In Situ Tissue Regenerative Biomaterials For Dynamic Modulation Of Key Cells In Tissue Regeneration

Tissue defect and dysfunction is one of the major hazards affecting human life and health.The tissue engineering strategy relying on allogeneic cells and bioactive scaffolds has been regarded as a well-established tool for the treatment of tissue damage.Yet,its use in clinical practice is hampered by the immunogenicity of exogenous cells,the phenotypic changes during ex vivo expansion and the low survival rate after cell implantation.Recent advances in body’s self-healing via endogenous cell homing in response to injury have shifted the focus from traditional tissue engineering to in situ tissue regeneration,which is to directly transplant the scaffold into the wound site without in vitro construction.The in situ tissue regeneration is aiming to modulate microenvironment of the wound site and the behaviors of autologous cells,and thus to stimulate the regenerative potential of body.In this thesis,two kinds of commonly used biomaterials,collagen and polycaprolactone(PCL),were used as substrates to design the in situ tissue regenerative scaffolds.The scaffolds were aiming to promote tissue repair and regeneration by modulating the behaviors of macrophages and mesenchymal stem cells(MSCs),which were regarded to be two key cells in tissue regeneration.In a normal healing process,macrophages exhibit a pro-inflammatory phenotype in the early stage and then shift to an anti-inflammatory phenotype in the later stage.The importance of the macrophage phenotypic balance between pro-and anti-inflammatory phenotypes is widely understood.The macrophage phenotype is closely related to its cell morphology.According to this,shape memory PCL films doped with gold nanorods were used to construct the dynamic surface topography.The original flat PCL films maintain the relatively proinflammatory phenotype of macrophages,after triggered by the near infrared light(NIR),the surface topography transformed from flat to microgrooved,so as to promote the elongation of macrophages and the phenotypic polarization toward the anti-inflammatory phenotype.The certain ratio of two-branced and four-branched PCL macromonomers were used to regulate the transition temperature of the PCL films to 41.8 ℃.After combining with the gold nanorods,the surface topography of the PCL film was able to change within 60 s under the exposure of NIR(808 nm,1.5 W/cm~2)with no cytotoxicity.Bone marrow derived macrophages(BMDMs)were extracted and purified,and the topographical transformation toward microgrooved was demonstrated to be effective to induce the BMDMs elongation and upregulation of anti-inflammatory factors,such as arginase 1(Arg-1),interleukin-10(IL-10)and CD163 in vitro and in vivo.The effect of bone marrow mesenchymal stem cells(BMSCs)on tissue repair has been widely demonstrated due to their self-renewal capacity and pluripotency.Homing of MSCs by chemokines is one of the most common strategies in in-situ tissue regeneration.However,the non-specific recruitment of chemokines is likely to promote tissue fibrosis.E7 peptides(EPLQLKM)were demonstrated to be specific affinity to BMSCs,collagen substrates with different E7 densities were constructed by modulating the concentration of sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC).NIH3T3 cells and RAW264.7 cells were used to represent the fibroblasts and macrophages in the wound sites.With the increasing E7 density,the adhesion rate,adhesion area and adhesion force of BMSCs on the E7 modified collagen substrate obviously increased,but no significant effect on the adhesion of NIH3T3 cells and RAW264.7 cells was observed,which proved that the E7modified collagen substrates could selective promote the adhesion of BMSCs.In addition,the flow environment in vivo was simulated by a self-designed flow model,which proved that e7-collagen membrane enhanced the dynamic capturing ability of BMSCs under flow conditions.In addition,the selective capture under flow circumstances was demonstrated by the co-culture of BMSCs,NIH3T3 cells and RAW264.7 cells in the flow model.Combining E7 peptide-modified collagen scaffold with SDF-1α ,which is commonly used as a chemokine to recruit BMSCs,an in situ tissue regenerative scaffold named SDF-1α /E7 modified collagen scaffold was constructed.Due to the short half-life of SDF-1α ,the nanoparticles contained SDF-1α constructed by negatively charged sulfonated chitosan(SCS)and positively charged poly-L-lysine(PLL)were effective to protect the activity of SDF-1α from contacting with acids,bases and organic solvents.The nanoparticles constructed by 3%SCS and 0.3%PLL showed the meost compact morphology and the highest encapsulation efficiency(75%).The SDF-1α in the nanoparticles was released rapidly in the first 48 hours,and the nanoparticles could protect over 80%of SDF-1α to maintain active.The SDF-1α /E7 modified collagen scaffold owned good biocompatibility,and its effective recruitment of BMSCs and inflammatory BMDMs has been verified in vitro.The recruitment effect of inflammatory BMDMs on BMSCs and the immune regulation effect of BMSCs on inflammatory BMDMs were verified through transwell model,which lays a theoretical foundation for the subsequent application of the SDF-1α /E7 modified collagen scaffold in tissue repair.Intrauterine adhesion(IUA)is a common disease in gynecology and easy to cause infertility.The traditional treatment of IUA showed a high recurrence rate and a low pregnancy rate.More than 90%of IUA are caused by pathologic repair after endometrial injury.The effect of MSCs on the repair of endometrial functions in tissue engineering has been widely demonstrated.Here,the cell-free SDF-1α /E7 modified collagen scaffolds could significantly promote the endometrium regeneration by increasing the thickness of endometrium,increasing the number of glands,and reducing the degree of fibrosis in the rat model.Compared with the natural repair group,when the dosage of SDF-1α was 400 ng per scaffold,SDF-1α /E7 modified collagen scaffolds significantly increased the pregnant rate from 6%to 81%.In addition,the rat endometrial fibrosis model was fabricated to simulate the clinical IUA.Compared with the fibrosis uterus undergoing traditional surgery only,the uterus implanted with SDF-1α /E7 modified collagen scaffold after surgery had thicker endometrium,more glands,lower degree of fibrosis,and the pregnant rate increased from 50%to 77%.The SDF-1α /E7 modified collagen scaffold and in situ tissue regeneration strategy showed a great performance in the endometrium regeneration.