A Preliminary Study Of Extracellular Matrix Scaffolds For Cartilage Regeneration And Bone Tissue Engineering

Research Background Regenerative repair of oral and maxillofacial bone or cartilage tissue defects remains a very difficult challenge that needs to be solved in oral medicine. A variety of diseases, including oral and maxillofacial tumors(mandibular ameloblastoma), trauma(condylar dysplasia) and congenital malformations(congenital absence of maxillary bone), could result in defects maxillofacial bone and cartilage. Currently, the gold standard of treatment of bone defects in clinical is still “autogenous bone graft”, but this approach requires opening a second front, causing secondary damage to patient, the suffering and spending also would be increased a lot. Fortunately, the emergence of tissue engineering and regenerative medicine as well as its vigorous development provides a perfect direction for repair of these defects. In addition, scaffold, as one of the three pillars of tissue engineering, plays a very important role in the success fabrication of engineered tissue. Right now, there are two main kind of scaffold that used in regenerative medicine: one is derived from natural tissue, such as collagen; the second is synthetic polymer scaffold,such as PCL and PLA. Both kinds of scaffold have their advantages and disadvantages. Moreover, extracellular matrix(ECM) scaffold is quite important for cell adhesion, proliferation, and differentiation in the process of organ formation and tissue regeneration. In addition, bioactive molecules derived from ECM also have been demonstrated to recruit a great variety of endogenous progenitor and stem cells in vitro and in vivo in mammals. Therefore, ECM as a biological scaffold has its unique advantages. This experiment is aimed to investigate the application of ECM scaffold in cartilage regeneration and bone tissue engineering, and then provide some reliable basis for the clinical application of ECM scaffold in the future.Part I: A preliminary study of extracellular matrix scaffolds derived from chondrocytes sheet for cartilage regenerationExperiment 1. Fabrication of chondrocytes sheetPurpose: The purpose of this study was to fabricate the chondrocytes sheet and investigate their characteristics. Methods: First we isolated chondrocytes from 4-week-old New Zealand rabbits and cultured them with H-DMEM consisted of 20% FBS continuously for 2 weeks. Then the characteristics of chondrocytes sheet were evaluated by gross observation, histology and micro-structure analysis like scanning electronic microscope(SEM) and transmission electron microscope(TEM). Results: Chondrocytes isolated from 4-week-old rabbits grew well and could be able to form sheet after culturing for 2 weeks. HE and safranin-O staining showed that cell sheet was composed of chondrocytes and their self-secreted extracellular matrix(ECM). SEM and TEM observation also showed the similar results with histology that there was a large amount of ECM existed in the chondrocytes sheet. Conclusions: After cultured continuously for 2 weeks, chondrocytes could be able to form sheet which could be used in the next study.Experiment 2. Preparation of extracellular matrix scaffolds derived from decellularized chondrocytes sheetPurpose: The purpose of this study was to investigate the optimal decellularization method of chondrocytes sheet for obtaining extracellular matrix(ECM) and evaluate their characteristics. Methods: First we cultured chondrocytes to form solid white chondrocytes sheet, then the sheets were decellularized using sodium dodecyl sulfate(SDS) that there has three different concentration, 1%, 5%, and 10%, followed by 1% Triton X-100, and deoxyribonuclease enzyme solution to remove cells. We then analyzed the characterization of decellularized ECM, including gross appearance, histology, ultra microstructure, cytotoxicity, maximum tensile stress, the content of collagen and sulfated glycosaminoglycan(GAG), DNA quantification, etc. Results: All the three concentrations, including 1%, 5%, and 10%SDS, could remove the chondrocytes existed in chondrocytes sheet completely, but 10%SDS led to the breakdown of the architecture and potential loss of surface composition of ECM, which was confirmed by gross appearance, histology, ultra microstructure and measurement of UTS, etc. However, such disadvantages didn’t appear in 1%SDS group. Conclusions: The results in vitro demonstrated that 1% SDS not only possessed the ability to remove chondrocytes completely, but also maintained the native architecture and composition of ECM, which can be regarded as the best decellularization method.Experiment 3. Extracellular matrix scaffolds derived from decellularized chondrocytes sheet for the regeneration of osteochondral defects in rabbitsPurpose: The purpose of this study was to verify whether ECM scaffolds derived from decellularized chondrocytes sheet could be used to repair osteochondral defects in rabbits. Methods: 35 adult New Zealand rabbits were randomly divided into 4 groups, including control group, 1%, 5% and 10%SDS groups, eight rabbits in each group; the left three were regarded as positive group. After the osteochondral defects were made, ECM scaffolds were implanted into rabbits. At 6 and 12 weeks after implantation, animals were humanly sacrificed and samples were harvested, respectively. Gross observation, Micro-CT scanning and histology were conducted to assess the repair effect. In addition, gross and histology scoring were also measured to analyze the differences among groups. Results: All of the three experimental groups showed defects were repaired to some extent, but there has the best repair result in 1%SDS group via gross appearance and histology. The defects were almost filled with newly formed cartilage tissue at 12 weeks after implantation. Furthermore, Micro-CT images also demonstrated that the subchondral bone was regenerated well in 1%SDS group. Conclusions: Implanted ECM scaffolds decellularized by 1% SDS exhibited better gross appearance and histological and immunohistochemical charactersitics as well as subchondral bone regeneration, which indicated that such ECM scaffolds can be suitable for cell-free cartilage regeneration.Experiment 4. A preliminary study of mechanism about the effect of ECM derived from chondrocytes sheet on cartilage regenerationPurpose: The purpose of this study was to investigate the mechanism about the effect of ECM derived from chondrocytes sheet on cartilage regeneration. Methods: First we analyzed the effect of VEGF and BMSCs on the migration of endothelial cells by using a co-culture system based on Transwell migration assay; and also compared the number of migrated endothelial cells after V1, which was a VEGF inhibitor, was added in the co-culture system. Then we further investigated the effect of ECM on BMSCs migration. Finally we discussed the effect of ECM on BMSCs differentiation via RT-PCR and Western blot. Results: VEGF, BMSCs and CB could increase the ability of endothelial cells migration. And there had a similar improved effect between BMSCs and VEGF at a concentration of 10ng/ml. At the same time, ECM also could promote the potential of BMSCs migration. Moreover, RT-PCR and western blot also revealed that ECM increased the expression level of SOX-9 of BMSCs, while decreased the expression level of COL-X of BMSCs when co-cultured BMSCs and ECM together. Conclusions: Thanks to the large amount of bioactive elements in the ECM or ECM-rich CB, they could recruit more stem cells from marrow into defects and then triggered the repair or regeneration process. In the other hand, the recruited stem cells also could promote more endothelial cells migrated into defects via VEGF signal pathway; besides, BMSCs also could secret more VEGF to facilitate cartilage regeneration. Furthermore, ECM increased the expression level of SOX-9 of BMSCs, while decreased the expression level of COL-X of BMSCs. And finally, cartilage regeneration was realized.Part II: A preliminary study of ECM-based bone marrow mesenchymal stem cells sheet for bone tissue engineeringExperiment 1. BMSCs sheet combined with PRF increases the bone formation in critical-size calvarial defects in rabbitsPurpose: In our previous study, we have proved that PRF could increase the osteogenic potential of BMSCs sheet in vitro and in nude mice. The purpose of this study was to investigate whether the combination of BMSCs sheet and PRF could improve bone healing in critical-size calvarial defects in rabbits. Methods: First we prepared PRF and BMSCs sheet from the same donor. Then the combination of BMSCs sheet and PRF was implanted in calvarial defects while BMSCs sheet was implanted as control. At 8 weeks after transplantation, Micro-CT and histology was conducted to evaluate bone restoration. Results: Cell sheet formed after BMSCs were cultured continuously for 2 weeks and they could easily be lifted with a cell scraper. Eight weeks later after implantation, there only had a little bone formed in control group, but in combined BMSCs sheet and PRF group, defects were almost filled with newly formed bone tissue. Bone volume / total volume(BV/TV) and half-quantitative analysis based on MTC staining also showed that bone regeneration in composite group was better than that in control group. Conclusions: The combination of osteogenic BMSCs sheet and PRF could increase bone formation in critical-size calvarial defects in rabbits, which could be considered as a new method to promote skeletal healing.Experiment 2.Combined adipose-derived stem cells with BMSCs sheet increases bone formation at an ectopic sitePurpose: In our previous study, we have demonstrated that platelet-rich fibrin(PRF) could increase the osteogenic potential of BMSCs sheet in severe combined immunodeficiency(SCID). The purpose of this study was to investigate whether adipose-derived stem cells(ADSCs) could improve bone formation at an ectopic site. Methods: First we isolated and cultured ADSCs and BMSCs sheet from the same rabbit. Then the complex of BMSCs sheet and ADSCs was prepared for transplantation subcutaneously into the back of mice while BMSCs sheet was transplanted as control. At 8 weeks after transplantation, Micro-CT and histology was conducted to evaluate bone restoration. Results: Osteogenic sheet formed after BMSCs were cultured in osteogenic medium continuously for 2 weeks. At eight weeks after implantation, there only had a little neo-bone formed in control group, while greater bony regeneration was observed in complex group, which was confirmed by histology and Micro-CT scanning. BV/TV and half-quantitative analysis also proved the results. Conclusions: ADSCs could significantly improve the osteogenic potential of BMSCs sheet, which indicated that the complex of ADSCs and BMSCs sheet may be considered as a promising way to stimulate bony regeneration.Experiment 3. Combined BMSCs sheet with ostrich true bone ceramic increases bony formation at an ectopic sitePurpose: In our previous study, we have demonstrated that sintered ostrich cancellous bone(OCB) could be regarded as a bone substitute for bone regeneration, but their biodegradation need to be augmented. The purpose of this study was to prepare a novel scaffold and assess their osteo-conductivity when they used with BMSCs sheet together. Methods: First we prepared sintered true bone ceramic(OTBC) scaffold based on the thermochemical reaction. Then the characteristics, including gross appearance and element composition, biocompatibility and biodegradability was assessed by SEM and MTT assay. In addition, the osteo-conductivity in vivo was also investigated. Results: OTBC scaffolds, was mainly composed of hydroxyapatite(HA) and β-tricalcium phosphate(β-TCP), was a novel scaffold due to their good biocompatibility, biodegradation and osteo-conductivity property. Conclusions: Combined OTBC with BMSCs sheet increased bone formation at an ectopic site, which indicated that OTBC scaffold was a good bone substitute and could be employed in bone tissue engineering in the future.