolds in bone tissue engineering because of its osteoinductive, plus biocompatibility, asepsis, porosity, and so on.3 BMSCs could differentiate into osteoblasts and EC in the same microenvironment when precisely induced.4 It is potential to form vascularized tissue engineered bone by transplantating the constructs of BMSCs/ alginate gels cultured with mix culture medium which contained of Dex-SαMEM and VEGF-SαMEM.
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