The Osteogenic Effect Of Human IPSCs On PLLA/SF Biomimetic Nanofiber Scaffolds Containing Osteoblastes-derived Extracellular Matrix

Due to the large area of bone defects can not be self-healing in body,bone transplantation can only be used to achieve bone repair and regeneration,at present.However,autologous bone or allograft and other clinical methods available for the treatment of severe bone defects is still difficult to obtain satisfactory therapeutic effect.Bone tissue engineering(BTE)can theoretically regenerate the original tissue completely and is considered as the most ideal method for repairing large area bone defects.BTE technique has broad application prospects in the future treatment of clinical bone injury.The main purpose of this study is to firstly prepare poly(L-lactic acid)/Silk Fibroin(PLLA/SF)loaded osteoblasts-derived extracellular matrix(O-ECM)electrospun bionic nanofiber scaffolds(O-ECM/PLLA/SF).Second,human induced pluripotent stem cells(hi PSCs)were induced into mesenchymal stem cells(MSCs)in vitro,which are called hi PSCs-derived MSCs(hi PSCs-MSCs).A novel “Bionic Scaffold + Stem Cell” BTE complex was constructed by inoculating hi PSC-MSCs onto O-ECM/PLLA/SF.Finally,in vitro and in vivo experiments explore the effect of complex-induced osteogenic differentiation in vitro and its ability to repair and regenerate bone in vivo.Electrospinning is the most concise and effective method for preparing scaffolds for the preparation of nano-or sub-micron size fibers by far.Clinical tests show that the mechanical properties of PLLA scaffolds are sufficient for tissue engineering research,but single due to higher crystallinity,poor hydrophilicity,slow degradation,sexual crisp and other defects,PLLA is difficult to use alone.SF protein has been widely used in various fields of tissue engineering because of its good biocompatibility and hydrophilicity,but its mechanical properties are not enough for bone repair alone.Therefore,in the choice of bionic scaffolds,in order to combine the excellent performance of both to achieve the effect of complementary advantages,this study blended the PLLA and SF combined with electrospinning technology.However,studies have shown that simple composite materials can only bark the structure of the extracellular matrix(ECM)and do not mimic the microenvironment in vivo.Therefore,in this study,we designed a PLLA/SF scaffolds with O-ECM strategy to modify the blend nanofiber scaffold,in order to achieve the same effect on the structure and function at the same time bionic.ECM is a three-dimensional network structure secreted by cells.Its main components are various adhesion proteins and polysaccharides,which provide a favorable growth environment for cell adhesion and proliferation.The present study assumes that O-ECM retains the ECM components secreted by osteoblasts and has very high biological activity.Therefore,the modified PLLA/SF bionic nanofiber scaffolds are expected to promote the proliferation and osteogenic differentiation ability of stem cells.The choice of seed cells is also the key to BTE applications.In this study,pluripotent stem cells(i PSCs)were used as research objects.Compared with adult stem cells MSCs,i PSCs possess strong self-renewal ability and multi-directional differentiation potential.They are derived from a wide range of sources and can be directly reprogrammed by adult cells.However,due to tumorigenicity,Problems such as uncontrollable differentiation have not yet been solved,making their progress in clinical application slow.For this reason,we designed hi PSCs to induce MSCs(hi PSC-MSCs)to construct BTE seed cells.The gain of hi PSC-MSCs is expected to make up for the limitations of i PSCs and MSCs.The hi PSC-MSCs not only maintain the strong proliferative activity of seed cells,biological safety and differentiation control,which makes hi PSC-MSCs in the field of BTE have better application prospects and research significance.In this context,the primary task of this study is to prepare O-ECM/PLLA/SF bionic nanofiber scaffolds.Firstly,the electrospinning method was used to prepare blended nanofiber scaffolds with PLLA/SF mass ratios of(100/0,70/30,50/50,30/70 and 0/100),respectively,and to explore their comprehensive performance.The fiber membranes were characterized from fiber morphology,diameter distribution,infrared spectrum analysis,TGA,mechanical properties analysis,hydrophilicity test and cell compatibility test.The results showed that the fiber diameter distribution of PLLA/SF = 50/50 group was the most uniform and the diameter range was within the range of 50-500 nm of natural ECM fiber.At the same time,the results of comprehensive infrared data,thermal stability,mechanical properties and cell compatibility showed that the PLLA/SF = 50/50 group had the best performance,so we chose this group as the scaffold for subsequent experiments.Secondly,we extracted and purified the rat osteoblasts(r OBs)from the skull of newborn SD rats and cultured the PLLA/SF blend nanofiber scaffolds by using P3 generation r OBs.0.5% Triton X-100 and 20 m M NH4 OH were decellularized to prepare O-ECM/PLLA/SF bionic scaffolds.Subsequently,the morphology,composition and cell compatibility of O-ECM/PLLA/ SF were examined by scanning electron microscopy,immunofluorescence staining and CCK-8 experiments.The results showed that there were almost no residual cells in the cells after decellularization.The major components of ECM,such as fibronectin,laminin and collagen secreted by cells,were uniformly attached to the scaffolds.O-ECM-loaded fibrous scaffolds could significantly promote cell growth.Through the induction of hi PSCs,hi PSC-MSCs seed cells were successfully obtained.The results showed that hi PSC-MSCs cultured to the seventh generation had achieved the purification effect.Flow cytometry showed that the MSCs markers CD90,CD44 and CD29 in hi PSC-MSCs were all positive,the expression levels were 99.87%,99.87% and 99.58%,both hematopoietic stem cell markers CD34 and CD45 were negative(0.85% and 0.87%),which were consistent with the expression of MSCs surface markers.The results of the three-line differentiation showed that both osteogenic,adipose and cartilage staining were positive.Quantitative PCR results showed that hi PSCs highly expressed pluripotent stem cell-related genes(Nanog,OCT-4 and MSX-1),while hi PSC-MSCs maintained a low level of pluripotency gene expression.We speculate that induced hi PSC-MSCs combine the characteristics of i PSCs and MSC cells with the advantages of self-renewal ability and good biosecurity.We planted and cultured hi PSC-MSCs on O-ECM/PLLA/ SF bionic scaffolds and explored their osteogenic differentiation in vitro for 14 days,the expression of osteogenesis-related genes,bone marker proteins and bone secretory proteins were detected.The results showed that q PCR could detect the expression of bone related genes(Ocn,Col,Alp and Runx2),immunofluorescence staining of bone marker proteins(OPN and RUNX2),alkaline phosphatase(ALP)and alizarin red staining(ARS)and Col quantitative analysis of experimental data show that compared to other experimental groups,the O-ECM/PLLA/SF group contributed to osteogenic differentiation significantly,indicating that our prepared O-ECM/PLLA/SF bionic scaffold can induce hi PSC-MSCs osteogenic differentiation.Finally,the effect of repairing bone defect was explored by implanting the “Bionic Scaffold + Stem Cell” complex into the experimental animal model of rat skull defects.Micro CT imaging analysis,bone mineral density measurement,HE staining,Masson staining and immunohistochemistry were used to detect the osteogenic effects of the defects at 4 and 8 weeks respectively.The experiment found that the “bionic scaffold + stem cell” complex in vivo implants 4 weeks after the formation of bone is not obvious,but there has been new angiogenesis,8 weeks have a significant osteogenic effect.The results showed that the osteogenesis effect of the hi PSC-MSCs-containing complex was more pronounced than that of O-ECM/PLLA/SF alone or PLLA/SF alone.In addition,the ectopic osteogenesis effect was detected by embedding in the back of the mice for 4 weeks,and the ectopic osteogenesis effect was detected by X-ray and HE staining,respectively.The results of the above defect repair confirmed that the constructed “hi PSC-MSCs + O-ECM/PLLA/SF” complex with excellent osteogenic ability.In this study,O-ECM/PLLA/SF bionic scaffolds were obtained successfully and hi PSC-MSCs were creatively combined with them to construct “hi PSC-MSCs + O-ECM/PLLA/SF” biomimetic complexes.The experimental results show that the composite of osteoblast differentiation and in vivo bone regeneration ability have significant effect.The prepared O-ECM/PLLA/SF bionic scaffold + stem cell complex can significantly promote the osteogenic differentiation of stem cells and the regeneration of bone damage repair,which will have important research prospects and clinical application value in the field of BTE research.