Experimental Study On PGS-PCL Porous Scaffold Combined With Bone Marrow Mesenchymal Stem Cells For Repairing Articular Cartilage Injury

Background: Articular cartilage injury is a common clinical disease.With the increase of sports injuries,the incidence of articular cartilage injuries is on the rise.Knee joint pain is the main clinical manifestation of articular cartilage injury.Some patients will suffer from arthrodesis and dysfunction,which will eventually lead to osteoarthritis.According to the depth of cartilage injury,articular cartilage injury can be divided into three types: superficial cartilage injury,full-thickness cartilage injury and osteochondral injury.Surface cartilage damage refers to the damage occurring on the surface of articular cartilage without involving the tidal line.Full-thickness cartilage damage refers to damage accumulation below the tidal line but not penetrating through the subchondral bone plate.Osteochondral injury refers to the injury penetrating through the subchondral bone plate.In general,superficial cartilage damage will not repair itself,while full-thickness cartilage damage will heal with fibrocartilage,which is less elastic and harder than hyaline cartilage.At present,the commonly used clinical treatment methods include joint cavity cleaning,microfracture,autologous osteochondral transplantation(such as mosaic transplantation),allogeneic osteochondral transplantation,etc.Although the symptoms can be relieved,the cartilage repair effect is not ideal and there are many limitations.Therefore,it is of great clinical significance to study a method to promote cartilage injury repair.Tissue engineering is a discipline that uses engineering and life science technology to research and develop tissues that can repair or replace damaged tissues.The basic principle is to inoculate high-concentration seed cells on biological materials for in vitro culture.The materials provide temporary three-dimensional growth space for cells,which is conducive to cell growth.Then,the cell / material composite is implanted into cartilage damage sites for repairing or replacing damaged tissues.In this process,the application of scaffold materials is essential.Tissue engineering scaffolds need to meet the following requirements: 1)need to have good tissue compatibility,which is conducive to cell adhesion and growth;2)to be able to degrade in vivo,and degradation products can be absorbed without causing inflammatory reactions and immune reactions;3)have a porous structure,which is conducive to cell growth and nutrient exchange.;4)have certain physical and chemical properties to maintain the strength and toughness at the initial stage of treatment.Poly(glycerol sebacate)(PGS)is a kind of synthetic polymer elastomer material,which has good biodegradability and tissue compatibility,but it has the defects of fast degradation speed and low mechanical strength.Polycaprolactone,PCL is a semicrystalline biodegradable polymer material with slow degradation rate and certain mechanical strength and toughness,which can just make up for the defects of PGS.In this study,PGS and PCL were mixed and copolymerized to obtain PGS-PCL composite material,and by varying the molar ratio of the two materials,the rigidity,toughness and degradation period of the material were adjusted,the good tissue compatibility of the material was ensured as much as possible.The selected PGS-PCL materials were prepared for cartilage tissue engineering scaffolds for experimental studies of cartilage damage repair.The specific experimental methods are as follows.Part ? Preparation and Performance Test of PGS-PCL Polymeric MaterialsObjective: 1.To extract primary bone marrow mesenchymal stem cells(BMSCs)and articular chondrocytes(ACCs)from New Zealand rabbits.2.In order to find the PGS-PCL material that is most suitable for cartilage tissue engineering,PGS and PCL are mixed and copolymerized according to a certain molar ratio to construct PGS-PCL composite material to evaluate the safety and practicability of the material.Methods: Bone marrow blood from femur and tibia of 1-month-old New Zealand rabbit was extracted,and bone marrow mesenchymal stem cells were isolated and cultured.Through multiple induction of adipogenesis,osteogenesis and chondrogenesis to prove its multiple differentiation ability.New Zealand rabbit femoral and tibial articular cartilage was scraped with a blade,primary chondrocytes were extracted and identified after culture and passage.PGS was prepared from sebacic acid and glycerol.The PGS and purchased PCL were mixed at molar ratios of 2: 1,1: 1 and 1: 2 respectively,and three groups of PGSPCL composite materials were obtained through copolymerization reaction,which were listed as M1,M2 and M3 in turn.The mechanical properties of the three groups of materials were tested by universal mechanical analyzer.In vitro degradation experiments were conducted to analyze the degradation performance and stability of the materials.Culture plates with M1,M2 and M3 coatings were prepared.BMSCs and ACCs were inoculated into the culture plates with material coatings respectively,and monolayer in vitro culture was conducted to analyze the cell compatibility of the materials.Results: 1.BMSCs extracted from bone marrow blood of New Zealand rabbits could differentiate into osteoblasts,cartilages and lipids,and had stem cell characteristics;ACCs was extracted from cartilage of knee joint of New Zealand rabbit,and allicin blue staining was positive.2.The three groups of PGS-PCL composite materials(M1,M2,M3)could be degraded slowly in PBS buffer solution,and the degradation period was significantly longer than that of PGS.During the degradation process,the material could maintain the integrity of its appearance and degrade in the form of surface erosion without fragmentation and collapse.The cumulative loss mass in 60 days was 23.63%,14.67% and 13.01% respectively.3.The mechanical properties of 3.PGS and PCL were complementary.As the proportion of PCL in the composite increases,the modulus compressed,tensile modulus and tensile strength increased,and the elongation at break decreased,which could meet the requirements of cartilage tissue engineering on mechanical properties.4.Three groups of PGS-PCL composite materials were cocultured with BMSCs and ACCs respectively in vitro.Observing the cell morphology,it was found that BMSCs and ACCs could maintain the typical spindle-shaped and roundlike morphology,the cells had good proliferation ability and cell activity in M2 material.Conclusion: PGS-PCL composite has good mechanical properties,biodegradability and cell compatibility.By changing the molar ratio of substrate PGS and PCL,the mechanical strength and degradation period of the composite can be adjusted.The PGSPCL material prepared by mixing PGS and PCL according to the molar ratio of 1: 1 has better comprehensive properties and has the potential to make cartilage tissue engineering scaffolds.Part ? In vitro evaluation of PGS-PCL composite stentObjective: To prepare three kinds of PGS-PCL composite scaffolds with different pore sizes,analyze the mechanical properties,degradation ability and cell compatibility of the scaffolds,evaluate the feasibility of PGS-PCL composite scaffolds in cartilage tissue engineering,and select the suitable pore size structure.Methods: PGS and PCL were mixed according to a molar ratio of 1: 1,and three groups of PGS-PCL composite scaffolds were prepared by salting-out method.The pore diameters were 0-50?m,50-150?m and 150-250?m respectively,which were denoted as S1,S2 and S3 in turn.The structure of the scaffold was observed by scanning electron microscope,the porosity was calculated,and the mechanical analysis and degradation performance of the scaffold were tested.Bone marrow mesenchymal stem cells were seeded on PGS-PCL porous scaffolds at a density of 1×106/ml and cultured in vitro for 3 days.Scanning electron microscopy was used to observe the growth of cells in 3D scaffold materials.CCK-8 method was used to detect cell growth rate,Live/Dead method was used to detect cell viability,and F-actin staining was used to observe cell morphological changes.Results: The PGS-PCL composite scaffolds(S1,S2,S3)in each group were observed to have three-dimensional porous morphology with pore diameters of 0-50?m,50-150?m and 150-250?m in sequence,with average porosity higher than 75%,good connectivity between pores,and convenient transportation of nutrients and excretion of metabolites.In vitro degradation test results showed that the three groups of PGS-PCL composite scaffolds were slowly degraded in PBS solution.During the degradation process,the materials could maintain the integrity of the porous structure of the scaffolds and degraded in the form of surface erosion without fragmentation and collapse.The cumulative loss mass in 60 days was 25.37%,15.87% and 13.05% respectively.Based on the mechanical tests,it was proved that after PGS-PCL material was processed into a porous structure,the mechanical strength decreased,while the toughness increased.In vitro cell experiments showed that the three groups of PGS-PCL scaffolds with different pore sizes showed good cell compatibility.PGS-PCL scaffolds with pore sizes of 50-150?m had good cell adhesion and rapid proliferation.Conclusion: The PGS-PCL porous scaffold prepared by salting-out method has good mechanical properties and biodegradability,and good cell compatibility.When the pore size of the scaffold is in the range of 50-150?m,it is more conducive to the proliferation and migration of BMSCs.Part ? In vitro induction and in vivo cartilage regeneration of BMSCs loadedon PGS-PCL composite scaffoldObjective: 1.To induce chondrogenesis of BMSCs in PGS-PCL cartilage tissue engineering scaffolds and test the chondrogenic ability of the scaffolds in vitro;2.In order to test the chondrogenic ability of PGS-PCL scaffold,PGS-PCL porous scaffold was loaded with BMSCs to repair cartilage defects of New Zealand rabbit knee joint in vivo experiment.Methods: BMSCs were planted on PGS-PCL scaffolds and co-cultured in vitro for 1 day to obtain PGS-PCL cell scaffolds carrying a certain amount of BMSCs.It was replaced by cartilage induction medium,BMSCs were induced to differentiate into cartilage under 3 days.Samples were extracted 28 days later.After embedding part of the samples,tissue sections were made,and the other part was tested for gene protein level.A 3-month-old New Zealand rabbit was selected and a full-thickness cartilage defect with a diameter of 3mm was made on the patellofemoral articular surface of the rabbit knee joint after successful anesthesia.The induced BMSCs/PGS-PCL cell scaffold was implanted into the defect area.The PGS-PCL scaffold without cells and the simple defect model were used as the control group.New cartilage tissue samples were extracted at 12 W and 24 w after operation.One part was used for mechanical analysis and the other part was used for tissue staining.Results: After induction of 1.BMSCs/PCL-PCL cell scaffold in vitro,cell toluidine blue staining and safranine O staining were positive,the GAG content in cells increased,and the expression of cartilaginous related genes(ACAN,COL2)significantly increased.2.In vivo experimental results showed that 12 weeks after BMSCs/PGS-PCL scaffold repair group,new " cartilage-like tissue" appeared at cartilage defect site,and the defect was partially filled.HE staining showed that the boundaries between new cartilage and surrounding cartilage were obvious,and there were still a small amount of polymeric materials remaining.Toluidine blue staining and safranine O staining were weakly positive,and COL2 staining was positive.Twenty-four weeks after the operation,the defect site was repaired,and the section staining showed that it was hyaline cartilage repair,and the effect was significantly better than that of the stent group and the blank control group.Conclusion: 1.By using the BMSCs in the PGS to the PGS-PGS,the BMSCs are successfully differentiated from cartilage to cartilage.2.PGS-PCL tissue engineering scaffold has the ability to repair cartilage defects of rabbit knee joint,and the new articular cartilage is hyaline cartilage.Part ? Preliminary Study on Heparinized PGS-PCL Scaffold Sustained Release Transforming Growth Factor-?3 for Cartilage RepairObjective: To prepare heparinized PGS-PCL scaffold(HEP-PGS-PCL scaffold)by mixing heparin with PGS-PCL scaffold,and then add transforming growth factor-?3(TGF-?3)to the Hep-PGS-PCL scaffold to study the adsorption and slow release ability of TGF-?3 on the scaffold and its effect on bone marrow mesenchymal stem cells.Methods: Hep-PGS-PCL scaffold was prepared by using heparin as a medium to enhance the binding ability of PGS-PCL scaffold with protein substances,and the ability of sustained release heparin of the scaffold was detected.TGF-?3 was loaded on HepPGS-PCL scaffold to evaluate the drug loading capacity and slow release capacity of the composite scaffold.BMSCs were loaded on Hep-PGS-PCL-TGF-?3 porous scaffolds for in vitro three-dimensional culture to evaluate the chondrogenic induction ability of the composite scaffolds.Results: TGF-?3/PGS scaffold could slowly release TGF-?3 for more than 30 days.Conclusion: Hep-PGS-PCL scaffold can adsorb and slow release TGF-?3.In vitro experiments prove that slow release scaffold can induce bone marrow mesenchymal stem cells to differentiate into cartilaginous squares.