Experimental Study Of Intellectual Repair Of The Rabbit Osteoarthritis By Functional Self-assembling Peptide Hydrogel Scaffold Combined With Adipose-derived Stem Cells

PART ISynthesis and assembling of a new-type self-assembling peptide hydrogelObjective:Design and compound self-assembled peptide hydrogelthe that promote cell proliferation and TGF-β adhesion, and detect the ultrastructure of self-assembly hydrogel scaffold.Methods:Dissolve the synthetic polypeptides powders of TB and PRG to deionized water respectively until the final concentration is1%. Then dilute the liquor to0.05%(m/v) and take out3ul peptide solution of PRG, TB and the equal volume mixture of them respectively. The atomic force microscope detects the ultra microstructure of peptide hydrogel.Results:PRG and TB peptide-amphiphile molecules were synthesized successfully. A hydrogel was formed when the PRG and TB peptide-amphiphile solution mixed. AFM images demonstrated that the nanofibers of PRG/TB hybrid hydrogel was larger than the PRG and TB, and the average diameter of the nanofibers of PRG, TB and PRG/TB were (16.7±4.7nm),(18.0±1.3nm) and (28.3±5.6nm).Conclusion:Peptide-amphiphile PRG and TB can be mixed and formed by self-assembly of nanoscale hydrogel scaffold (PRG/TB), and they can be used as biological scaffolds for cartilage tissue engineering. PART2Study of new model of osteoarthritisObjective:To investigate a method by injection that can establish a model and the model can simulate the natural course of the early and mid osteoarthritis and can be used in the research of evaluating the tissue engineering to repair articular cartilage.Method:40New Zealand white male rabbits were randomly divided into three groups. After anesthesia, by way of joint puncture, injected different concentrations of MIA(lmg/ml,3mg/ml and6mg/ml)100ul into each right knee and injected PBS into left knee joint as a comparation. Shoot knee X-ray respectively at the second week, fourth week and at the sixth week, compare the gross morphology by ink staining and histopathological staining to observe the degeneration of cartilage.Result:Degeneration of the rabbit knee joint of1mg/ml MIA injection group was not obvious, the3mg/ml and6mg/ml MIA group rabbit knee joint of the pathology staining score and the comparation group have statistically significant (P<0.05), but the degeneration of the6mg/ml MIA injection group was serious.Conclusion:Rabbit knee joints can cause mild to moderate osteoarthritis of the joint degeneration within six weeks by injecting3mg/ml MIA, the result provide a new model to evaluate the tissue engineering to repair of cartilage. PART3Biology effects of fusion protein TGF-β3with lentiviral vector and peptide hydrogel to adipose-derived stem cellObjective:Constructed a new type of fusion protein LAP-MMP-mTGF-β3by lentiviral vector with targeted therapy function, and cultured the adipose-derived stem cells (ADSCs) in the peptide hydrogel scaffold after transfected, to verify its feasibility and targeting specificity.Method:ADSCs were obtained by digestion method isolation. Identified the ADSCs’ surface antigen by the flow method and identified the the ADSCs’ multi-directional differentiation capacity by a special staining method. Inserted PLGLWA restriction sites of LAP, mTGF-β3and matrix metalloproteinase (MMP) respectively into the eukaryotic plasmid expression vector GV287and then get the plasmids of recombinant TGF-β3fusion protein LAP-MMP-mTGF-β3. The plasmid was packaged by lentiviral vector and transfected to ADSCs, and then incubated in a PRG/TB peptide hydrogel. CCK8assay detected proliferation of cells, surviving cells were detected by Calcein/propidium iodide (Calcein-am/P1) in the gel inside, and mTGF-β3in supernatant was detected by Elisa assay after stimulated by MMP enzymes.Results:Isolated and cultured ADSCs successfully and the ADSCs proliferation can be improved when cultured in the PRG/TB hybrid peptide hydrogels. Lentiviral vectors and PRG/TB peptide hydrogel has weak toxicity on ADSCs, and can activate recombinant TGF-β3fusion protein releasing activity mTGF-p3with the presence of MMP enzymes.Conclusion:Constructed a new type fusion protein LAP-MMP-mTGF-β3with packaged lentiviral vector successfully, the ADSCs which transfected with novel fusion protein which packaged by Lentiviral vectors combined PRG/TB peptide hydrogel together has potential applications in repairing cartilage defects. PART4The research of regulation of LV-mTGF-p3transfected adipose-derived stem cells differentiation to chondroblasts in functionalized self-assembling peptide scaffoldObjective:To dicuss the influence of the cartilaginous which induced by functionalized self-assembles peptide scaffold and the transfected rabbit adipose-derived stem cells (ADSCs), and the cells was packaged by lentiviral packaging and transfected from fusion protein LAP-MMP-mTGF-β3.Methods:After transfected the lentiviral packaging fusion protein LAP-MMP-mTGF-β3to a third generational ADSCs, coculture it in cartilage cells induced by fluid together with the functional PRG/TB hydrogels, and then add matrix metalloproteinase to improve the release TGF-β3and improve the differentiation of cartilage cells. Three days later, detect the expression of TGF-β3in ADSCs by Western Blot. When ADSCs induced differentiationto cartilage cells for twenty-one days, detect the expression of Mrna and proteic of cartilage cells specific genes (ACAN), type Ⅱ collagen (COL2A1) and SOX-9by Real-time PCR and Western blot.Results:The efficiency of the ADSCs transfection was up to90%. Three days later, the expression of green fluorescent protein (EGFP) was observed under the fluorescent microscope. Compared to the non-transfection ADSCs when transfected with lentiviral packaging fusion protein LAP-MMP-mTGF-β3, the transfection group ADSCs can improve the cell differentiation very obviously. Twenty-one days later, compared the non-transfection group and the transfection group, the expression of ACAN, COL2A1and SOX-9mRNA were timed by2.7,2.4and1.0respectively and the protein expression was timed by2.4,1.57and0.89respectively.Conclusion:The novel fusion protein LAP-MMP-mTGF-β3can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future. PART5The research of repair of osteoarthritis by combined functionalized self-assembling peptide scaffold and LV-mTGF-p3transfected adipose-derived stem cellsObjective:To illustrate the ability of LV-mTGF-β3transfected ADSCs were repair cartilage in vivo combined with functionalized self-assembling PRG/TB peptide hydrogels.Methods:The transfected ADSCs with fusion protein LAP-MMP-mTGF-β3by lentiviral were cultured in PRG/TB hydrogels to chondroblasts in addition with10ng/ml MMP-1. After21day, transplanted the mixture to the back of the nude and observed the toxicity, non-transfected group mixture as control. Four weeks later, detected the cartilage formation by Alcian blue staining and immunohistochemistry. The osteoarthritis aminal model of New Zealand White rabbit by MIA injection, and injected the mixture of peptide PRG/TB hydrogels and translated ADSCs to the articular cavity every week, injected PBS as control. Eight weeks later, the cartilage repair was observed by pathological section staining.Results:The cartilage formation of transfected ADSCs mixture was more evident detected by naked-eyed, Alcian blue staining and immunohistochemistry in contrast to the control.The degeneration induced by artificial osteoarthritis was prevented by injection of transfected ADSCs mixture.Conclusions:The mixture of functionalized self-assembling peptide scaffold and LV-mTGF-β3transfected ADSCs can induction differentiation cartilage in vivo and prevent the degeneration induced by osteoarthritis, which would open up prospects for target therapy of cartilage damage repair in future.