| Background:Cartilage defect on the craniofacial is a common clinical disease in plastic and cosmetic surgery.Common cartilage defects including microtia,nasal deformity,and cartilage defects on eyelid,nose,and ear caused by various traumas.Due to the limited regeneration capacity of chondrocytes,it is difficult to treat cartilage defects.Especially for large cartilage defects,which eventually leads to pain and even deformity.The existing clinical methods for the treatment of cartilage defects are mainly autologous and allogeneic cartilage transplantation.However,due to the limited source of cartilage,large damage,many complications in the donor site,absorption,deformation after transplantation,and immune rejection of allograft,the clinical application of these treatment methods is limited.Thus,tissue engineering cartilage came into being.The process of constructing tissue engineering cartilage is to transfer chondrocytes into biodegradable,good histocompatibility biomaterials to form a chondrocytes-scaffold composite,and then culture in vivo and in vitro to form new composite cartilage to cure cartilage defects.Tissue engineering cartilage has developed rapidly in recent years.As one of the three major elements of tissue engineering,scaffold is a key factor in determining whether tissue engineering cartilage can be successfully constructed.Requirements for scaffold materials not only include the controllable degradation rate,good biocompatibility,and no side effects of metabolites,etc.,also include certain mechanical requirements due to the special role of cartilage in the body,especially on craniofacial.Therefore,it is difficult to achieve this goal with only natural materials at present.Thus,constructing the composite scaffolds with various materials,especially natural materials and synthetic polymer materials,is still a research hotspot in tissue engineering scaffolds.In fact,whether the composite scaffold can be used in clinics mainly depends on the severity of the host’s immune response after its transplantation in vivo.This is also the main problem that currently prevents the clinical application of various tissue-engineered cartilage successfully constructed in vitro.Therefore,a clear understanding of the changes in the immune response caused by the composite scaffold in vivo,especially the stability of its long-term transplantation in vivo,is also a prerequisite for obtaining the ultimate ideal tissue engineering scaffold.Therefore,in this study,we constructed natural degummed silk/silk fibroin/gelatin(S/SF/G)scaffolds with different gelatin concentrations,and degummed silk was used to increase the mechanical strength of the scaffold.At the same time,we incorporated polylactic acid porous microspheres to construct degummed silk/silk fibroin/gelatin/polylactic acid porous microspheres(S/SF/G/PLLAPMs)scaffolds to further increase its mechanical strength,while constructing a better cartilage by taking advantage of its multi-level pores.Also the RGD sequence on gelatin was applied to improve the surface hydrophobicity of polylactic acid.With the help of the IEP effect of gelatin to neutralize the inflammatory stimulus produced by the metabolites of polylactic acid,an ideal scaffold that met the requirements of tissue engineering cartilage scaffolds was finally constructed.We comprehensively evaluated the possibility of constructing tissue engineering cartilage and clinical application of the scaffold through detailed evaluation of the physical and chemical properties of the composite scaffold,cartilage formation in vitro and in vivo,and short-term and long-term immune response after implantation in vivo.Objective:1.To evaluate the physical and chemical properties of S/SF/G and S/SF/G/PLLA PMs scaffolds under different gelatin concentrations.X2.To evaluate the cartilage formation ability of S/SF/G and S/SF/G/PLLA PMs scaffolds under different gelatin concentrations in vitro.3.To evaluate the ability to construct cartilage of S/SF/G and S/SF/G/PLLA PMs scaffolds with different gelatin concentrations in nude mice and pigs.4.To evaluate the short-term and long-term stability of S/SF/G and S/SF/G/PLLA PMs scaffolds in vivo.Methods:Part 1.Characterization of S/SF/G and S/SF/G/PLLA PMs scaffolds1.Light and electron microscopes were used to observe the structure of the scaffolds,including the connection of pores,the size of the pores,and the distribution of microspheres.2.Swelling rate,degradation rate,mechanical properties,and chemical structure were analyzed to clarify the differences in physical and chemical properties between different scaffolds.3.Chondrocytes were isolated and cultured,and the biocompatibility,toxicity,cell adhesion,etc.were tested of the scaffolds to determine the possibility of each scaffold as cartilage scaffold.Part 2.Study on tissue engineering cartilage formation of S/SF/G and S/SF/G/PLLA-PMs composite scaffolds in vitro1.Light and electron microscopes were used to observe the growth of cells on the scaffolds at different culture time.2.DNA quantification kit,hydroxyproline detection kit,DMMB method were applied to detect the secretion of total collagen and glycosaminoglycan of composite cartilage cultured in vitro.3.Real-time PCR was applied to detect the expression of chondrogenesis-related genes in vitro.4.Western Blot and histochemical staining were used to detect the expression of chondrogenesis-related proteins in vitro.Part 3.Study on tissue engineering cartilage formation of S/SF/G and S/SF/G/PLLA-PMs composite scaffolds in vivo1.The scaffold-cell complexes cultured in vitro were transplanted into nude mice and pigs.2.Changes of the scaffold-cell complexes at different times in vivo were observed.3.HE,Safranin O-fast green staining,immunohistochemistry were applied to evaluate cartilage formation.Part 4.Acute and chronic immune response in S/SF/G and S/SF/G/PLLA-PMs scaffold-cell complexes1.The scaffold-cell complexes were transplanted into pigs.XII2.Real-time PCR was used to detect the expression of inflammation-related genes at different times in vivo.3.Immunofluorescence and histochemical staining were used to detect the expression of immune cell surface markers and fibrosis markers at different times in vivo.Results:Part 1.Characterization of S/SF/G and S/SF/G/PLLA-PMs composite scaffolds1.S/SF/G-7% average pore diameter was 307±142um,S/SF/G-9%average pore diameter was 273±167um.The average particle size of PLLA porous microspheres was 100-300μm,with an average of245±35μm,and the pore size was 20-40μm,with an average of27±4.8μm.The pores of the scaffolds were connected and evenly distributed.2.The scaffolds containing PLLA porous microspheres had lower swelling rate,degradation rate,and porosity than those without porous microspheres.With the increase of gelatin concentration,the swelling rate and degradation rate did not change significantly,while the porosity decreased to a certain extent.FTIR analysis showed PLLA chemical groups and β-sheet characteristic peaks.3.The cell compatibility of each scaffolds was good.The presence or absence of microspheres had no significant effect on cell adhesion and proliferation.The ability of cell proliferation and adhesion was improved to a certain extent with the increase of gelatin concentration.Part 2.In vitro study of S/SF/G and S/SF/G/PLLA-PMs composite scaffolds to construct tissue engineering cartilage1.In general observation,cartilage formation got better with time,and the effect of cartilage formation was better with the increase of gelatin concentration in vitro.The presence or absence of porous microspheres had little effect on cartilage formation.2.A large amount of extracellular matrix secreted after scaffoldcell complexes cultured for 4 weeks in vitro,and the presence or absence of microspheres had little effect on collagen and glycosaminoglycans secretion,but the secretion of extracellular matrix and chondrogenesis-related genes and protein expression were significantly promoted with the increase in gelatin concentration.The extracellular matrix secretion of each group of scaffolds in vitro cultured for 8 weeks increased to a certain extent compared with 4weeks,especially S/SF/G/PLLA-PMs-9%.And at 9% gelatin concentration,the extracellular matrix secretion of the composite scaffold containing porous microspheres was more vigorous than that of the group without microspheres.XIVPart 3.Study on tissue engineering cartilage formation of S/SF/G and S/SF/G/PLLA-PMs composite scaffolds in vivoAfter 8 weeks of transplantation in nude mice,the morphology of the four groups of scaffold-cell complexes was intact,histology showed strong positive expression of chondrogenesis-related protein.The scaffolds appeared to a certain degree of contracture 2 weeks after transplantation in pigs,but the expression of extracellular matrix and chondrogenesis-related protein was still present.And the scaffold-cell complexes survived better at 9% gelatin concentration.The scaffolds without PLLA microspheres degraded significantly after 8 weeks in vivo,and the scaffolds containing PLLA microspheres could maintain its integrity,but there was no obvious chondrogenesis marker expression in each group.Part 4.Acute and chronic immune response in S/SF/G and S/SF/G/PLLA-PMs scaffold-cell complexes1.The expression of inflammation-related genes at different times in the body showed that the expression of inflammatory factors in the group containing PLLA microspheres was significantly higher than that in the group without microspheres within 2 weeks of transplantation,and the inflammatory factors in each group were significantly decreased after 2m transplantation in vivo.2.The results of immunofluorescence and immunohistochemical staining showed that the immune response was dominated by neutrophils and macrophages within 1 week,and macrophages for about 1-2 weeks.B lymphocytes cells were also involved after 2 weeks transplantation in vivo.The immunohistochemical results within 2weeks showed that the presence or absence of PLLA porous microspheres had little effect on the types and distribution of immune cells.The expression of fibrosis-related markers appeared at 2 weeks,and the expression gradually increased over time.There was a significant difference in distribution of fibrosis-related markers in the groups with or without microspheres with rarely expressed inside the microspheres.And histochemical results showed that the expression of COL1A1 decreased significantly in the scaffolds with PLLA microspheres.Conclusions:1.S/SF/G and S/SF/G-PLLA-PMs scaffolds have good physical and chemical properties and biocompatibility and can be used as ideal scaffolds for tissue engineering cartilage.2.The addition of PLLA porous microspheres can increase the mechanical properties of the composite scaffold and delay the degradation of the scaffold.3.The higher the gelatin concentration,the better the cartilage formation of the composite scaffold in vitro and in vivo.The 9% gelatin concentration composite scaffold is more suitable as a tissue engineering cartilage scaffold than the 7% gelatin composite scaffold.4.In the early stage of transplantation in vivo,the S/SF/G-PLLAPMs scaffolds have more severe inflammation than the S/SF/G scaffolds.5.S/SF/G-PLLA-PMs scaffold is more stable than S/SF/G after long-term culture in vivo.The existence of PLLA porous microspheres can delay blood vessel invasion and fibrosis after long-term culture in vivo. |
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