To Fabricate A Novel Biological Cartilage Scaffold With Wharton's Jelly Of Human Umbilical Cord And The Preliminary Research Of The Construction Of Tissue Engineering Cartilage With The Scaffold In Vitro

The treatment of articular cartilage and osteochondral defects has been a problem in orthopedic surgery up to now.The defects may be caused by trauma, osteoarthritis and other related diseases. Because cartilage has a limited capacity for self-repair and numerous strategies currently in clinical treatment inculding microfracture techniques, subcondral drilling, periosteal or perichondrial grafts, transplantation of osteochondral autografts or allografts, many shortcomings limit the repair procedure and compromise long-term results. Tissue engineering of cartilage and osteochondral constructs might be regarded as one with the biggest potential to make a significant clinical impact in the future. Tissue engineering include three main fators:cell, scaffold and cytokine. The goal of present study is to fabricate a novel biological cartilage scaffold with Wharton's jelly of human umbilical cord.To test its composition, physical and chemical properties and biological performance,to explore the possibility of construction tissue-engineering cartilage through the human umbilical cord mesenchymal stem cells(HUMSC) compound with the novel biological cartilage scaffold in vitro so as to provide certain theoretical basis and the experimental basis for the cartilage tissue engineering research and development.Chapter1To fabricate a novel biological cartilage scaffold with Wharton's jelly of human umbilical cordObjective To research the feasibility of fabricate a novel biological cartilage scaffold with Wharton's jelly of human umbilical cord. And to observe the appearance and microstructure of scaffold, see if it conform to the basic requirements of the cartilaginous framework so as to provide the experimental basis for the research of cartilage tissue engineering scaffold.Methods We obtained the fetus umbilical cord, then separated out the Wharton's jelly from umbilical cord.We used the method of enzyme digestion combined with physical freeze-thaw to take off the cells, then used the method of freeze-drying to fabricate cartilage scaffold.The scaffold was prepared and served for the HE stain and the scanning electron micfoscope (SEM)observation.Result Gross observation showed the scaffold had a loosely porous. The histological staining (haematoxylin-eosin) showed that there were no cell residues in the scaffold. The scaffold was porous surface crisscross network of three-dimensional porous cylindrical structure and looked like sponge under SEM observation;The composite matrix presented an open pore ultrastructure with a high degree of interconnectivity. The pore distribution was more even, of material relization gap.Conclusion This part of the experiment was successfully achieved three-dimensional porous sponge appearance scaffold.The method took off cells thoroughly.The scaffold had a three-dimensional mesh structure porous, pore distribution was more even, it can be used as a appropriate candidate for scaffold's carrier of cartilage tissue engineering. Chapter2The component analysis of the novel biological cartilaginous scaffold and the test of its general physical and chemical propertiesObjective Through the component analysis of the novel biological cartilaginous scaffold and the test of its general physical and chemical properties, we studied whether its component was similar to cartilage matrix components, and met to the requirements of the physical and chemical properties of cartilage scaffold.Methods The quantitation of GAGs and type Ⅱ collagen of the novel biological cartilaginous scaffold were detected through Dimethyl methylene blue(DMB)method and ELISA method. Then the pore diameter, porosity,density, swelling ratio and degradation ratio analysis of the scaffold were investigated.Result The content of GAGs in the scaffold was254.1±18.8ug/mg, and the type Ⅱ collagen was742.8±74.4ug/mg. The scaffold had a good pore interconnectivity.The pore diameter was105.2±12.8um; the porosity was90.3±1.2%; the density was0.0848±0.0091mg/mm3, the swelling ratio was1042.0±38.2%and the degradation ratio was17.2±1.3%after2weeks and37.6±2.5%after4weeks.Conclusion:The novel biological cartilaginous scaffold was abundant and contains significant amounts of type Ⅱ collagen and GAGs, and its component is similar to ECM of cartilage.The scaffold met to the requirements of the physical and chemical properties of cartilage scaffold. Chapter3Biological evaluation of the novel biological cartilaginous scaffoldObjective To explore the Biological properties and biocompatibility of the scaffold for investigating its possibility application in the cartilage tissue engineering.Methods The experiments of intracutaneous stimulation, pyrogen, hemolysis and implantation into muscles were investigated to evaluate the biocompatibility of the leaching liquor of scaffold at different concentrations. And to detect the leaching liquor of scaffold to cell of toxicity By MTT test. MTT test was also done to assess the growth and proliferation of HUMSC combined with the novel biological cartilaginous scaffold in vitro.Result The results of experiments on intracutaneous stimulation, pyrogen, hemolysis and implantation into muscles met the regulations mentioned above. MTT detection suggested that there were no significant differences among groups in terms of absorbance value in1,3,5,7and9days after culturing with the leaching liquor (P>0.05).MTT results revealed that the leaching liquor of scaffold had no adverse effect on the cell proliferation (P>0.05). MTT result revealed that the scaffold had no adverse effect on the cell proliferation after the scaffold combined with HUMSC (P>0.05). The cells adhesion ratio were observed gradually increase, and were above95%after12h in the scaffold.Conclusion The scaffold had a higher cells adhesion ratio, produced no adverse effect on the cell proliferation and had a low cytotoxicity.The novel biological cartilaginous scaffold had right biological characteristics which met the basic conditions demanded in cartilage tissue engineering. Chapter4The preliminary research of the construction of tissue engineering cartilage by combined HUMSC with the novel biological cartilaginous scaffold in vitroObjective To explore the possibility of construction tissue-engineering cartilage by HUMSC compound with the novel biological cartilaginous scaffold in vitro.Methods The P3HUMSC were seeded on scaffolds which had be pre-treated. Chondrogenic inductor or general media were respectively used according to different groups; group A:HUMSC compound with the novel biological cartilaginous scaffold in vitro, and chondrogenic inductor; group B:HUMSC compound with the novel biological cartilaginous scaffold in vitro, and general media; group C:HUMSC compound with P(LLA-CL) scaffold in vitro, and general media.After3weeks, the compounds were harvested and HE stain,toluidine blue stain and collagen II stain were applied. RT-PCR was used to detect the expression of Sox-9,Col2al and aggrecan mRNA; Western blot was applied to detect the expression of Col2al and aggrecan protein.Result After3weeks, the HE stain demonstrated that cartilage-like tissue been formed in the group A and B which was not observed in the group C; A strong positive staining for toluidine blue stain was investigated in group A cells and extracelluar matrix, so were for collagen Ⅱ immunohistochemistry;A positive staining for toluidine blue stain was investigated in group B cells and extracelluar matrix, so were for collagen Ⅱ immunohistochemistry; But in group C, the outcome was weak positive for toluidine blue and collagen Ⅱ staining; The RT-PCR revealed that the gene expressions of mRNA for Sox-9, Col2al and aggrecan gradually up-regulation at7,14,21days after cultured in the group A and B. The level of the mRNA expression in group A were significant higher than group B and C(P<0.05); And group B were significant higher than group C at the same time point (P<0.05); The result of westem-blot were concordant with RT-PCR which indicated that the productions of Col2al and aggrecan protein also increase by degrees in group A and B, but significant difference was found compare the group A with the group B (P<0.05), and there were no experssion in group C.Conclusion HUMSC combined with the novel biological cartilaginous scaffold induced in vitro can successfully construct tissue-engineering cartilage which may be a new way to repair the injuried cartilage. HUMSC compound with the novel biological cartilaginous scaffold no pass through chondrogenic inductor in vitro had a tendency to spontaneous cartilage.