Self-assembling Peptide Hydrogel As TGF-β Mimetic For Cartilage Repair

Chapter 1.Preparation of RAD/CM hydrogel and material characterizationObjective: In self-assembling peptide,the amino acids with positive,neutral and negative charges are arranged at intervals,and the nanofibers come out with non-covalent and hydrophobic bonds.The functional peptides could be grafted directly behind the self-assembling sequence,to endow it with biological activity.On this basis,we try to graft TGF-β mimetic peptide,CM10(LIANAK),onto RAD-I.In this part,we analyzed the preparation process,macroscopic morphology,microstructure and rheological properties of self-assembling peptide hydrogels.Method: We designed and synthesized self-assembling peptide RAD and RAD-CM.RAD and RAD-CM were dissolved by ultrasonic in Milli-Q water and mixed at 1:1 volume ratio.The mixed peptide solution was characterized by Atomic Force Microscopy(AFM)and Circular Dichroism(CD)to evaluate the assembly performance.After neutralized by medium,hydrogel was acquired.The macroscopic morphology was observed,and microstructure as well as micropore was detected by Scanning Electron Microscopy(SEM).In addition,Rheological test(Rheology)was carried out to evaluate the viscoelasticity of hydrogels and analyze their mechanic property.Results: AFM results showed that the peptide molecules in RAD/CM and CM groups both assembled into typical interwoven nanofibers,about 7nm in diameter.CD tests both showed a positive peak at 195 nm and a negative peak at 216 nm,which is a typical spectrum curve of anti-parallel β-sheet structure.After neutralization,a more stable hydrogel was acquired.After overturned,it could stay at the bottom of the centrifuge tube.SEM showed dense nanofibers network.Rheological results show that in both groups the storage modulus is significantly higher than the loss modulus,and the trends remains stable within 1% deformation,while the storage modulus and loss modulus converge after about 5% deformation.Conclusion: RAD/CM peptide can form stable nanofiber hydrogel after dissolution and neutralization.Rheological properties indicated that hydrogel can withstand a certain pressure,and when loaded heavily,it would turn into viscous fluid to adapt to the defect morphology,which might be a promising application in individually customed repair.Chapter 2.In vitro chondrogenic differentiation inducing study of RAD/CM hydrogel.Objective: In this part,rabbit BMSCs were cultured on RAD/CM peptide hydrogel to verify its biocompatibility.At the same time,the role of CM-grafted RAD would be explored in the differentiation of stem cells into chondrocytes,so as to evaluate the function of TGF-β mimetic peptide hydrogel.Methods: We prepared RAD/CM self-assembling peptide hydrogel and set up RAD hydrogel as control.For proliferation test,the absorbance of CCK-8 working fluid at 450 nm was measured at 1/4/7 days and the curve was graphed.For Live/dead staining,cells were incubated with Calcein-AM /Propidium Iodide dyes on the 1/4 day.The number and proportion of living and dead cells were observed with laser scanning microscope at excitation wavelengths of 488 and 545 nm.For cytoskeleton staining,Sytox Green/Rhodamine-Phalloidin staining was performed at 2/6 days.The distribution of microfilaments and nucleus was observed under laser scanning microscope at excitation wavelength of 488 and 545 nm.To analyze the chondrogenic bioactivity of grafted CM,BMSCs went through 21-day induced chondrogenic differentiation without TGF-β on peptide hydrogels,and then Alcian Blue staining was performed to evaluate the cartilage matrix,as well as RT-q PCR to examine the expression of GAPDH,Sox9,aggrecan,collagen II and collagen I.Results: The results of CCK-8 showed ideal growth curve for 1/4/7 days.On Day1 and Day4,the density of living cells was both significantly higher than dead cells,which remained extremely rare.Cytoskeleton staining showed that the microfilaments were indistinct and the cells were well spread on the Day2.On Day6,the microfilaments were densely distributed into network,indicating that the cells spread better.After 21-day inducing without TGF-β,RT-q PCR results showed that the expression of examined genes(Sox9,Agg,Col II and Col I)in RAD/CM group was higher than that in RAD group.Meanwhile,Alcian Blue Staining results revealed that larger blue cell mass exists on RAD/CM group,which is close to RAD/TGF-β group.On the contrary,only separate cells on RAD group were seen,instead of blue cell mass.Conclusion: CCK-8,Live/Dead staining,and Cytoskeletal staining confirmed the excellent viability of RAD/CM hydrogel.RAD/CM self-assembling peptide hydrogel turned out the chondrogenic function of TGF-β in vitro,which provides theoretical and experimental basis for further in vivo studies.Chapter 3.RAD/CM hydrogel combined with decellularized cartilage matrix for rabbit knee defect repair.Objective: In this part,RAD/CM hydrogel was compounded with DCM scaffold for strength,and the in vivo function of RAD/CM/DCM composite scaffold for rabbit knee cartilage defect repair was evaluated.Methods: Cylindrical DCM that 3.5mm in diameter and about 1.2mm in height was made from porcine cartilage,and then soaked in peptide solution before PBS neutralizing overnight.20 New Zealand rabbits got operations to construct the full-thickness cartilage defect model,and different groups of scaffold materials,(RAD/CM/DCM,RAD/DCM,DCM,Control),were implanted.4 weeks later,repair samples were acquired and gross morphology,ICRS scores,micro-CT as well as pathological staining of Hematoxylin Eosin,Safranin O/Fast Green,Immunohistochemical and Sirius Red were performed to analyze the repair effects.Results: DCM is elastic and loose,and turned into puffy after combined with hydrogel,so as to stabilize the morphology of soft peptide hydrogel.4 weeks later,the neocartilage of RAD/CM/DCM groups showed the texture and transparency closest to normal tissue,meanwhile the best fusion with surrounding tissue,which is consistent with ICRS scores.The micro-CT results revealed that the three groups with DCM showed lower density at the bottom of cartilage defect than the control group.The HE and safranin O/Fast Green staining showed plump repair of proteoglycan and great integration with surrounding tissue,which is better than the other three groups.In addition,the Immunohistochemical and Sirius Red staining results showed the most specific and significant distribution collagen type II,while quite less collagen type I or III.Conclusion: In vivo researches confirmed the strong chondrogenic inducing bioactivity of RAD/CM,and DCM scaffold retained time and space,which showed a great synergism for cartilage repair.