Construction Of Poly (Amino Acid) Biomimetic Scaffolds And Cartilage Repair Research By Tissue Engineering

The articular cartilage tissue lacks lymphatic vessels and blood vessels,and cartilage cells have low migration and proliferation capabilities,so it is difficult for cartilage defects to repair themselves.The use of cell-containing cartilage tissue engineering scaffolds can achieve in situ repair of cartilage defects,but this method still faces the problem of insufficient cell numbers and difficulty in regulating their differentiation behavior.At the same time,cartilage tissue engineering often involves complex and time-consuming in vitro construction processes,so it is difficult to achieve clinical promotion and application.By recruiting cells in situ,the in situ induced regeneration of cartilage tissue can be achieved,and this method can avoid the problem of insufficient numbers when using exogenous cells.So far,there have been few reports on biological scaffolds that can induce regeneration of cartilage defects in situ.The construction of scaffolds to promote in situ regeneration of cartilage tissue is an urgent problem to be solved.In this thesis,poly-γ-glutamic acid(γ-PGA)and hyaluronic acid(HA)are selected as the main research materials to prepare a variety of hydrogel scaffolds,and to study the gelation time,swelling behavior,and swelling behavior of the hydrogel scaffolds.Degradation behavior,thermal stability,mechanical properties,cell compatibility and other properties.In addition,its application in mediating the directional differentiation of stem cells and repairing cartilage defects has also been studied.The main research contents are as follows:1.γ-PGA-GMA/DTT hydrogel used in rabbit ear cartilage repair researchThe methacrylation of γ-PGA was modified to γ-PGA-GMA,and the hydrogel was prepared by the click chemistry reaction between dithiothreitol(DTT)and γ-PGA-GMA.The stent can withstand nearly 90% strain,and the compressive modulus can reach nearly 1MPa.Moreover,the cell compatibility is good,and the cell survival rate is close to 100%.And in vitro reverse transcription quantitative polymerase chain reaction(RT-q PCR)results show that the hydrogel scaffold can well mediate the differentiation of stem cells into chondrocytes,and the expression of related genes is more than 3 times that of the control group.After transplanting the hydrogel scaffold containing stem cells to the rabbit ear cartilage defect,the new tissue at the defect has a good combination with the surrounding tissues,and the cartilage layer is regenerated,and the cartilage layer has obvious GAGs and type II collagen(COL II)Deposition.The above experimental results show that this hydrogel scaffold can well mediate the directional differentiation of stem cells and realize the in situ regeneration of cartilage defects,and is an ideal scaffold material for cartilage tissue engineering.2.γ-PGA/HA/n HAP hydrogel mediates osteochondral differentiation of stem cellsAfter the cartilage tissue is damaged,it may cause secondary damage to the subchondral bone area.Using γ-PGA and HA as raw materials,we have prepared a hydrogel scaffold material that is expected to realize the integrated repair of osteochondral bone.After the two polymers were modified by methacrylation,DTT and nano-hydroxyapatite(n HAP)were added to construct a hydrogel scaffold.The test results show that the γ-PGA/HA/n HAP hydrogel has good toughness(70% strain,nearly 1MPa compressive modulus),and its degradation time can reach several weeks.In addition,in vitro cell experiments proved that the cell survival rate was≥ 100%.The results of RT-q PCR show that the hydrogel scaffold can mediate the differentiation of stem cells into cartilage.In addition,the n HAP in the hydrogel can induce osteogenic differentiation of stem cells to a certain extent due to its good bone-promoting ability,so that the hydrogel scaffold loaded with stem cells is expected to realize the integrated repair of cartilage/subchondral bone.3.CS/γ-PGA gradient hydrogel promotes chondrogenic differentiation of stem cellsAdipic acid dihydrazide(ADH)and GMA were modified on the backbones of γ-PGA and chondroitin sulfate(CS),and sodium periodate(Na IO4)was used to oxidize the vicinal diol on CS to vicinal dialdehyde.The Schiff base reaction is used to initially form a gel,and then a hydrogel with a gradient crosslinking density is prepared by the photomask method and double bond radical polymerization reaction.The gelation time of the hydrogel can be controlled between 100-1000 s.Swelling,SEM and other tests prove that there is indeed a gradient structure inside the hydrogel.In addition,in vitro cell 3D culture and stem cell differentiation experiments show that this γ-PGA/CS gradient hydrogel not only has good cell compatibility(survival rate ≥100%),but also can effectively mediate the differentiation of stem cells into chondrocytes.It is a kind of hydrogel material with the potential of cartilage tissue engineering scaffold.4.Injectable hydrogel recruits stem cells,inhibits inflammation and mediates stem cell differentiationUsing γ-PGA and HA as raw materials,γ-PGA and HA were modified into PGA-SH-NH2 and Me HA-CHO,respectively.The dual cross-linking method of thiol-olefin Michael addition reaction and Schiff base reaction was used to prepare a dual-covalent cross-linked dynamic hydrogel.The results of CCK-8 experiment(cell survival rate ≥100%),cell death staining and3 D culture experiment all showed good cytocompatibility of the hydrogel.Cell migration and chemotaxis experiments have proved that the cell migration and transmembrane induction ability of the hydrogel extract is more than 10 times that of the control group.The results of these two experiments proved that the hydrogel has the potential to recruit stem cells.In addition,under the action of hydrogel,bone marrow-derived macrophages(BMDM)are more likely to be polarized into M2 type macrophages,showing the effect of inhibiting inflammation.The results of RT-q PCR experiments show that after hydrogel,macrophages will secrete nearly2 times more M2 macrophage-related factors,such as VEGF,TGF-β,etc.These results indicate that the hydrogel can induce macrophages to polarize to the M2 type,reduce the inflammatory response,and help alleviate arthritis.5.PCL/γ-PGA/HA composite scaffold promotes integrated repair of osteochondralUsing γ-PGA,HA and PCL as raw materials,we prepared a hydrogel/polycaprolactone(PCL)composite scaffold.The experimental results prove that the free-radical polymerized hydrogel has high mechanical strength(close to 700 k Pa),and can withstand 70% strain,and the hydrogel/PCL composite scaffold has good cell compatibility.The results of cell 3D culture also indicate that cells can survive well in the composite scaffold and achieve proliferation,so the composite scaffold can meet the basic requirements of tissue engineering scaffolds.In addition,in the RT-q PCR experiment,after the stem cells were co-cultured with the hydrogel/PCL composite scaffold for 2 weeks,the stem cells differentiated into two types of cells: chondrocytes and osteoblasts.The PCL scaffold has a large modulus and can mediate the differentiation of stem cells into osteoblasts.Therefore,this hydrogel/PCL scaffold can not only act as an extracellular microenvironment,but also mediate the two-way differentiation of stem cells.It is a good material for osteochondral tissue engineering.