Therapeutic Effects Of GDF-5Gene Transfected Adipose-derived Stem Cells Loaded In Thermosensitive Collagen/Hydrogels In Rat Disc Degeneration Models

Part. ⅠEstablishing and purifying GDF-5gene over-expressed rat ASCsObjective:To explore the optimal multiplicity of infection (MOI) of lentivirus-mediated GDF-5gene over-expressed rat adipose stem cells (ASCs-GDF5) and obtain high-purified ASCs-GDF5using flow cytometry (FCM).Methods:Rat ASCs were isolated and cultured using collagenase digestion method. The cells morphology was observed and growth curve was tested. Bseides, cell phenotypes were identified. Lentiviral vector system with GDF-5/GFP chimeric gene was prepared and the transfection efficiency was explored under series of MOI (=1,5,10,20,40,60,80,100). The optimal MOI was determined and transfection efficiency was tested using FCM. High-purified ASCs-GDF-5were obtained through fluorescence activated cells sorting with FCM. The positive rate of transfected cells was verified further with DAPI staining. The viability of cells transfected and non-transfected was evaluated with CCK-8assay.Results:Rat ASCs were successfully isolated and cultured. The markers (CD90, CD29, CD44, CD105), expressed in mesenchymal stem cells, were positive in cultured cells, while the hematopoietic cells surface antigen (CD45, CD34) and bone marrow stem cells surface antigen (CD106) were negative. GDF-5gene over-expressed lentiviral vector system was successfully constructed. The optimal MOI was40, with a transfection rate of65%. The positive rate of transfected cells was improved to96%through fluorescence activated cells sorting using FCM. There was no significant decrease in the rate of positive cells after3passages in vitro. There was no significant difference in the viability and growth curve between transfected and non-transfected cells with CCK-8assay.Conclusion:The optimal MOI of GDF-5gene transfected rat ASCs, mediated by lentivirus, is40with a transfection rate of65%. The positive rate of transfected cells can be significantly improved through fluorescence activated cells sorting using FCM, without significant effect on cell viability. Part. IIThe identification of GDF-5gene over-expressed rat ASCs and assessment of the differentiation towards nucleus pulposus-like cells.Objective:To assess the validity of GDF-5gene transfected rat ASCs expressing GDF-5gene and protein. To evaluate its differentiation towards the nucleus pulposus-like cells in vitro.Methods:GDF-5gene transfected ASCs were collected. Gene and protein expression of GDF-5was tested wiht Real-time PCR and Western Blot analysis. The content of GDF-5in culture supernatant was detected with Elisa method. After cells was cultured without any additions of inducers for7days in vitro, the gene and protein expression of pulposus-like cell markers (Sox9, Collagen II, Aggrecan) was tested with Real-time PCR and Western Blot respectively. Some cells were seeded in the slides for two days and immunofluorescence stains was performed to detect the expression of Sox9, Collagen Ⅱ, Aggrecan and observe their intracellular localization.Results:The gene and protein levels of GDF-5were significantly increased in transfection group, compared with blank control group (P<0.001). The GDF-5content in culture supernatants was (151.7±33.2) ng/ml, which was significantly higher than the control group (P<0.001). Real-time PCR and Western Blot results showed the gene and protein expression levels of Sox9, Collagen II, Aggrecan in GDF-5transfected group were significantly increased than the black control group after cultured for7days in vitro (P<0.001). Immunofluorescence results indicated that the protein of Sox9, Collagen Ⅱ, Aggrecan was strongly expressed in GDF-5over-expressed ASCs, with Sox9mainly expressed in the nucleus, Collagen II mainly expressed in the cytoplasm and Aggrecan mainly expressed in both the nucleus and the cytoplasm.Conclusion:The gene and protein of GDF-5expressed strongly in GDF-5gene transfected rat ASCs and GDF-5protein can be secreted into the extracellular supernatant. The GDF-5gene over-expressed ASCs can differentiate towards nucleus pulposus-like cells on gene and protein levels, without adding inducers. Part.ⅢSynthesis of collagen modified thermosensitive hydrogels and and biocompatibility assessmentObjective:To synthesize a thermosensitive hydrogel with type II collagen modification and assess the characterization and biocompatibility.Methods:Triblock copolymer PCLA-PEG-PCLA hydrogels were synthesized via ring-opening polymerization and then was modified with0.1%(m/v) type II collagen. The modified hydrogels were sterilized with cobalt-60irradiation and molecular weight was measured using1H-nuclear magnetic resonance (1H-NMR) spectrum and size exclusive chromatography. The temperature of sol-gel transition was determined with tube inversion and rheometer method. The pH changes of hydrogels degradation in PBS were measured and morphology was observed. Viability of cells loaded in hydrogels was tested with MTT assay. FCM Annexin V/PI was performed to determine whether the medium with collagen/hydrogels would cause cells apoptosis or death. Hydrogels were injected into rats subcutaneously to assess the histocompatibility.Results:Triblock copolymer PCLA-PEG-PCLA hydrogels were successfully synthesized via the ring-opening polymerization. There was no significant difference in molecular weight of hydrogels after Cobalt60irradiation sterilization. Both the irradiation sterilization and collagen modification had no effect on temperature of sol-gel precipitation transition. MTT results showed that the cell viability was significantly increased in collagen/hydrogels than in hydrogels alone. The pH was neutral and morphology of collagen modified hydrogels was maintained better than simple hydrogels during degradation in PBS in vitro. The culture medium containing collagen modified hydrogels did not cause cells apoptosis or death according to FCM Annexin V/PI results. HE stains of subcutaneous implantation showed the collagen modified hydrogels had excellent histocompatibility in vivo.Conclusion:The viability of cells loaded in collagen/hydrogels was significantly higher than hydrogels alone. The collagen modification had no significant effect on temperature of sol-gel precipitation transition. Collagen modified hydrogels are nontoxic to cells and have excellent histocompatibility in vivo. Part. IVRepair of rat intervertebral disc degeneration with GDF-5gene over-expressed ASCs loaded in collagen modified thermosensitive hydrogelsObjective:To assess the therapeutic effects of GDF-5transfected adipose-derived stem cells loaded in thermosensitive collagen/hydrogels in rat disc degeneration modelsMethods:Co5/Co6、Co7/Co8、Co8/Co9discs were punctured from annulus fibrosus to the nucleus pulposus center under X-ray fluoroscopy with21G puncture needles, leaving Co6/Co7discs as normal controls. After puncture,2μl PBS was injected into Co5/Co6discs as negative controls (PBS Group). According to different treatments of Co7/Co8, Co8/Co9discs, all of the rats were divided into groups as follows:(1) ASCs-GDF5+Col/Gel Group:collagen modified hydrogels containing GDF-5gene transfected ASCs;(2) ASCs-GDF5+Gel Group:hydrogels containing GDF-5gene transfected ASCs;(3) ASCs-GDF5+PBS Group:PBS containing GDF-5gene transfected rat ASCs;(4) ASCs+Col/Gel Group:collagen modified hydrogels containing ASCs;(5) ASCs+Gel Group:hydrogels containing ASCs;(6) ASCs+PBS Group:PBS containing ASCs;(7)Col/Gel Group:collagen modified hydrogel alone (8) Gel Group:hydrogel alone. All the injection volume was2μl. Frozen sections were made to observe the GFP-labeling cells with laser confocal microscopy every two weeks after transplantation to determine the survival of the cells implanted. At30days,60days and90days, radiologic examination was performed on rat caudal vertebra to calculate the disc high index(DHI(%)) in each group, and HE stain, safranin O-fast green stain and toluidine blue stain were used to assess the histological changes.Results:The GFP-labeling cells can be observed at4weeks after transplantation, with morphologic changes towards nucleus pulposus-like cells. The DHI(%) of ASCs-GDF5+Col/Gel Group, ASCs-GDF5+PBS Group, ASCs+Col/Gel Group was significantly higher than the PBS Group (P<0.05) at30days,60days and90days after implantation. Furthermore, the DHI(%) of ASCs-GDF5+Gel Group and ASCs+PBS Group was also significantly increased than that of PBS Group (P<0.05) at90days after treatment. The DHI(%) of ASCs-GDF5+Col/Gel Group and ASCs+Col/Gel Group was significantly higher at90days than at30day after implantation (P<0.05). There was no significant change of the DHI(%) among the three time points in ASCs-GDF5+Gel Group, ASCs-GDF5+PBS Group and ASCs+PBS Group (P>0.05). However, the DHI(%) was significantly decreased during the three time points follow-up in ASCs+Gel Group, Col/Gel Group, Gel Group and PBS Group (P<0.05).Intervertebral disc degeneration models in PBS Group were well established at30days after needle puncture. According to histological grading scores (HE stain) of AF and NP, the scores of ASCs-GDF5+Col/Gel Group, ASCs-GDF5+Gel Group, ASCs-GDF5+PBS Group and ASCs+Col/Gel Group were significantly lower than that of negative control PBS Group at the three time points(P<0.05). Moreover, the scores of ASCs-GDF5+Col/Gel Group were significantly lower than other groups (P<0.05) at each time point and maintained well during the whole follow-up (P>0.05), while other groups got higher scores at90days than at30day after implantation (P<0.05). All discs included in this study did not demonstrate any histological features of inflammatory cell infiltration, both acute and chronic.Conclusion:GDF-5gene over-expressed rat ASCs, which were loaded in type Ⅱ collagen modified PCLA-PEG-PCLA hydrogels, can survive and differentiate toward nucleus pulposus-like cells morphologically. Both the ASCs loaded in collagen modified hydrogels and the GDF-5gene over-expressed ASCs can reverse or prevent the rat caudal disc height loss and improve the disc degeneration histologically. GDF-5gene over-expressed ASCs loaded in collagen modified hydrogels can achieve the best therapeutic results among all the groups.