| IntroductionThe injury of ACL inevitable results the instability of the knee joint, which will lead the repetitive damage to the cartililage. Under this kind of situation, the cartilage of the knee of this patient will degrade more quickly than normal people. It is publicly recognized that the process of the healing of the bone-ligament after the reconstruction for ACL is complicated and takes time. It has been always the hot topic in the field of sports medicine. Now there are many strategies trying to stimulate the healing of the bone-ligament after the reconstruction of the ACL, but the long-standing outcome are not very satisfactory. As an alternative substitute therapy, tissue engineering based on stem cell offers a promising approach for the bone-ligament healing. Three main requirements should be met when tissue engineering method is considered to this problem:first, sufficient number of properly functioning seed cells and osteogenic capacity. Second, a three-dimensional scaffold with biocompatible, non-toxic degradation, suitable hole size and porosity. Third, appropriate cytokines should be used which would regulate the seed cell proliferation and differentiation and maintain their phenotypic.Periosteum-derived stem cells are adult cells originating from the inner layer of the periosteum. Compared with other adult stem cells, the resources for obtaining the periosteum-derived stem cells are very sufficient and the procedure for harvest the PDSCs is convenient. Also the PDSCs have the better proliferation and stable determined differentiation. PDSCs have very similarly biological characteristics and potential differentiation and better osteogenic differentiation compared with BMSCs. The PDSCs can adjust themselves to the host environment very well, and maintain their potential for the bone formation and finally lead to the bone defect repair through the mechanism of enchondral ossification. Studies showed that the PDSCs have been the ideal source of seed cells in the tissue engineering and become the hot issue in the field of orthopaedic surgery research. The new biomaterial which emerges recently is called Gelatin-Hpa. Gelling stiffness and velocity of Gelatin-Hpa are not controlled by the concentration of the primers of polymers. The gelling enzyme HRP and oxidant H2O2regulate the velocity and degree of the gel independently. This kind of injectable hydrogel Gelatin-Hpa is now emerging as completely new scaffold in the application of bong tissue engineering.PDGF-bb is a polypeptide which produced by different kind of cells. It has the effect of mitosis and chemotaxis on lots of cells including MSCs, osteoblasts, fibroblasts, endothelial cell. PDGF-bb is the powerful chemotaxis factor in the family of cytokines. PDGF-bb can stimulate the osteoblast to synthesize DNA, cell replication, and collagen or non-collagen protein synthesis. PDGF-bb can stimulate the proliferation of endothelial cell, induce the mRNA expression of VEGF, reconstructed the blood circulation system. As for the BMP-2, a lot of studies have been investigated in the function the cytokine. BMP-2can initiate the process of osteogenic differentiation and affect the whole course of bone formation. It is widely used in the application of tissue engineering.Although studies have showed that PDSCs has the function of repairing the bone defect, but it is unclear to use the non-inducing PDSCs for repairing bone defect. Meanwhile, as the number of the periosteum-derived stem cells is low, so it is essential to stimulate the cell to proliferation and osteogenic differentiation. Based on the unique function of the cytokines PDGF-bb and BMP-2, this study aims to apply both of the two cytokines PDGF-bb and BMP-2on the periosteum-derived stem cells to see the proliferation and osteogenic differentiation. Now there is no such study to investigate the combined effects of PDGF-bb and BMP-2on the osteogenic differentiation of periosteum-derived stem cells within2D and injectable Gelatin-Hpa hydrogel matrices. After the completion of this study, our next step aim to implant this kind of compound hydrogel system containing dual cytokines PDGF-bb and BMP-2through a simple injection into the ACL reconstruction animal, hoping this can enhance the healing of bone-ligament in the bone tunnel and finally can translate this technique into the clinical practice. One of the big challenges of applying the stem cell in the bone-ligament healing is that there is not sufficient stem cell in the local target areas. As for the periosteum-derived stem cells, there are three questions we should address first before this strategy applies in the field of bone-ligament healing. First, there is no specific definition for periosteal cell, more accurate expression of this kind of cells is periosteum-derived stem cells. Their potential differentiation and efficiency is still under further investigating. Second, the periosteum-derived stem cells are located in the inner layer of the periosteum, the number is very low. It is essential to have extraneous factors to stimulate the proliferation and osteogenic differentiation of the periosteum-derived stem cells. Third, an appropriate scaffold is needed when the periosteum-derived stem cells are employed in the injury area. This biomaterials should provide an optimal environment for the differentiation and osteogenic differentiation of periosteum-derived stem cells.In order to resolve the above issues encountered in the application of periosteum-derived stem cells, this study are divided into three parts (1) The first Part:The isolation, culture and definition of periosteum-derived stem cells:using the femoral periosteum of the swine and the primary cell culture method, the periosteum-derived stem cells were isolated based on indirectly Immune magnetic bead first-choice method with the biomarker of STRO-1.The morphology of the periosteum-derived stem cells and the cell growth curved are observed by phase contrast microscope under the expansion medium. Under the osteogenic culture for periosteum-derived stem cells, the activity of ALP, calcium deposition, and mRNA expression level of the osteogenic differentiation biomarkers such as ALP, Runx-2, Col-1and OCN were recorded. Results showed that all of the different passages of P3, P5, P7have the good osteogenic differentiation capacity. The capacity of osteogenic differentiation of the periosteum-derived stem cells decreases as the passages increase. The various osteogenic differentiation indicators for P7are significantly weaker than that of P3.(2) To investigate the effect of PDGF-bb alone or combined with BMP-2on the proliferation and osteogenic differentiation of periosteum-derived stem cells. The effect of different concentration of PDGF-bb on the proliferation of the periosteum-derived stem cells was observed with the method of MTT. Results showed that the proliferation capacity of periosteum-derived stem cells increase as the concentration of the PDGF-bb increase, reaching its peak when the concentration of the PDGF-bb is50ng/ml. But the proliferation capacity of periosteum-derived stem cells decreases when the concentration of the PDGF-bb increased furtherly. The difference among the different concentration of the PDGF-bb on the proliferation of periosteum-derived stem cells is not significant (P>0.05). Then, Using the methods of RT-PCR and von-Kossa calcium deposition staining, the osteogenic differentiation of the periosteum-derived stem cells among the different groups OM, OM+PDGF-bb, OM+BMP-2, OM+BMP-2+PDGF-bb, compared with the cytokine PDGF-bb alone or BMP-2alone on the osteogenic differentiation of the periosteum-derived stem cells, the combination of PDGF-bb and BMP-2have no superior effect (P>0.05) under the2dimensional culture system. PDGF-bb has the inhibitory effect on the stimulating effect of BMP-2on the osteogenic differentiation of the periosteum-derived stem cells within monolayer culture.(3) Based on the above two parts work, we select periosteum-derived stem cells as seed cells and Gelatin-Hpa as scaffold, adding with PDGF-bb alone or combination with BMP-2. The biocompatibility, proliferation and osteogenic differentiation of periosteum-derived stem cells within the injectable hydrogel system Gelatin-Hpa were evaluated. Results showed that the periosteum-derived stem cells have the good biocompatibility within the Gelatin-Hpa. There are synergistic effect between the cytokine PDGF-bb and BMP-2on the osteogenic differentiation of periosteum-derived stem cells within the injectable hydrogel system Gelatin-Hpa. Part1The isolation, culture and definition of periosteum-derived stem cellsObjective:Isolation and purify the periosteum-derived stem cells from the swine, and test the proliferation and osteogenic differentiation of the periosteum-derived stem cells from the different passages(P3, P5, P7).Methods:Take the periosteum which we harvested from the femur of swine out of from the50ml tube, then cut the periosteum into pieces and digested them with specific enzyme. Culture the mixture cells according to principle of the primary cell culture. Based on the indirectly of the Immune magnetic bead first-choice method using STRO-1as the biomarker, the periosteum-derived stem cells were purified out. Under the culture of the osteogenic medium, the ALP activity, the mineral deposition and the osteogenic related marker such as ALP, Runx-2, Col-1, and OCN were observed between the different passages of the periosteum-derived stem cells (P3, P5, P7).Results:Under the common expansion culture medium, the morphology of periosteum-derived stem cells grows well and showed a classic cell growth curve. Three different passages of the periosteum-derived stem cells all had a great osteogenic potential. As the passage of the cells increase, the ability of the osteogenic differentiation potential of the periosteum-derived stem cells decrease. As seen on the results section, the various indicators of the osteogenic differentiation of the passage7are significantly weaker than that of the passage3of the periosteum-derived stem cells.Conclusion:periosteum-derived stem cells have the strong osteogenic differentiation potential and simple culture as well as induction conditions. It is one of the promsing seed cells in the field of the bone tissue engineering. It is better to select the passage less than5to be the seed cells when taking the periosteum-derived stem cells.Part2The effect of PDGF-bb alone or combined with BMP-2on the proliferation and osteogenic differentiation of periosteum-derived stem cells within2D culturesObjective:To investigate the effect of PDGF-bb alone or combined with BMP-2on the proliferation and osteogenic differentiation of periosteum-derived stem cells.Methods:Using the MTT technique, add the differentiation of10,20,50,100,150ng/ml of PDGF-bb into96-well culture plate and evaluated the effect of PDGF-bb on the proliferation of periosteum-derived stem cells. Then the passage3of periosteum-derived stem cells was selected, investigated the osteogenic differentiation of periosteum-derived stem cells under the different culture medium (OM,OM+PDGF-bb, OM+BMP-2, OM+BMP-2+PDGF-bb) using the method of RT-PCR and von-Kossa staining.Results:The stimulating effect on the proliferation of the periosteum-derived stem cells increase as the concentration of periosteum-derived stem cells increase. It reaches its peak when the concentration of periosteum-derived stem cells is50ng/ml. But the stimulating effect on the proliferation of the periosteum-derived stem cells decrease when the concentration of periosteum-derived stem cells increase further. The difference among the different concentration of the PDGF-bb on the proliferation of periosteum-derived stem cells is not significant (P>0.05). Then, Using the methods of RT-PCR and von-Kossa calcium deposition staining, the osteogenic differentiation of the periosteum-derived stem cells among the different groups OM, OM+PDGF-bb, OM+BMP-2, OM+BMP-2+PDGF-bb, compared with the cytokine PDGF-bb alone or BMP-2alone on the osteogenic differentiation of the periosteum-derived stem cells, the combination of PDGF-bb and BMP-2have no superior effect (P>0.05) under the2dimensional culture system. PDGF-bb has the inhibitory effect on the stimulating effect of BMP-2on the osteogenic differentiation of the periosteum-derived stem cells within monolayer culture. Conclusion:The effect of PDGF-bb on the proliferation of periosteum-derived stem cells is very powerful. It reaches its peak when the concentration of PDGF-bb is50ng/ml. But the effect on the proliferation of periosteum-derived stem cells decreases while the concentration of PDGF-bb increases further. PDGF-bb has the inhibitory effect on the stimulating effect of BMP-2on the osteogenic differentiation of the periosteum-derived stem cells within monolayer culture.Part3The combination of the dual effect of DGF-bb and BMP-2on the proliferation and osteogenic differentiation ofperiosteum-derived stem cells within3D scaffolds Gelatin-HpaObjective:To evaluate the biocompatibility, proliferation and osteogenic differentiation of periosteum-derived stem cells within the injectable hydrogel system Gelatin-Hpa.Methods:First the rheologoly and degradation of the Gelatin-Hpa was evaluated. Then the adhesion and cell viability of the periosteum-derived stem cells were investigated. The periosteum-derived stem cells were challenged within the Gelatin-Hpa using different H2O2. Finally, the periosteum-derived stem cells are cultured within the Gelatin-Hpa under different osteogenic medium containing different cytokines (OM, OM+PDGF-bb, OM+BMP-2, OM+BMP-2+PDGF-bb). The mRNA expression level of the osteogenic differentiation biomarkers (ALP, Runx-2, Col-1, and OCN) and calcium deposition were recorded using the RT-PCR and von-Kossa staining.Results:The periosteum-derived stem cells have the good biocompatibility within the Gelatin-Hpa (the cell viability>90%, cell adhesion>90%). The periosteum-derived stem cells have the high H2O2resistance. After the periosteum-derived stem cells were embedded in the Gelatin-Hpa24hours later, the cells were challenged by different concentration of H2O2and the collage group set as control group. Results showed the periosteum-derived stem cells in the collagen were more subjected to influence by the challenges of H2O2, with lots of cells dead, especially at the higher H2O2concentration. On the contrary, the periosteum-derived stem cells seeded in the Gelatin-Hpa were not influnced by H2O2. At day7and14, the mRNA expression of early osteogenic differentiation gene such as ALP and Col-1in the group with OM+BMP-2+PDGF-bb culture medium, its level are significantly higher than other groups. The statistically difference are observed between the group of OM+BMP-2+PDGF-bb and OM+BMP2. There are synergistic effect between the cytokine PDGF-bb and BMP-2on the osteogenic differentiation of periosteum-derived stem cells within the injectable hydrogel system Gelatin-Hpa. This issue is totally different from osteogenic differentiation of periosteum-derived stem cells when the cells cultured on the monolayer.Conclusion:The periosteum-derived stem cells have the high H2O2resistance. The periosteum-derived stem cells have the good biocompatibility within the Gelatin-Hpa. There are synergistic effect between the cytokine PDGF-bb and BMP-2on the osteogenic differentiation of periosteum-derived stem cells within the injectable hydrogel system Gelatin-Hpa. |
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