Preparation Of Chondrocyte-differential Hydrogel Scaffold And Its Application For Nasal Septum Defect

Backgroud: Perforation of the nasal septum can lead to structural and functional disorders of the nasal cavity,characterized by symptoms such as pain,epistaxis,and even chronic sinusitis.The clinical treatment for nasal septal perforation is mainly flap repair.If the perforation area is large and the stability of the cartilage supporting structure is reduced,the nasal septum may deviated,and even the external nasal deformity will occur in severe cases.Therefore,it is necessary to restore the support performance of the nasal septum.But the septal cartilage is a special connective tissue without nerves,blood vessels and lymphatic vessels,and has a relatively low cell density(composed of about 1%chondrocytes and 99% extracellular matrix),so its self-repairing ability is very weak,resulting to a lack of effective treatments.Bone marrow mesenchymal stem cells(BMSCs)Cells with different differentiation ability can differentiate into muscle cells,nerve cells,osteoblasts,chondrocytes,etc.TGF-β1 can promote the chondrogenic differentiation ability of BMSCs.Gelatin Methacrylate(GelMA)hydrogel has good biocompatibility and stiffness adjustability.The biological function and mechanical stiffness of GelMA hydrogel can provide a suitable living microenvironment for a variety of cells.Based on the properties of GelMA and the characteristics of BMSCs,this study applied the application of TGF-β1-loaded GelMA(GelMA-T)hydrogel loaded with BMSCs to repair nasal septal cartilage defects.Objective: To investigate the repair effect of GelMA-T hydrogel-loaded BMSCs on cartilage defect of nasal septumMethods: According to the content of GelMA,GelMA-T hydrogels with concentrations of 5%,10% and 15% were prepared,and the rheological properties,mechanical properties,degradation and release properties of GelMA-T hydrogels with different concentrations were measured.BMSCs from the bone marrow of rabbits were cultured,isolated and identified.Cell culture was carried out on the GelMA-T hydrogel or with the extract of GelMA-T hydrogel.The cytotoxicity and adhesion of GelMA-T hydrogel were observed on the 1st,3rd,and 7th days.GelMA hydrogels with different concentrations were used as controls.BMSCs were cultured on GelMA-T hydrogels of different concentrations for 21 days.Then the cells were subjected to Sox9 immunofluorescence staining for Sox9 and q RT-PCR for Col2,Sox9,and ACAN gene expression to screen out the optimal material concentration.A rabbit model of nasal septal defect was established,and the GelMA-T hydrogel loaded with BMSCs was implanted into the defect site.The nasal septum of experimental animals was taken out at 4,12,and 24 weeks after surgery,and histological sections were stained with hematoxylin & eosin,toluidine blue and Safranin O staining was used to observe the repair of nasal septal defect in animals.Results: The storage moduli(G’)of the 5%,10% and 15% GelMA-T hydrogels at 1Hz were 574.0,3564.6 and 12030.3 Pa respectively.And the dissipation moduli(G’’)were20.4,147.2 and 248.8 Pa.And the loss factors(G’’/G’)are 0.04,0.04 and 0.02 respectively.The storage moduli of the three materials are higher than the energy dissipation moduli,indicating that the three materials are all in the viscoelastic solid state.The compressive elastic moduli of 5%,10% and 15% GelMA-T hydrogels were 11.0 ± 0.6,54.7 ± 1.6 and122.6 ± 2.7 k Pa respectively.The above results show that with the increase of GelMA concentration the stiffness(storage modulus/elastic modulus)of the material also increases,indicating that the stiffness of GelMA-T can be regulated by adusting the composition of the material.At day 28,the mass loss rates of 5%,10% and 15% GelMA-T hydrogels were91.0%,77.1% and 55.3% respectively.Finally,the cumulative release amounts of 5%,10%and 15% GelMA-T hydrogels were about 30%,70% and 90%,which could meet the concentration requirements for TGF-β1 to play a good differentiation performance.The in vitro cytological experiments of 5%,10% and 15% GelMA-T hydrogels indicated that GelMA-T hydrogels at various concentrations had low cytotoxicity and good biocompatibility.Its immunofluorescence and q RT-PCR experiments showed that both 5%,10% and 15% pure GelMA hydrogels and GelMA-T hydrogels have the ability to induce chondrogenic differentiation of BMSCs,but GelMA-T hydrogels have the ability to induce chondrogenic differentiation.The induction effect was significantly stronger than that of pure GelMA hydrogel,and the expression of cartilage-related genes was up-regulated more.GelMA hydrogel group also had the best induction effect at the concentration of 10%,indicating that the elastic modulus at this concentration is suitable for BMSCs to differentiate into cartilage.The best induced differentiation 10% GelMA-T hydrogel group screened in vitro and the 10% GelMA hydrogel group as a control loaded with the same amount of BMSCs were implanted into the defect of the nasal septum for 4,12,and 24 weeks observation.The results showed that,consistent with the results of in vitro culture,the 10% GelMA-T hydrogel group repaired the cartilage defect of the nasal septum better.The addition of TGF-β1 made the cell distribution more uniform,the cell matrix more uniform,and the overall shape of the nasal septum.Intact,material groups all showed normal septal morphology compared to the severe septal deviation in the simple defect group.Conclusion: The GelMA-T hydrogel developed in this research exhibits excellent biocompatibility and effectively induces cartilage differentiation of BMSCs.10%GelMA-T hydrogel has the best effect on promoting cartilage differentiation,leading to improved cartilage defect repair and enabling physiological and structural restoration of nasal septal perforation..