Decellularized Cartilage Matrix Hydrogel Encapsulated Urine-derived Stem Cells For Articular Cartilage Repair

Objective:Articular cartilage defects caused by exercise,trauma and degeneration are common diseases in orthopedics.With the emergence of national sports upsurge,the incidence of articular cartilage injury will increase year by year.Once articular cartilage is damaged,due to the lack of blood supply and nerve nutrition,its self-repair ability is limited.In order to delay and avoid osteoarthritis after cartilage injury,timely regeneration and repair of cartilage defect is particularly important.Traditional methods of cartilage defect repair,such as articular cartilage drilling,microfracture and autologous and allogeneic osteochondral transplantation,still have some shortcomings,such as fibrocartilage,donor site complications and immunogenicity.Cartilage tissue engineering technology provides a new idea for the regeneration and repair of cartilage defects.Tissue engineering mainly includes seed cells,scaffold materials and growth factors.Tissue engineered cartilage is a kind of biomaterial composite constructed by seed cells and scaffolds,which is implanted into the cartilage defect in vivo.In the process of material degradation,the new cartilage tissue constantly replaces the biodegradable biomaterials,so as to complete the regeneration and repair of cartilage.In order to achieve the complete regeneration and repair of articular cartilage,appropriate seed cells and scaffold materials are extremely important.In 2008,Zhang et al.firstly reported that urine derived stem cells(USCs)isolated from human urine showed good proliferation and multiple differentiation potential.In recent years,USCs have become the research focus of seed cells in tissue engineering due to its advantages of isolation and extraction from clean midstream urine,strong proliferation ability,and no ethics involved.Some studies have shown that USCs have chondrogenic differentiation potential under specific culture conditions in vitro,and can promote cartilage regeneration and repair in vivo.However,the ability of USCs to repair cartilage defects remains to be further confirmed.Decellularized matrix material has become a hot biomaterial in tissue engineering due to the removal of immunogenic cells and partial retention of extracellular matrix components with biological characteristics.Many kinds of decellularized matrix materials have been reported to be used in tissue engineering.Hydrogel is an ideal material for simulating extracellular matrix because of its rich water content and three-dimensional porous structure.Decellularized matrix hydrogel has the advantage of decellularized matrix material theoretically,and it has the characteristics of hydrogels forming by polymerization and physiological temperature through injections by catheter or syringe.It has been reported that a variety of decellularized matrix materials can promote macrophage polarization to M2 type,and M2 type macrophages can promote the regeneration and repair of damaged tissue.M2 type macrophages can also promote the synthesis and metabolism of cartilage extracellular matrix,and play an important role in the repair of cartilage defects.The aim of this study was to investigate the preparation of injectable decellularized cartilage matrix(DCM)hydrogel and to combine USCs cells to repair cartilage defects of the knee joint,and to explore the effect of DCM hydrogel on macrophage polarization.Therefore,the purpose of this study is as follows:1.The purpose of this study is to extract USCs from urine as seed cells of cartilage tissue engineering to explore their chondrogenic differentiation potential;2.DCM hydrogel was prepared to investigate its characterization,cytocompatibility and chondrogenic induction properties.3.To investigate the role of DCM hydrogel in regulating macrophage polarization.4.To explore the effect of DCM hydrogel composite USCs on repairing articular cartilage defects in rats.Materials and Methods:1.Primary USCs cells were isolated from human urine by centrifugation.The proliferation ability of USCs was determined by CCK-8 method,and the expression of cell surface antigen was detected by flow cytometry.The chondrogenic differentiation potential of USCs was identified by three-dimensional culture,histological staining and fluorescent quantitative PCR.2.Porcine articular cartilage was decellularized by freeze-drying,DNA and RNase digestion,and digested by pepsin under acidic condition.Different concentrations of DCM hydrogels were prepared by adjusting p H,temperature and decellularized articular cartilage powder concentration.The swelling rate of DCM hydrogel was measured by freeze-drying weighing method.Scanning electron microscope(SEM)was used to observe the morphology of the material.Fourier transform infrared spectroscopy(FTIR)was used to observe the functional groups of the materials before and after decellularization,and the gel formation of DCM hydrogel was observed by rheometer.DAPI and he staining were used to observe the condition of articular chondrocytes before and after decellularized,and the changes of DNA content,glycosaminoglycans(GAGs)and hydroxyproline were measured quantitatively.The biocompatibility of the materials was observed by scanning electron microscopy(SEM)and confocal dying.DCM hydrogel composite USCs was used to investigate the chondrogenic induction properties of DCM hydrogels in vitro by histological staining,collagen and GAGs content determination and PCR.3.DCM hydrogel was co-cultured with mouse macrophage cell line(RAW 264.7)transwell,and divided into 5 groups: M0 macrophage control group,M1 macrophage positive control group,M2 macrophage positive control group,DCM hydrogel group and pepsin group.The morphology and skeleton of macrophages in each group were observed by inverted microscope and phage peptide.Immunofluorescence and PCR were used to explore the effect of DCM hydrogel on macrophage polarization in vitro.Subcutaneous injection of DCM hydrogel was used to investigate the regulation of macrophage polarization in vivo.4.The rats were divided into five groups: sham surgery group(simple incision joint capsule),defect group,USCs group,DCM hydrogel group(group DCM),and DCM combined USC group(DCM-USCs group).The effects of DCM hydrogel combined with USCs on repair of full-thickness cartilage defects in rats were investigated by gross observation,histological staining and Wakitani histological score after 6W and 12 W respectively.Results:1.Primary USCs were successfully isolated from human urine.Under the inverted phase contrast microscope,USCs was plastic-adherent and colony growth cells.Most of cells were rice like polygonal cells with regular edges and high refractive index.CCK-8 results showed that USCs had good self-proliferation and replication ability.Flow cytometry showed that USCs expressed mesenchymal stem cell specific antigens CD29,CD44 and CD90,but negative expressed hematopoietic stem cell antigens CD19,CD34 and CD45.ALP staining showed a lot of blue precipitates and alizarin red staining showed a lot of red calcium nodules.Oil red O staining showed that the red staining substance was located in the cytoplasm.After 28 days of three-dimensional spheroidization culture,the formation of spheroids was observed in general.By paraffin embedded section staining,safranin O and Alcian blue staining,extracellular matrix secreted a lot of cartilage specific glycosaminoglycans.Immunohistochemical results showed that a lot of type II collagen and aggrecan were formed.In addition,RT-PCR results showed that the m RNA expressions of COL2A1,ACAN and Sox9 in chondrogenic induction group were significantly higher than those in control group.2.10,20,30,and 40mg/ml concentrations of articular cartilage decellularized matrix hydrogel were successfully prepared,which had an injectable temperature of4 ℃ and could be spontaneously in-situ glued at 37 ℃,the concentration of 30 mg/m L required the shortest time to form the gel,and the gel formation was stable.The swelling rates were 1405.7% ± 189.4%,1451.3% ± 162.3%,1543.0% ± 77.9% and1453.0% ± 107.9%,respectively.There were no significant differences among the groups(P > 0.05).SEM results the collagen fibers in different concentrations of hydrogels were cross-linked into a porous network structure,and the concentration of DCM hydrogel increased,and the structure became denser.The results of FTIR showed that there was no significant change in the structure of articular cartilage before and after decellularization.The results of rheology show that the storage modulus is greater than the loss modulus at 33 ℃.DNA quantitation showed that the DNA content was significantly reduced to 23.5 ng / mg after decellularization.He and DAPI staining showed that decellularization was successful.The content of collagen before and after decellularized cells was 233.5 + 15.29 ng/mg vs 259.1 + 12.45 ng/mg(P > 0.05).The GAGs content before and after decellularized cells was 15.38 + 2.22ng/mg vs 65.53 65.53 2.10 2.10(P <0.0001).The results of DCM hydrogel scanning electron microscopy and living staining showed that the DCM hydrogel had good cytocompatibility.USCs was cultured in DCM hydrogel for 28 days.HE and toluidine blue staining showed that chondrocytes could induce cartilage formation in normal culture medium and chondrogenic induction medium,similar to typical chondrocytes and cartilage lacuna like structures.However,the quantity of collagen and gags and the m RNA expression of COL2A1,ACAN and SOX9 in chondrogenic induction medium group were significantly higher than those in conventional medium group.3.Inverted phase contrast microscope and phallic peptide staining showed that macrophages in DCM hydrogel group showed long fusiform M2 macrophage morphology.Immunofluorescence showed that compared with control group,CD206 expression in DCM hydrogel group increased significantly,while i NOS expression did not significantly changed.RT-PCR results showed that the DCM hydrogel group could significantly increase the expression of CD206 and ARG-1(P < 0.05),and had no significant effect on the expression of i NOS and TNF-α.CD86 and CD206 staining of subcutaneous injection of DCM hydrogel showed that the macrophages were mainly M1 type on the 7 day after operation,and 14 days after the operation.4.Gross observation showed that 12 weeks after operation,obvious defects could still be seen in the model group.Partial repair of defects could be seen in the simple USCs and DCM groups,which was significantly improved compared with 6 weeks.The defects in DCM-USCs group were repaired better,and the defects were filled with new hyaline cartilage.In DCM-USCs group,12 weeks after operation,the defect was repaired with smooth and intact tissue surface and good cartilage repair.He,safranin O,toluidine blue staining and aggrecan,II,I collagen immunohistochemical results showed that compared with the model group,USCs group,DCM group and DCM-USCs group could partially repair cartilage defects,among which DCM-USCs group had the best repair effect,DCM-USCs group showed weak positive staining for type I collagen and strong positive staining for type II collagen,indicating that the new repair tissue was hyaline cartilage like tissue.Wakitani histological score showed that 12 weeks after operation,USCs group,DCM group and DCM-USCs were lower than those in the model group,and DCM-USCs group had the lowest score and good repair effect.Conclusion:1.USCsis a kind of mesenchymal stem cells with abundant sources,simple materials and chondrogenic differentiation potential,which can be used as seed cells for cartilage tissue engineering.2.DCM hydrogel retained some extracellular matrix components of articular cartilage,and had good biocompatibility and good chondrogenic induction properties.It could be used as scaffold for cartilage tissue engineering.3.in vitro and in vivo,DCM hydrogel can modulate the polarization of macrophages to anti-inflammatory M2 macrophages,which may contribute to the regeneration and repair of tissues.4.the use of USCs alone,DCM hydrogel alone and DCM hydrogel combined with USCs can promote the regeneration and repair of rat knee cartilage defects.Among them,DCM hydrogel combined with USCs has the best repair effect,and hyaline cartilage like tissue can be observed in cartilage defects.