| BackgroundArticular cartilage is an important weight-bearing tissue of the human body,which plays a physiological role in buffering joint stress,lubricating joints,etc.Articular cartilage injury caused by sports injury and other reasons is more common in clinical practice,seriously affecting the quality of life of patients.Previous studies have shown that articular cartilage injuries have a high incidence in the population.However,due to the lack of blood supply of articular cartilage,the regeneration and repair ability is very low after injury,and after cartilage injury,the joint is still under great pressure,further aggravating cartilage injury.At present,the treatment methods for articular cartilage injury mainly include: conservative treatment with drugs,arthroscopic debridement,microfracture,autologous chondrocyte transplantation and osteochondral transplantation,but these treatment methods have many defects and are difficult to form a long-term effective repair effect for cartilage injury and cannot regenerate hyaline cartilage.With the deepening of tissue engineering research,the advent of cartilage tissue engineering technology may shed light on cartilage injury repair.Among them,hydrogel materials have strong application potential in cartilage repair.Most materials of hydrogel scaffolds are derived from natural polysaccharides and have good biocompatibility,and the three-dimensional network structure of multiple pores inside hydrogel scaffolds is similar to that of cartilage extracellular matrix,which is conducive to maintaining the proliferation activity of seed cells loaded in hydrogels and providing conditions for nutrient exchange.Stem cells are undifferentiated cells with multilineage differentiation ability and self-renewal ability,which have therapeutic value in the treatment of various clinical diseases.In previous studies,mesenchymal stem cells have been used in the treatment of knee osteoarthritis,which significantly improves joint pain symptoms and joint movement disorders in patients.This suggests that stem cells can also play a huge therapeutic role in osteoarticular diseases.The use of stem cells as seed cells loaded in hydrogels may provide new therapeutic strategies for the regeneration and repair of cartilage injuries.The good biocompatibility of hydrogel can provide favorable conditions for the growth of stem cells,and the chondrogenic differentiation ability of stem cells can promote the regeneration of chondrocytes.PurposeBased on the above research background,chondroitin sulfate and hyaluronic acid were selected as matrix materials in this study to explore the feasibility of using two natural polysaccharide materials and type Ⅱ collagen to construct hydrogel scaffolds,and the pore structure,mechanical properties,swelling rate and biocompatibility of COL Ⅱ-OHA-OCS hydrogels were assessed by relevant tests to investigate the proliferation activity and chondrogenic differentiation ability of stem cells in hydrogels.To study the regeneration and repair effect of COL Ⅱ-OHA-OCS hydrogel scaffold loaded with stem cells on full-thickness cartilage defects by establishing a full-thickness cartilage defect model in piglets,and to provide a basis for further subsequent studies.Section I Preparation and Characterization of Biomimetic Hydrogel Scaffolds Materials and methods1.Oxidation of hyaluronic acid and chondroitin sulfateAppropriate amount of hyaluronic acid and chondroitin sulfate were weighed by electronic balance,oxidized with Na IO4,and oxidized hyaluronic acid(OHA)and oxidized chondroitin sulfate(OCS)were obtained after dialysis and lyophilization.2.Fourier transform infrared spectroscopy(FTIR)OHA and OCS obtained after oxidation were subjected to FTIR to analyze whether aldehyde groups formed upon oxidation for subsequent Schiff base cross-linking reactions3.Hydrogel preparation of different componentsHydrogels constructed from three different compositions were prepared: OHA-CS-COL Ⅱ,HA-OCS-COL Ⅱ,OHA-OCS-COL Ⅱ.4.Hydrogel-associated performance characterizationThe hydrogels constructed from the three different materials were characterized,including compressive properties testing,scanning electron microscopy,swelling rate determination,and in vitro degradation ability.ResultsOHA and OCS were successfully prepared by oxidation of hyaluronic acid and chondroitin sulfate,and FTIR results showed that aldehyde groups were present in both OHA and OCS.Three groups of hydrogels were successfully constructed using materials with different compositions,and the performance characterization results showed that the hydrogels in the OHA-OCS group were superior to the hydrogels in the OHA-CS and HA-OCS groups in compressive resistance,swelling performance,and in vitro degradation performance,which were more suitable for cartilage injury repair.Section Ⅱ Construction of hydrogel loaded with stem cells and co-culture in vitro Materials and methods1.Isolation and identification of human umbilical cord mesenchymal stem cellsFrom neonatal umbilical cord,Walton ’s colloid was isolated,minced and added to culture medium into a cell incubator,and primary umbilical cord mesenchymal stem cells could be obtained after incubation.Umbilical cord mesenchymal stem cells were subsequently sub-cultured and surface antigen identification was performed using flow cytometry.2.Cytocompatibility of hydrogel scaffolds with different componentsAccording to the method,three groups of hydrogels were prepared,and umbilical cord mesenchymal stem cells were added to the hydrogel after disinfection and sterilization to show that the cell survival status in the hydrogel was observed by live dead cell staining after a total of 1 day,4 days,and 7 days of culture in the cell incubator.3.Assessment of the proliferation ability of stem cells in hydrogelsAfter preparing three groups of hydrogels,they were lyophilized and ground into powder,sterilized by irradiation and mixed with stem cell medium,umbilical cord mesenchymal stem cells were added to the prepared medium,and CCK-8 assay was used at 1 day,4 days,and 7 days after culture to assess the proliferation ability of stem cells in hydrogel-containing materials.4.rt-PCR to detect chondro-related gene expression in stem cells in hydrogelsUmbilical cord mesenchymal stem cells(UMSCs)were mixed with three hydrogels and co-cultured in vitro for 14 days.The expression of chondrogenic-related genes COL Ⅱ,ACAN and SOX9 in the hydrogel was detected by rt-PCR.5.Staining of composite hydrogel sectionsUmbilical cord mesenchymal stem cells and three hydrogels were mixed and co-cultured in vitro for 14 days,and the composite hydrogel was sectioned using frozen sections,followed by HE staining and COL Ⅱ immunofluorescence staining.ResultsUmbilical cord mesenchymal stem cells(MSCs)were successfully extracted and isolated from human umbilical cord Walden ’s colloid and showed a long spindle shape.The results of viable dead cells showed that there was no significant difference in the survival of stem cells in the three groups of hydrogels,and all of them could survive effectively;CCK-8 assay showed that there was no significant difference in the proliferation ability of stem cells in the three groups of hydrogels,and different hydrogel materials had no effect on the viability and proliferation of stem cells.The results of PCR showed that COL Ⅱ,ACAN and SOX9 genes were expressed more strongly in stem cells in OHA-OCS group,which was superior to the other two hydrogels,indicating that in OHA-OCS group,the chondrogenic differentiation of stem cells was higher,which was conducive to cartilage regeneration and repair.In the HE staining results of frozen sections of hydrogel,hyperchromatic nuclei were observed in the three groups of composite hydrogels,showing a cell cluster state,and there was no significant difference in staining among the three groups;COL Ⅱ immunofluorescence staining showed that in the OHA-OCS group,fluorescence staining was the strongest,indicating that type Ⅱ collagen was secreted the most,which was conducive to the formation of a microenvironment for chondrocyte regeneration.Section ⅡI Hydrogel scaffolds loaded with stem cells for repair of full-thickness cartilage defectsMaterials and methods1.Selection and grouping of experimental animalsIn this experiment,12 adult Guizhou piglets were selected as experimental animals to prepare full-thickness cartilage defect models at the trochlea of their knees,and all piglets were divided into three groups by random number method,which were blank control(group A),hydrogel repair alone(group B),and hydrogel composite stem cell repair(group C).2.Autologous MSCs extractionMSCs were extracted from the selected Guizhou minipigs by autologous extraction,and the anterior superior iliac spine was selected from the bone marrow puncture site and extracted using stem cell separation solution for cartilage repair loaded in hydrogel.3.Cartilage defect preparation and surgical proceduresIn the medial side of one knee joint of the piglet,an incision of about 5 cm in length was made to expose the trochlea of the knee joint,and two full-thickness cartilage defect models were made perpendicular to the articular surface using a trephine.In group A,no repair measures were taken,in group B,OHA-OCS hydrogel was used for filling repair,and in group C,OHA-OCS hydrogel loaded with autologous MSCs was used to repair cartilage defects;subsequently the joint capsule and skin were sutured layer by layer.4.Evaluation of cartilage defect repair effectAt 6 and 12 weeks after surgery,three groups of piglets were sampled for gross external observation and ICRS scoring,followed by paraffin sections of osteochondral samples for HE staining,fast green safranin O staining,toluidine blue staining,and immunohistochemical staining of type Ⅱ collagen,and the cartilage defect repair effect was analyzed using the O’Driscoll scoring criteria.ResultsIn the knee joint of piglets,the full-thickness cartilage defect model was successfully completed,and the hydrogel loaded with stem cells was significantly better than the hydrogel alone group in the repair of cartilage defects,showing a better integration state in the cartilage defect area;and the hydrogel alone repair effect was better than the blank control,indicating that the supporting effect of hydrogel alone was also conducive to the repair of cartilage defects.ConclusionUsing oxidized hyaluronic acid and chondroitin sulfate,Schiff base can react with type Ⅱ collagen to form a stable cross-linked structure.Among the three hydrogels with different components,the hydrogels in the OHA-OCS group showed more stable physicochemical properties,with higher crosslinking strength,better compressive properties and degradation properties.Umbilical cord mesenchymal stem cells survived and proliferated effectively in all three groups of hydrogels,indicating that the matrix material used to prepare the hydrogel did not have any negative impact on the viability of stem cells.The chondrogenic differentiation ability of stem cells were strongest in the OHA-OCS group,indicating that OHA-OCS material is most suitable for preparing hydrogel scaffold materials.In the repair of full-thickness cartilage defects in piglets,OHA-OCS hydrogel loaded with stem cells showed the best repair effect,indicating that the chondrogenic differentiation of stem cells promoted cartilage defect repair.OHA-OCS hydrogel alone also had a certain repair effect on cartilage defects,indicating that the supporting effect of the hydrogel material itself can repair cartilage damage to a certain extent. |
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