Mesenchymalstem Cells Combine With Porous Scaffolds For Repairing Bone And Cartilage Defects

Articular cartilage defect is common in sport injury.In clinical, it will cause knees pain. If this injury treated inapproately, they may cause joint deformity, disability, and finally patients will develop osteoarthritis. As the cartilage has no vessel and nerve, chondrocytes have limited proliferation self repair. Currently, a variety of methods have been applyed for the treatment of cartilage defects. However; the long-term result was unsatisfactory. Especially, when the lesion involeved in subchondral bone, the treatemt became more difficult. Bone defect caused by trauma, osteomyelitis or bone tumor is another question in orthopedic practice. Autogenous bone graft is still the gold standard for the treatment of bone defect, but the limited sorce of autogenous bone limited it application. Allogenous bone graft has the problems of immunological rejection and bone healing slowly.In recent years, tissue engineering technology provides a new approach for the treatment of bone and cartilage defects. Bone marrow mesenchymal stem cells(MSCs) because of its good differentiation capability is currently the most popular seed cells. Adipose derived mesenchymal stem cells (ADSCs) with rich resources, strong prelifiration, and multi-directional differentiation have become the research hotspot in the field of tissue engineering. The3D porous scaffolds in tissue engineering gives a supporting for stem cell proliferation and provides a three-dimensional construction as the template for stem cell regeneration. In recent years, the application of synthetic material is gradually increasing. PLGA has been approved by the FDA and can be used as a scaffold for tissue engineering. It could be adjusted the speed of degradation and posease good mechanical properties, but poor histocompatibility. Many researchers try to modify PLGA to improve its biocompatibility and the results is encouging. Gelatin sponge is a commercial aviable products and widely used in clinical. Because of its porous structure, good biocompatibility and biodegradability, it is also good scaffold for tissue engineering.In the repairing of cartilage defect, the subchondral bone plays an important role. In this study, we usedCartilage subchondral bone plays an important role. In this study, we using hydroxyapatite (HA) to improve the biocompatibility of PLGA. Also the HA has the property of bone induction whici will premote the regeneration of subchondral bone. We also used stem cells in vivo tracer technique to study the in vivo fates of transplanted stem cells.Bone formation is divided into2ways:intramembranous osteogenesis and endochondral ossification. Researches shows that endochondral ossification can accelerate bone healing. In this study, we used gelatin sponge as scaffolds. In vitro chondrogenic induction of ADSCs was performed. Then the induced ADSCs was transplanted in the the tibial defect of rats, and to further observation of endochondral ossification during bone defect healing.This study is divided into three parts:(1) the construction of PLGA/NHA scaffold and the proliferation and distribution of MSCs in PLGA/NHA scaffolds;(2) the experimental study of PLGA/NHA porous scaffolds for the repair of rat articular cartilage defects;(3) gelatin sponge scaffold combine with chondrogenic ADSCs for the repair of large segmental bone defects through endochondral ossification in a rat model.Part I The construction of PLGA/NHA scaffold and the proliferation and distribution of MSCs in PLGA/NHA scaffoldsObjective:To fabricate the PLGA/NHA scaffolds and investigate the proliferation and distribution of MSCs on the scaffolds.Methods:We used thermal phase separation technology for fabricating PLGA/NHA porous scaffolds. We used scanning electron microscopy(SEM) and mechanical testing to observe the characters of scaffolds. The third generation of MSCs were used. We seeded the cells on the scaffolds, and MTT and DNA quantitative tests were performed to investigate the proliferation of MSCs. The SEM scan and CM-Dil fluorescence staining were used to investigate the adhesion and distribution of MSCs on the scaffolds.Results:The PLGA/NHA scaffolds is porous and the average porosity is88.3%±2.8%. The biomechanical test results show that the elastic modulus of the PLGA/NHA scaffolds is superior to the PLGA scaffolds. MTT and DNA quantitative tests showed that the numbers of MSC in PLGA/NHA porous scaffolds was significantly higher than that in PLGA porous scaffolds. Scanning electron microscopy showed that MSCs attached very well in the PLGA/NHA scaffolds. The laser confocal microscopy showed that the number of MSCs in PLGA/NHA scaffolds is more than that in PLGA porous scaffolds.Conclusion:adding nano HA particles could improve the growth and adhesion of MSC in the PLGA scaffolds. The HA particles could also improve the mechanical properties of PLGA scaffolds. The the NHA modified PLGA scaffold can be used as tissue engineering scaffolds for the treatment of osteochondral defect.Part II:The experimental study of PLGA/NHA porous scaffolds for the repair of rat articular cartilage defectsObjective:To investigate the effects of PLGA/NHA porous scaffolds combined with MSCs for repairing rat articular cartilage defects and the final fate of transplanted MSCs.Methods:We separated MSCs and then marked with CM-Dil fluorescence staining. The marked MSCs were implanted in PLGA/NHA and PLGA scaffolds. The cell-scaffolds constructs were then implanted in the distal cartilage defects of rat femur respectively. After6weeks and12weeks, we harvested the distal femur of rats. We performed gross observation, histological evaluation and immunohistochemical tests to compare the repair results.Results:The results showed that12weeks post operation, the regenerated cartilage in PLGA/NHA-MSC group was smooth,rich in GAG and collagen type Ⅱ, without type Ⅰ collagen. We tracked MSCs by CM-Dil, we found the CM-Dil labeled cells were stil in the repair area12weeks post operation.Conclusion:PLGA/NHA scaffolds combined with MSCs could effectively repair rat articular cartilage defects and improve the quality of cartilage. The transplanted MSCs could survive during early osteochondral repair. They could improve the local micro-environment without adding growth factors. PLGA/NHA scaffolds combined with MSC can be used as a good tissue engineering materials for repairing osteochondral defects.Part Ⅲ:Gelatin sponge scaffolds combined with chondrogenic ADSCs for the repair of large segmental bone defects through endochondral ossification in a rat modelObjective:To study the gelatin sponge scaffolds combined with chondrogenic inducted ADSCs for repairing segmental bone defects through endochondral ossification in a rat model.Methods:We seprated rat ADSCs and then confirmed the sternness by osteogenic, adipogenic and chondrogenic differentiation. The gelatin sponge was cut into square as scaffolds. Then we performed scanning electron microscope to observe the surfice characters of the scaffold. The ADSCs were implanted in gelatin sponge scaffolds. The DNA quantitative test was performed to evaluate the proliferation of ADSCs on the scaffolds to form gelatin-ADSCs composites. Then we createed a2mm bone defects of tibia in a rat model. We performed chondragenic induction of gelatin-ADSCs composites for14days to form gelatin-ADSC-CD composites. The bone defects were filled with blank, gelatin sponge, gelatin-ADSCs composites, gelatin-ADSC-CD. After2weeks,4weeks and8weeks of operation, we performed X-ray, microCT imaging and pathological staining were used to evaluate the osteogenesis and chondrogenesis of regenerated bone.Results:The isolated ADSCs can be induced ti multiple differentiation. The ADSCs could proliferate well on the gelatin sponge scaffolds. In the bone defects model, we found that the defect region was rich in GAG matrix and cartilage cells in gelatin-ADSC-CD group. This means the endochondral ossification was started. But there was no GAG matrix and cartilage cells in control and gelatin sponge scaffolds groups.8weeks post operation, the X-ray and CT showed the bone defects healed completely in gelatin-ADSC-CD group, and safranin O staining showed there was completing endochondral ossification. The CT scan showed there was still fracture line in the tibial in gelatin-ADSC group, and safranin O staining showed there was still GAG in bone defect indicting remodeling process incomplete. In the blank group and gelatin sponge group, the fracture lines were obvious. Safranin O staining showed the defect rich in GAG matrix, and the fractures were still in the process of endochondral ossification.Conclusion:Gelatin sponge combined with chondrogenic induction of ADSCs can accelerate the endochondral ossification, promote fracture healing, and improve the quality of repaired bone. Due to gelatin sponge is clinical used widely and commercial available, clinical application has broad prospects.