Study On The Chondrogenic Capability Of PCL/PTHF/COL Composite Nanofiber Scaffolds

Objective: PCL/PTHF/COL composite nanofiber scaffolds were prepared using electrospinning techniques.Ability of bone marrow mesenchymal stem cells differentiate into chondrocytes by composite nanofiber scaffolds in vitro was perpormed.Further investigation was implement to find out the mechanism of how nanofiber stiffness affect chondrogenesis.And the repair of knee joint cartilage defects in Sprague-Dawley(SD)rats using the nanofibers were also measured.Methods: In the first part,Poly(PCL/PTHF urethane)was synthesized using PCL-diol and PTHF-diol.Poly(PCL/PTHF urethane)was mixed with collagen type I from rat tail(R),collagen type I from calf skin(C)and chondroitin sulfate sodium salt from shark cartilage(S)separatly.And the mixture were made into composite nanofiber scaffolds through electrospinning.The surface topographies of the electrospunfibers were characterized by scanning electron microscopy(SEM).The mechanical properties of the composite nanofiber scaffolds were texted by uniaxial tensile testing technique.The thermal behaviors of the composite nanofiber scaffolds were investigated by Thermo Gravimetric Analysis.Differential Scanning Calorimeter(DSC)thermal analysis was performed to test the Melting Temperatures and Enthalpy Change(?Hm).The crystallinity(Xc)was calculated using the equation,Xc =?Hm/ ?Hm0 × 100%.The second part is to investig6+ate the chondrogenic differentiation of mesenchymal stem cells and its mechanism by composite nanofiber scaffolds in vitro.The experimental group was divided into control group(group K),group P,group C,group R and group S.Bone mesenchymal stem cells were isolated from Sprague-Dawley(SD)rats.The third passage cells were seeded on the composite nanofiber scaffolds and cultured for 7,14,21 days.The MTT assay,Fluorescein diacetate/propidium iodide(FDA/PI)staining,scanning electron microscopy(SEM),glycosaminoglycan(GAG)secretion,immunofluorescence staining of collagen type II and cartilage marker gene expression detection(q RT-PCR)were performed to investigate the induction of bone marrow mesenchymal stem cells differentiate into cartilage.Microarray sequencing(mRNA sequencing)was measured to investigate the mechanism of the different composite nanofiber scaffolds inducing the bone marrow mesenchymal stem cells differentiate into chondrocytes.In the third part,the experiments were performed to repair of knee joint cartilage defects by composite nanofiber scaffolds combined with mesenchymal stem cells.The experimental group was divided into control group(group K),group P,group C,group R,group S,group BP,group BC,group BR and group BS A 2*1.5mm of knee joint cartilage defects were cut by a scalpel in intercondylar fossa.The composite nanofiber scaffolds with and without the stem cells were cover to the defects,and the control group was without any treatment.After 4 and 8 weeks,the joints were taken from the rats.The repair of engineered cartilage were scaled by the gross observation,gross scores and histological scores.The fixed joints were used to HE staining,Masson staining and immunohistochemicalstaining to observe the repair of articular cartilage defects.Results: In this paper,we successfully complete the preparation of the cancellated composite nanofiber scaffolds which have similar structure to collagen fibers.The melting temperature of nanofibers amount the group P,group C,group R and group S were 34?,35?,39? and 36? respectively,the crystallinities were 28.8%,24.3%,22.8%,25.9% respectively and the Young's modulus were 6.8 ± 1.5 Mpa,4.3 ± 0.7 Mpa,7.5 ± 0.9 Mpa,3.6 ± 0.6Mpa respectively.In the results of chondrogenesis in vitro,MTT assay and FDA/PI staining were performed to detect the proliferation and viability of the stem cells.The group C showed the best curative effect.The cells' growth and binding with the material were observed by SEM.The group C and the group R showed the good compatibility with the material,and the cell in these two groups performed tendency to differentiate into chondrocytes.The Immunofluorescence staining showed that the material groups could induce the mesenchymal stem cells differentiation into chondrocytes.COL-2,ACAN and SOX9 were upregulated in the material groups and the group C showed the best result.Microarray sequencing data shown that there were 863-1927 significant difference genes compared to the 4 different material groups and the control group.Uniformly,there were significant differences between the material groups and the control group in the biological process through enrichment analysis,included the development process,the course of development,cell system development,regulation of pressure,stimulus response regulator and organ development etc.al.Which showed that the composite nanofiber scaffolds could influence the biological processes of the mesenchymal stem cells,and reveal the chondrogenic ability to n of mesenchymal stem cells.Compared to group P,group C and group R,the transduction signal of nanofiber scaffolds mayinducing chondrogenic differentiation included the TGF-? ? PI3 K and MYC signal pathway.Analyzing the results of the gross observation and histological staining in vivo,the nanofibers combined with stem cells shown the better effect on the cartilage repair than the control group,and the group BC shown the best effective on the cartilage defect.Conclusion: We successfully completed the preparation the 4 composite nanofiber scaffolds with different structures and Young's modulus by electrospinning.The PCL/PTHF/COL composite nanofiber scaffolds can induce mesenchymal stem cells into chondrogenic differentiation in vitro and showed the good curative effect on the articular cartilage repair in vivo.The signal transduction between different nanofibers with different Young's modulus affects chondrogenic differentiaton through MYC,TGF-? and PI3 K signal pathway.This study can provide a basic theoretics for the design of nanofiber scaffold materials and the repair of articular cartilage defects in clinical use.