The Study On Tissue Engineering Technique Based On Human Mesenchymal Stem Cells

The concept of tissue engineering was first proposed in1987by U.S. National Science Foundation. It is an interdisciplinary field that the principles of engineering and life sciences are applied toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ. The basic principle and method is that cells expanded in vitro are often implanted or ’seeded’ into biocompatible and biodegradable scaffold for preparation of the tissue engineered constructs, and then the constructs were transplanted into the damaged area. In vivo, as the degradation of scaffold, the continual proliferation and differentiation of cells could result in the new tissue formation and finally be close to the goal of tissue reconstruction. Due to its great scientific significant, clinical application prospect and potential developmental value, tissue engineering has become of major interest for tissue reconstruction. In fact, tissue engineering technique has been employed for tissue reconstruction in many clinical fields, such as heart, bone, cartilage, liver, skin and so on.Bone defect resulted from trauma and tumor resection is a common clinical problem. Traditional therapies that include autogenous bone; allograft bone and artificial bone grafting have their problems and limitations, resulting in the difficulty in meeting the clinical requirement. To conquer these issues, the tissue-engineered bone graft is being explored. In recent years, bone tissue engineering research has been developed rapidly, for bone defect is very common and in strong need for treatment. In addition, their structure is relatively single, which makes the tissue-engineered bone to be one of the earliest tissue engineering products applied for clinical therapy. Mesenchymal stem cells (MSCs) derived from bone marrow are an ideal seeding cells for cell therapy and tissue engineering due to its strong regeneration potential, multi-differentiation potential and immunosuppressive properties. Therefore, in our study we focus on the bone tissue engineering research based on hMSCs in the first and second part (Chapter2and Chapter3). Part One:we evaluates the reconstruction effects of hMSCs and osteoblast-like cells differentiated from hMSCs in poly-lactic-co-glycolic acid (PLGA) scaffolds on the calvarial defect of rats. Two bilateral full-thickness defects (5mm in diameter) were created in the calvarium of Sprague-Dawley (SD) rats. The defects were filled by PLGA scaffolds with hMSCs (hMSC Construct) or with osteoblast-like cells differentiated from hMSCs (Osteoblast Construct). Meanwhile, fluorescent carbocyanine CM-Dil was used to track the implanted cells in vivo during transplantation. Evaluation was performed using macroscopic view, histology and immunohistochemical analysis after transplantation. The results showed that the implanted human cells could survive in vivo for10weeks. Both hMSC Construct and Osteoblast Construct led to an effective reconstruction of critical-size calvarial defects, and the bone reconstruction potential of hMSC Construct was superior to that of Osteoblast Construct in non-autogenous applications. Our findings verify the feasibility of the use of xenogenic MSCs for tissue engineering and demonstrate that undifferentiated hMSCs are more suitable for bone reconstruction in xenotransplantation models.Part Two:In order to improve the technical system of bone tissue engineering and find out the best scaffold for bone tissue engineering, the following research was carried out. Copolymer composite scaffolds and bioceramic/polymer composite scaffolds are two representative forms of composite scaffolds used for bone tissue engineering. Studies on the comparison of biocompatibility and bone-repairing effects between these two scaffolds are significant for selecting or improving the scaffold for clinical application. We prepared two porous scaffolds comprising poly-lactic-acid/poly-glycolic-acid (PLGA) and poly-lactic-acid/nano-hydroxyapatite (nHAP/PLA) respectively, and examined their biocompatibility with human bone marrow-derived mesenchymal stem cells (hMSCs) through evaluating adhesion, proliferation and osteogenic differentiation potentials of hMSCs in the scaffold. Then, the PLGA scaffold with hMSCs (PM construct) and the nHAP/PLA scaffold with hMSCs (HPM construct) were transplanted into the rat calvarial defect areas to compare their effects on the bone reconstruction. The results showed that the nHAP/PLA scaffold was in favor of adhesion, matrix deposition and osteogenic differentiation of hMSCs. For in vivo transplantation, both HPM and PM constructs led to mineralization and osteogenesis in the defect area of rat. However, the area grafted with PM construct showed a better formation of mature bone than that with HPM construct. In addition, the evaluation of in vitro and in vivo degradation indicated that the degradation rate of nHAP/PLA scaffold was much lower than that of PLGA scaffold. It is inferred that the lower degradation of nHAP/PLA scaffold should result in its inferior bone reconstruction in rat calvaria. Therefore, the preparation of an ideal composite scaffold for bone tissue engineering should be taken into account of the balance between its biocompatibility, degradation rate, osteoconductivity and mechanical property.Part Three:As well as performing the bone tissue engineering research, we had been pondering that the technical system of bone tissue engineering could also be applied for other tissue reconstruction. Thus, we noted that with the advent of laparoscopic biliary procedures, there was an increase in the number of reports of benign biliary injury and complicaitons. However, the current treatments still have some weakness, so the tissue-engineered bile duct is also expected to use for treatment of bile duct injury. The study was conducted to evaluate the feasibility of using hMSCs-scaffold construct for bile duct repairing. We designed a novel PCL/PLGA double-layer bile duct scaffold and prepared the hMSC-PCL/PLGA scaffold constructs (MPPCs) by the preparation device of cell-tubular scaffold construct. Then, the blank PCL/PLGA scaffolds and MPPCs were transplanted into the porcine bile duct injury area for evaluation of their effects on the bile duct repairing. The results showed that the PCL/PLGA double-layer bile duct scaffold could support the adhesion, proliferation and matrix deposition of hMSCs. Compared to the blank PCL/PLGA scaffolds, MPPCs had the superior repairing effect on the bile duct injury. Therefore, MPPCs could not only support the bile flowing in the biliary tract, but also have positive effects on the repairing of damaged bile duct with no need of reoperation to remove the foreign materials. The present strategy to employ the double-layer bile duct scaffold with hMSCs for reconstruction may lead to develop a new treatment form for biliary diseases.