Construction And Application Of Photo-crosslinked Pullulan/Polyethylene Glycol Hydrogel With Regulatory Capacity Of Inflammatory Microenvironment For Cartilage Repair

With the increase of obese population,the acceleration of aging society,and the development of national sports,the incidence of articular cartilage injury is increasing year by year.Cartilage defect can lead to osteoarthritis(OA),causing joint pain and even physical disability.Cartilage injury has become the second largest chronic disability disease in the world,second only to cardio cerebrovascular disease,causing significant family and socio-economic burden.Articular cartilage has no blood vessels and nerves,and lacks of stem cells or cartilage progenitor cells,therefore it cannot self-repair when the defect size is more than 2 mm.Current treatment methods mainly include autologous/allogeneic osteochondral transplantation,bone marrow stimulation-microfracture,autologous chondrocyte transplantation,high tibial osteotomy,artificial joint replacement,drug treatment such as sodium hyaluronate injection,and conservative treatment such as weight loss and exercise.Although these treatments can relieve pain and improve joint function to varying degrees,they also have some limitations,such as limited sampling sites,donor site injury,and immune rejection;the repaired cartilage is mostly fibrocartilage,and its mechanical and biological properties are difficult to reach the level of normal hyaline cartilage;there is a limited repair for large defect;it is difficult to reverse the further degeneration and necrosis of articular cartilage.Therefore,the clinical efficacy is not completely satisfactory.Cartilage repair is still a huge problem in the field of clinical orthopedics and sports medicine.Tissue engineering provides a new strategy for cartilage repair and regeneration.Hydrogel is an infinite three-dimensional network structure with good biocompatibility,controllable degradation rate,and good elasticity.Because of its physical and chemical properties similar to extracellular matrix,it is widely used in cartilage tissue engineering.Pullulan polysaccharide is a non-charged linear polysaccharide,which is biodegradable,non-toxic,non-immunogenic and non-carcinogenic.Studies have shown that pullulan can be used as a new cell adhesion agent for cartilage repair.The pullulan hydrogel has good biocompatibility and is beneficial to cell growth and proliferation.However,due to its fast degradation speed and poor mechanical properties,it cannot meet the requirements of articular cartilage repair.Synthetic polymers can be manufactured on a large scale,and the microstructure,mechanical strength,and degradation time can be easily controlled and manipulated.The concept of preparing composite hydrogels with synthetic polymers and natural biomaterial with cellular recognition sites has attracted much attention.Polyethylene glycol,as a synthetic polymer material approved by FDA for drug delivery and tissue engineering,has good mechanical properties and supports the adhesion and proliferation of chondrocytes and mesenchymal stem cells.Thus it has been widely used in the preparation of cartilage tissue engineering hydrogels.Accordingly,the first part of this thesis is to prepare crosslinked methyl propionic acid(Pul MA)/PEGDA composite hydrogels.The effects of PEGDA on the mechanical properties,rheological properties,degradation rate,morphology,and swelling equilibrium of Pul MA/PEGDA composite hydrogels were studied.Bone marrow mesenchymal stem cells(BMSCs)were selected as seed cells to study cellular behavior in hydrogels.The ability of Pul MA/PEGDA composite hydrogels to regenerate cartilage was evaluated by cell survival rate,cell proliferation,glycosaminoglycan content and cartilage related gene expression.Cartilage tissue engineering has been widely used in the regeneration and repair of cartilage defects,but the control of the inflammatory microenvironment is rarely considered in the design process of scaffold.Cartilage defect can increase the inflammatory factors around tissue,such as interleukin-1(IL-6),tumor necrosis factor α(TNF-α),and interferonγ.These inflammatory factors will inhibit the chondrogenic differentiation of seed cells and reduce the generation rate of cartilage ECM,hinder the regeneration and growth of new tissues,so it is difficult to achieve the ideal regeneration of hyaline cartilage.Therefore,it is of great significance to study the control of inflammatory microenvironment in the process of scaffold design.Accordingly,the second part of the thesis is load anti-inflammatory peptide KAFAK in metal organic frameworks(MOFs)and encapsulate them in Pul MA/ PEGDA hydrogel.The modified hydrogel can regulate the inflammatory microenvironment of cartilage regeneration by controlling the release of KAFAK,which achieves a better effect of cartilage defect repair.MethodsPart 1: Fabrication of photo-crosslinked pullulan/polyethylene glycol composite hydrogel for cartilage tissue engineering1.Synthesis and characterization of Pul MAPruland polysaccharide was modified with methacrylic acid through esterification,and then the structure of the prepared pulma was identified by 1H NMR and FT-IR.2.Fabrication and characterization of Pul MA/PEGDA composite hydrogelPul MA and PEGDA solutions can rapidly form hydrogels under UV irradiation in the presence of photo-crosslinker LAP.The physicochemical properties of Pul MA/PEGDA composite hydrogel,including morphology,swelling,degradation,rheological properties,and mechanical properties were characterized.3.Encapsulation,survival,and proliferation of rabbit BMSCs in Pul MA/PEGDA composite hydrogelThe prepolymer of Pul MA/PEGDA formed hydrogel in the presence of photo-crosslinker LAP,and the BMSCs were encapsulated in the hydrogel.The morphology of the cells within the 3D hydrogel was observed under microscope.The live/dead staining was performed using Calcein-AM/Double Stain Kit.4.Determination of glycosaminoglycan in Pul MA/PEGDA composite hydrogelThe content of glycosaminoglycan in each group of hydrogels was measured after 7 days and 14 days chondrogenic induction.Then the value was homogenized by the DNA content in each group.5.Expression of chondrogenic markers(COL2A1,ACAN,and SOX9)and hypertrophic gene(COL10A1)in Pul MA/PEGDA composite hydrogelsIn order to compare the ability of Pul MA,Pul MA/PEGDA,and PEGDA hydrogels to maintain chondrogenic phenotype of BMSCs,chondrogenic markers COL2A1,ACAN and SOX9 genes and hypertrophic marker COL10A1 gene were measured using RT-PCR after 7days and 14 days chondrogenic induction.6.Subcutaneous embedding experiment of Pul MA/PEGDA composite hydrogelIn order to compare the degradation and biosafety of Pul MA,Pul MA/PEGDA and PEGDA hydrogels,three hydrogels were embedded in the skin of mice for 28 days to observe their degradation,and HE staining was used to verify the biosafety.Part 2: Anti-inflammatory peptides loaded zirconium metal-organic frameworks encapsulated in pullulan/polyethylene glycol composite hydrogels promote the production of cartilage extracellular matrix1.Fabrication and characterization of nano zirconium metal organic framework(Zr MOF)and Zr MOF@KAFAKThe nano Zr MOF was successfully prepared by thermal solution method.The morphology,structure,and particle size of the product were observed and analyzed by scanning electron microscope,transmission electron microscope,and particle size analysis.2.Characterization of Pul MA/PEGDA composite hydrogel encapsulated with Zr MOFThe Zr MOF was mixed in Pul MA and PEGDA solutions,and the hydrogel was rapidly formed by UV irradiation in the presence of photo-crosslinker LAP.Then,the physical and chemical properties of the Pul MA/PEGDA composite hydrogel encapsulated with Zr MOF were characterized by IR spectrum,morphology and structure(elemental energy spectrum analysis),swelling ability,degradation property,rheological mechanics,and mechanical properties.3.Encapsulation,survival and proliferation of BMSCs in Pul MA/PEGDA composite hydrogelsencapsulated with Zr MOFThe prepolymer of Pul MA/PEGDA formed hydrogel in the presence of photo-crosslinker LAP,and Zr MOF and BMSCs were encapsulated in the hydrogel.The morphology of the cells within the Gel group,Gel-100 Zr MOF group,and Gel-100 Zr MOF@KAFAK was observed under microscope.The live/dead staining was performed using Calcein-AM/Double Stain Kit.4.Determination of glycosaminoglycans in Pul MA/PEGDA composite hydrogel encapsulated with Zr MOFThe content of glycosaminoglycan in each group of hydrogels was measured after 7 days and 14 days chondrogenic induction.Then the value was homogenized by the DNA content in each group.5.Expression of chondrogenic markers(COL2A1,ACAN,and SOX9)and hypertrophic gene(COL10A1)in Pul MA/PEGDA compositehydrogelencapsulated with Zr MOFIn order to compare the ability of Gel,Gel-100 Zr MOF,and Gel-100 Zr MOF@KAFAK groups to maintain chondrogenic phenotype of BMSCs,chondrogenic markers COL2A1,ACAN and SOX9 genes and hypertrophic marker COL10A1 gene were measured using RT-PCR after 7 days and 14 days chondrogenic induction.ResultsPart 1 : Fabrication of photo-crosslinked pullulan/polyethylene glycol composite hydrogel for cartilage tissue engineering1.The results of 1H NMR and FT-IR showed that methacrylic acid group was successfully grafted into pullulan polysaccharide,and the degree of methacrylation was 10%.2.The physicochemical properties of Pul MA/PEGDA composite hydrogels,including morphology,swelling,degradation,rheological properties,and mechanical properties were characterized.The results showed that PEGDA not only regulated the morphology,swelling,and degradation properties of hydrogels,but also improved the rheological and mechanical properties of hydrogels,proving that the prepared Pul MA/PEGDA composite hydrogel has controllable physical and chemical properties.3.Live/dead staining ofrabbit BMSCs showed that most of cells in Pul MA group,Pul MA+15%PEGDA group,and PEGDA group were alive,indicating that the prepared hydrogel had no obvious cytotoxicity.4.Quantitative measurement ofcartilage ECM glycosaminoglycan showed that Pul MA/PEGDA hydrogel had better ability to promote MSCs secreting cartilage ECM.5.The chondrogenic markers COL2A1,ACAN,and SOX9 were detected by RT-PCR.They were highly expressed in Pul MA/PEGDA hydrogel,indicating that Pul MA/PEGDA composite hydrogel had the ability to maintain cartilage phenotype.6.After 28 days of subcutaneous injection of hydrogel,no signs of edema or infiltration of neutrophils showed that the implanted hydrogel scaffolds did not cause acute or chronic inflammation,further indicating that Pul MA/PEGDA composite hydrogels had good histocompatibility in vivo.Part 2: Anti-inflammatory peptides loaded zirconium metal-organic frameworks encapsulated in pullulan/polyethylene glycol composite hydrogels promote the production of cartilage extracellular matrix.1.The preparation of nano Zr MOF was completed by thermal solution method.The prepared nano Zr MOF materials were long shuttle shaped and uniformly distributed under transmission electron microscope and scanning electron microscope.The hydrated particle size was 332.03 ± 37.75 nm and the zeta potential was-23.77 ± 3.33 m V.After anti-inflammatory peptide KAFAK was loaded into nano Zr MOF(Zr MOF@KAFAK),the hydrated particle size was 350.50 ± 8.19 nm and the zeta potential was 18.27 ± 0.55 m V.2.The physicochemical properties of Pul MA/PEGDA composite hydrogel encapsulated with nano Zr MOF including morphology,swelling,degradation,and rheological propertieswere characterized.The results showed that the introduction of nano Zr MOF could regulate the morphology,swelling,degradation,and rheological properties of the hydrogel system.3.The prolonged release of anti-inflammatory peptide KAFAK in Pul MA/PEGDA composite hydrogel encapsulated with Zr MOF@KAFAK was 21 days.4.IL-1 βpretreatmentwas used to simulate the inflammatory microenvironment.Determination of glycosaminoglycan content showed that BMSCs in Pul MA/PEGDA composite hydrogel encapsulated with Zr MOF@KAFAK hadbetterability to deposite cartilage ECM.5.The expression of chondrogenic markers COL2A1 and ACAN,and hypertrophic marker COL10A1 gene were determined by RT-PCR.The results showed that COL2A1 and ACAN were highly expressed in Gel-100 Zr MOF@KAFAK group,while the hypertrophic gene was decreased in Gel-100 Zr MOF@KAFAKgroup,which indicatedthat Gel-100 Zr MOF@KAFAK hydrogels released the anti-inflammatory peptide KAFAK slowly,thereby maintaining the chondrogenic phenotype and inhibiting the hypertrophy of BMSCs.Conclusion1.The hydrogels with natural polymers pullulan and synthetic polymer PEGDA were successfully prepared by photo-crosslinking.The physical and chemical properties of the hydrogel system could be controlled by changing the concentration of PEGDA.2.The BMSCs encapsulated in the Pul MA/PEGDA composite hydrogel had good cell viability,more ECM glycosaminoglycan secretion,and maintenance of chondrogenic phenotype.The hydrogel provided a new composite hydrogel system for cartilage tissue engineering.3.The nano Zr MOF was successfully encapsulated in the Pul MA/PEGDA composite hydrogel,and the physical and chemical properties of the whole system could be controlled by changing the nano Zr MOF material.4.The anti-inflammatory peptide KAFAK was loaded into Zr MOF(Zr MOF@KAFAK),and the prolonged release of KAFAK was achieved in Pul MA/PEGDA hydrogelencapsulated with Zr MOF@KAFAK.5.Better cartilage regeneration was achieved by regulating the inflammatory microenvironment,which provides a new strategy for constructing scaffold materials with both cartilage regeneration and inflammatory microenvironment regulation for cartilage tissue engineering.