Experimental Study On The Regeneration Of Annulus Fibrosus Using DAFM/PECUU Blended Electrospun Scaffold Based On Controllable Elastic Modulus

Part ? The preparation of DAFM/PECUU blended electrospun scaffold and the study of DAFM on the anabolism of rabbit annulus fibrosus-derived stem cells Purpose:To fabricate the decellularized annulus fibrosus matrix(DAFM).DAFM/poly(ether carbonate urethane)urea(PECUU)blended fibrous scaffold was manufactured by coaxial electrospinning technology.Besides,we also investigate whether DAFM could promote the expression of annulus fibrosus anabolism related genes,and to provide reference for the selection of scaffold of annulus fibrosus tissue engineering.Methods:The DAFM was prepared by enzymatic hydrolysis from porcine annulus fibrosus.It was characterized by fourier transform infrared reflection(FTIR)and scanning electron microscope(SEM)in terms of the main components and microstructure.PECUU fibrous scaffolds were prepared by electrospinning,and DAFM/PECUU blended fibrous scaffolds were prepared by coaxial electrospinning technology.Both two kinds of scaffolds were tested by transmission electron microscope and scanning electron microscope,contact angle test and mechanical test.BET test was used to detect the specific surface area,pore size and pore volume of the two kinds of scaffolds.Rabbit annulus fibrosus-derived stem cells(AFSCs)were cultured on PECUU fibrous scaffolds and DAFM/PECUU blended fibrous scaffolds,and cytoskeleton staining and scanning electron microscopy were used for cell morphology observation.The proliferation ability of AFSCs on the two kinds of scaffolds was detected by MTS assay.Quantitative reverse transcription PCR(RT-qPCR)was performed to analyze the AF anabolism related gene expression of Collagen type ?(Col-?),Collagen type ?(Col-?)and Aggrecan and inflammatory mediator genes such as Interleukin-6(IL-6)and Tumor necrosis factor(TNF)-? of AFSCs on the two kinds of scaffolds.The enzyme linked immunosorbent assay(ELISA)method was used to determine the secretion of AF-related extracellular matrix such as Col-?,Col-?,Aggrecan and glycosaminoglycan(GAG)and inflammatory mediator synthesis(IL-6 and TNF-?)of AFSCs on the two kinds of scaffolds.The PECUU fibrous scaffolds and the DAFM/PECUU blended fibrous scaffolds were implanted into the back muscles of rats.Four weeks later,the scaffolds were removed from the rats and the hematoxylin-eosin staining(HE)staining was performed to observe inflammatory reaction of the two kinds of scaffolds in vivo.Results:PECUU fibrous scaffolds and DAFM/PECUU blended fibrous scaffolds were prepared by electrospinning technology.Transmission electron microscopy showed that the inner layer of DAFM/PECUU blended fibrous scaffolds was PECUU and the outer layer was DAFM.Scanning electron microscopy results showed that both kinds of scaffolds had high specific surface area.The diameter of electrospun fiber was between 0.2 ?m to 1.6 ?m,and most of the fiber diameter were between 400 nm and 600 nm.The mechanical test results showed that the tensile modulus of PECUU fibrous scaffolds and DAFM/PECUU blended fibrous scaffolds were 2.23±0.23 MPa and 1.51±0.14 MPa,respectively.The difference was statistically significant(P<0.05).The specific surface area,pore size and pore volume of PECUU fibrous scaffolds were 28.59 m2/g,32.55 nm and 0.058 cm3/g,respectively.The specific surface area,pore size and pore volume of DAFM/PECUU blended fibrous scaffolds were 27.42 m2/g,34.71 nm and 0.049 cm3/g,respectively.The contact angle test showed that the PECUU fibrous scaffolds was 107.0°±6.9°,which was significantly higher than the contact angle of the DAFM/PECUU blended fibrous scaffolds,namely 29.2°±7.4°,and the difference was statistically significant(P<0.05).The results of cytoskeleton staining and scanning electron microscopy showed that AFSCs adhered well on the two kinds of scaffolds.MTS results indicated that AFSCs proliferated well on the two kinds of scaffolds.RT-qPCR results showed that the AF anabolism related gene expression of AFSCs such as Col-?,Col-? and Aggrecan on the DAFM/PECUU blended fibrous scaffolds were significantly higher than those of the PECUU fibrous scaffolds.The expression of inflammatory mediator related genes(IL-6 and TNF-?)of AFSCs on the DAFM/PECUU blended fibrous scaffolds was significantly lower than that of the PECUU fibrous scaffolds.ELISA results showed that the Col-?,Col-?5 Aggrecan and GAG secreted by the AFSCs on the DAFM/PECUU blended fibrous scaffolds were 5.45±0.06 ng/?g DNA,6.35±0.29 ng/?g DNA,and 595.02±46.34 pg/?g DNA and 5.10±0.33 ?g/?g DNA,respectively,which were significantly higher than those on PECUU fibrous scaffolds,namely 3.81±0.17 ng/?g DNA,4.32±0.22 ng/?g DNA,411.82±6.84 pg/?g DNA and 3.79±0.20 ?g/?g DNA.TNF-? and IL-6 secreted by AFSCs on DAFM/PECUU blended fibrous scaffolds were 121.60±1.72 pg/?g DNA and 28.70±1.58 pg/?g DNA,respectively,which were significantly lower than 165.00±5.02 pg/?g DNA and 39.45±3.31 pg/?g DNA inPECUU fibrous scaffolds.The difference was statistically significant(P<0.05).HE staining showed that the number of inflammatory cells around DAFM/PECUU blended fibrous scaffolds was significantly less than PECUU fibrous scaffolds.Conclusion:The DAFM/PECUU blended fibrous scaffolds with high hydrophilicity and specific surface area were successfully manufactured.AFSCs on DAFM/PECUU blended fibrous scaffolds expressed higher AF anabolism related genes and secreted more AF-related extracellular matrix than those on PECUU fibrous scaffolds.The inflammatory response of DAFM/PECUU blended fibrous scaffolds implanted in vivo was lighter than that of PECUU fibrous scaffolds.These results indicate that DAFM could promote AFSCs to express higher levels of AF anabolism related genes and generate more extracellular matrix.At the same time,DAFM/PECUU blended fibrous scaffolds could simulate the matrix composition of actual annulus fibrosus and may have broad application prospects as scaffold materials for annulus fibrosus tissue engineering.Part ?:Experimental study of DAFM/PECUU blended fibrous scaffolds with different elastic modulus on annulus fibrosus regenerationPurpose:The DAFM/PECUU blended fibrous scaffold with different elastic modulus were prepared by coaxial electrospinning technology using the DAFM and PECUU with different elastic modulus to simulate the gradient difference of elastic modulus of the inner and outer regions of the actual annulus fibrosus.Besides,we also investigate the effect of the elastic modulus of the scaffolds on the differentiation of rabbit AFSCs,as well as the role of implantation in the repair of annulus fibrosus defects,and provide a certain reference for annulus fibrosus tissue engineering.Methods:Coaxial electrospinning technology was used to prepare DAFM/PECUU blended fibrous scaffolds with different elastic modulus,and the transmission electron microscopy,scanning electron microscopy,contact angle test and the mechanical test of the two kinds of scaffolds were carried out.Rabbit AFSCs were cultured on DAFM/PECUU blended fibrous scaffolds with different elastic modulus,cytoskeleton staining and scanning electron microscopy were used to observe cell morphology.MTS assay was used to test the proliferation ability of AFSCs on the two kinds of scaffolds.RT-qPCR was used analyze the expression of AF anabolism related genes(Col-?,Col-? and Aggrecan)and inflammatory mediator genes(TNF-? and IL-6).ELISA method was used to determine AF-related extracellular matrix such as Col-?,Col-?,Aggrecan and GAG of AFSCs cultured on the two kinds of scaffolds.DAFM/PECUU blended fibrous scaffolds with different elastic modulus were implanted subcutaneously in rats for 3 months,and the degradation of the two kinds of scaffolds was observed by HE staining at 1 month and 3 months after implantation.At the same time,DAFM/PECUU blended fibrous scaffolds with different elastic modulus were implanted into the annulus fibrosus defect of the rat caudal intervertebral disc.Magnetic resonance imaging was used to observe the height of the rat caudal intervertebral disc and the signal of the intervertebral disc.HE staining and immunohistochemical staining were used to observe the repair of annulus fibrosus.Results:DAFM/PECUU blended fibrous scaffolds with different elastic modulus were manufactured,which were defined as DAFM/PECUU1 blended fibrous scaffolds and DAFM/PECUU2 blended fibrous scaffolds.The mechanical test results showed that the tensile modulus of DAFM/PECUU 1 and DAFM/PECUU2 blended fibrous scaffolds was 1.26±0.25 MPa and 2.53±0.12 MPa,respectively.The difference was statistically significant(P<0.05).Transmission electron microscopy showed that the inner layer of the scaffolds was PECUU,and the outer layer was DAFM.The contact angle test showed that the contact angle of the DAFM/PECUU1 and DAFM/PECUU2 blended fibrous scaffolds were 34.6°±5.3 °and 35.1°±7.0°,respectively.The difference was not statistically significant(P>0.05).The results of cytoskeleton staining and scanning electron microscopy showed that AFSCs adhered well on the two kinds of scaffolds.MTS results showed that AFSCs proliferated well on the two kinds of scaffolds.RT-qPCR results showed that the expression of Col-I gene of AFSCs on DAFM/PECUU1 blended fibrous scaffolds was significantly lower than that of DAFM/PECUU2 blended fibrous scaffolds;while the gene expression of Col-? and Aggrecan were significantly higher than that of DAFM/PECUU2 blended fibrous scaffolds.The difference was statistically significant(P<0.05).The expression of TNF-? and IL-6 in DAFM/PECUU 1 and DAFM/PECUU2 blended fibrous scaffolds was similar,the difference was not statistically significant(P>0.05).ELISA results showed that the Col-? protein secreted by the AFSCs on the DAFM/PECUU1 blended fibrous scaffolds was 4.72±0.11 ng/?g DNA,which was significantly lower than the 6.22±0.27 ng/?g DNA in the DAFM/PECUU2 blended fibrous scaffolds;while the Col-? protein,Aggrecan and GAG were 5.58±0.09 ng/?g DNA,535.67±21.01 pg/?g DNA and 5.17±0.25 ?g/?g DNA,respectively,which were significantly higher than the DAFM/PECUU2 blended fibrous scaffolds of 3.82±0.22 ng/?g DNA,403.84 ± 13.32 ng/?g DNA and 3.65±0.21 ?g/?g DNA.There were statistically significant difference(P<0.05).TNF-? and IL-6 secreted by AFSCs on DAFM/PECUU1 blended fibrous scaffolds were 123.96±2.69 pg/?g DNA and 30.78±1.19 pg/pg DNA,respectively,while TNF-? and IL-6 secreted on AFSCs on DAFM/PECUU2 blended fibrous scaffolds were 120.28±2.13 pg/?g DNA and 30.30±1.08 pg/?g DNA,respectively,with no significant difference(P>0.05).HE staining of DAFM/PECUU1 and DAFM/PECUU2 blended fibrous scaffolds implanted subcutaneously of rats showed that there were few inflammatory cells around the two kinds of scaffolds,and both kinds of scaffolds were degraded after three months.Three months after the DAFM/PECUU1 and DAFM/PECUU2 blended fibrous scaffolds implanted into the annulus fibrous defect of the rats,the magnetic resonance imaging showed that disc height of the annulus fibrous defect group was significantly lost,and the signal of the intervertebral disc was significantly changed.While the height of the intervertebral space in the DAFM/PECUU1 and DAFM/PECUU2 blended fibrous scaffold groups was not significantly lost,and the signal of the intervertebral disc was not significantly changed.After 3 months,both HE staining and immunohistochemical staining showed that the defect in the DAFM/PECUU1 and DAFM/PECUU2 blended fibrous group had been partially repaired.Conclusion:DAFM/PECUU blended fibrous scaffolds with different elastic modulus were successfully fabricated,which could simulate the difference of the mechanical properties of the inner and outer region of the annulus fibrosus tissue.At the same time,it was found that the scaffolds can regulate the differentiation of AFSCs into areas with similar mechanical properties of the annulus fibrosus tissue and direct AFSCs secret corresponding extracellular matrix.The DAFM/PECUU blended fibrous scaffolds with different elastic modulus could be degraded in vivo and could repair the annulus fibrosus defects at the same time.The results of this study could provide a basis for the study of annulus fibrosus tissue engineering,and also provide a reference for the theory that the mechanical properties of the scaffold could regulate the differentiation of stem cells.