Study Of Modeling Early Traumatic Changes Using Engineered Cartilage Based On Stem Cells And Hydrogel Scaffolds

Background: Post-traumatic osteoarthritis(PTOA)is mainly characterized by cartilage degeneration,which is secondary to joint trauma.It is of great significance to intervene PTOA at early stage but there is no effective approach to prevent the onset and development of PTOA.While animal-based systems have been instrumental in understanding pathogenic progression of PTOA,the difference between human and animal make it difficult to apply the discoveries in animal to human.The limited progress in the development of PTOA disease-modifying medications may be due to insufficient models for disease and therapeutic modeling.3D models have advantages of 2D cell models such as high reproducibility and controllable conditions and similar cell microenvironments with in vivo experiment.In this study,we are developing engineered cartilage constructs based on adult human mesenchymal stromal cells and methacrylated gelatin hydrogel and examining the effects of traumatic impacts on engineered cartilage constructs.We speculate that engineered cartilage will respond to traumatic impacts in a manner congruent with early PTOA pathogenesis.Purpose:(1)To develop an PTOA model using traumatic impact and engineered cartilage based on adult human mesenchymal stromal cells and methacrylated gelatin hydrogel.(2)To assess the phenotype of the PTOA models developed.(3)To test therapeutic effect of Urolithin A to PTOA on our in vitro PTOA models.Methods:(1)We encapsulated h BM-MSCs into photocrosslinked methacrylated gelatin hydrogel to make in vitro models.Hydrogel constructs differentiated in chondrogenic medium for 28 days and we analyzed the chondrogenic biomarkers and ECM.(2)We designed an impact system for hydrogels to deliver impact of different strains to hydrogel constructs.And we analyzed the survival of cells in hydrogels and elastic modulus of hydrogels vertically after impacts.(3)We treated engineered cartilage with traumatic impact or IL-1β and assessed survival and metabolic activities,biomechanical property,gene expression,production and destruction of extracellular matrix and activation of metalloproteinase.(4)We tested therapeutic effects of urolithin A for osteoarthritis using our in vitro PTOA model.We assessed osteoarthritisrelated genes and chondrocyte extracellular matrix between urolithin A treated engineered cartilage and impacted engineered cartilage.Results:(1)Compared with the undifferentiated group,the expressions of chondrogenic genes(COL2A1,SOX9,ACAN)in the engineered cartilage after 21 days of differentiation in chondrogenic medium were significantly up-regulated(p<0.05).In the undifferentiated hydrogel constructs,the percentages of cells with positive Safranin O/ fast green staining and cells with positive COL2 immunohistochemical staining were 0.00%,and in the engineered cartilage,the percentage of cells with positive Safranin O/ fast green staining was 33.52%(p<0.0001),the percentage of cells with positive COL2 immunohistochemical staining was11.48%(p<0.001).The vertical elastic modulus of the acellular hydrogel structures was significantly lower than that of the engineered cartilage(p<0.0001).(2)Compared with the control group,the percentage of surviving cells in the impacted hydrogel structures with cells after impact of 10% strain did not change significantly,while the percentage of surviving cells in the impact hydrogel structures with cells after impact of20% strain and 30% strain significantly decreased(p<0.05).The percentage of surviving cells in the hydrogel structures with cells decreased by 20% to 30% after impact of 30% strain(p<0.001).Compared with the control group,the vertical elastic moduli of cell-free hydrogel structures did not change significantly after impacts of 10% strain group,20% strain and 30% strain,while compared with the control group,the vertical elastic moduli of engineered cartilage constructs after impact of 10% strain did not change significantly but the vertical elastic modulus of engineered cartilage constructs after impacts of 20% strain and 30% strain decreased significantly(p<0.01).(3)After treatments of traumatic impact sand IL-1β,compared with control group,absorbances of CCK8 assay of engineered cartilage constructs significantly decreased(p<0.001),chondrogenic genes(COL2A1,ACAN)were significantly down-regulated(p<0.01),the percentages of cells with positive Safranin O/ fast green staining and cells with positive COL2 immunohistochemical staining in paraffin sections of engineered cartilage constructs decreased significantly(p<0.05),matrix metalloproteinases(MMP-13,ADAMTS-4)were significantly upregulated(p<0.05),and inflammatory genes(INOS,PTGS2)were significantly up-regulated(p<0.05).(4)Compared with the untreated group,the expression of COL2A1 was increased and the expression of ADAMTS-5,IL-8 and P16 was decreased in the traumatic engineered cartilage constructs after urolithin A treatment.The percentages of cells with positive Safranin O/ fast green staining and cells with positive COL2 immunohistochemical staining in paraffin sections of engineered cartilage constructs increased significantly(p<0.05).Conclusions:(1)Engineered cartilage constructs can mimic human cartilage tissue in vivo to some extent,which are ready for modeling PTOA.(2)The impact system for hydrogels is able to deliver impacts of different strains and protect hydrogel constructs from being crushed.Impact of 30%strain is able to induce traumatic changes.(3)Traumatic impacts to engineered cartilage induced PTOA-like changes and compared to IL-1βtreatment,PTOA models induced by traumatic impact can mimic onset of OA better.(4)Urolithin A attenuates engineered cartilage PTOA-like changes induced by traumatic impacts and it proved that our model of PTOA can involve in development of disease-modifying drugs.