Cartilage-Healing Capicity Of PCL/Ascorbic Acid Scaffold In Articular Cartilage Defects In A Rabbit Model

BACKGROUNDTrauma,infection,tumor,surgery,and congenital diseases are the main causes of the cartilage defects,and these problems has been the challenges of the clinical ortheopaedic surgeons for a long time.Articular cartilage defect is the clinical common disease,because the articulae have no vessel supply,innervation and lymphatic circumfluence,insufficient cell response,leading to cartilage's intrinsic inability to heal.It was seriously affect the patient'life when the patient suffer the joint pain and movement disorders caused by the cartiage defects,and ultimately lead to osteoarthritis,causing a vicious circle.In recent years,the research of cartilage defect repair methods have the leap development.Cartilage repair materials increasingly rich;With "biological treatment" for the concept of the new repair method,provides cartilage defect repair with numerous theoretical basis and experimental basis.Biological material,seed cells,biological factors of the different combination of factors in cartilage defect repair shows different repair ability,but it is still controversial.Therefore,deepening the experimental research of cartilage biological materials,so as to build the new cartilage repair biological materials,observe the effect of cartilage and repair,has been the most important job of the current scientific research.It not only deepen the depth of cartilage tissue engineering research,practice,new technology,can also provide new ideas and new methods for the trentment to symptomatic osteochondral defects.OBJECTIVEThis research used the original generation of cartilage cells derived from New Zealand white rabbit knee,sterile in vitro amplification,combined with different concentrations of ascorbic acid nutrient solution for the original generation cluvitation,evaluating the effect of ascorbic acid to New Zealand white rabbit chondrocytes 's proliferation and differentiation.In addition,PCL biomaterials combined with AA activity factor to repair articular cartilage defects in New Zealand white rabbit model,to evalute the capacity of this novel material in cartilage regeneration.It can provide experimental data for the research on the basis of cartilage tissue engineering and scientific basis.METHODS1.The effect analysis of ascorbic acid in New Zealand rabbits chondrocytes 's Proliferation and Differentiation.The 6-weeks-old New Zealand white rabbit,were killed directly through aie embolism,sterile separation epithelial tissue of knee joint and exposed the knee joint clearly.then sterile blades cut in articular cartilage,separated by two-step enzyme digestion method for the generation of cartilage cells,with 25 cm2 culture flask culture respectively 1,4,7,14 days,each plant cell number is 2.5*105 experimental group adopted complex ascorbic acid nutrient solution culture(concentration of 20 ug/mL,50 ug/mL100ug/mL)and control group using common medium without ascorbic acid,the rest is the same.We identify the cells by Toluidine blue staining,which could detect the extracellular matrix to identify the cells.According to the experiment plan,we performe the observation of cell morphology,growth density at differenct time point.Hydroxyproline kit cells secrete digestive method of hydroxyproline content detection.By reverse transcription polymerase chain reaction(rt-pcr)determination of its gathered proteoglycans(Aggrecan),type I collagen(COL1),type ii collagen(COL2),and other related gene expression of cartilage.Analysis of the different groups of cartilage differentiation.2.Cartilage-Healing Capicity of PCL/Ascorbic Acid Scaffold in Articular Cartilage Defects In a Rabbit ModelThe PCL scaffolds combined with the ascorbic Acid(Ascobic Acid,AA)were implanted into the articular cartilage defects,each group of materials with the same concentration of ascorbic Acid,divied into three group,including the PCL+AA group,PCL only groups.,the Blank group.Group A:PCL+AA;Group B:PCL only;Blank control group C:Blank,meanwhile A,B two groups were filling with preaped fibrin glue(fibrin gel 10 mu g)and thrombin raw mixture(thrombinogen 10 mu g)for fixing the materials.Eight male New Zealand white rabbits were randomly divided into 3 groups,each animal to bilateral knee joint femoral patella femoral articular surface preparation of diameter of 3.5 mm,3 mm deep defects.Then sacrificed the animals 6 weeks and 12 w after surgery respectively.Histological analyses and quantitative assessments were conducted at 6 and 12 weeks respectively after implantation.ResultsWhite ratbbit's articular chondrocytes showed a strong growth in vitro abserved under inverted phase contrast microscope.After 4d's culture,a spindle and a small protuberant,the nucleus is larger,closely packed cells,.A typical cell morphology called "stone road" form was abserved at day 14.According to the drawing parts and cell morphology in vitro,and the positive toluidine blue staining,which indicated the the chondrocyte cell secretion ECM of the cells and synthesis of proteoglycans.These factors can be determined that cultured cells were chondrocytes.In 1/4/7/14 days each time point of AA group and the control group cell secretion of HYP were measured,the result shows that with the extension of incubation time,AA group HYP secretion significantly greater than the control group,the difference is statistically significant.(P<0.05).As for AA50 group,The gene production is greater than the other groups at each time point RT-PCR results suggest a peak detection genes in different time points,osteogenesis gene expression Col 1 at the beginning of the training is higher,then decreases,and the expression of various points in AA group were lower than that of pure cultures of groups.Into cartilage differentiation genes Col 2 express peak at day 7,group were higher than the control group,including AA50 with simple culture group was statistically difference(P<0.05)Aggrecan gene recch peak at day 4,then remained high expression,expression of AA group were higher than that of pure cultures of groups.There is neither accidental death because of anesthesia during the operation nor infection post-operation.After a 6-weeks repair period,from macroscopic appearance,the experiment group showed more remarkable filling of cartilage defects in comparison to the control group,while only a sligt section of regenerated fibrous-like tissue was observed in the blank group.In addition,significant improvements in Wakitani scores were assessed in experiment group(4.05±1.11)compared with the control group(7.05±0.98)(p<0.05).HE staining revealed the presence of new cells in and around the PCL-AA scaffolds without inflammatory cells.For the safranin O staining,the ECM of the repair tissue was significantly greater in experimental and control group than in blank group(p<0.05).The regenerated cartilage and new cells volume was significantly greater in PCL-AA group than PCL group(p<0.05).Samples harvested at 12weeks showed more hyalione-like cartilage formation than 6 weeks in experimental group.ConclusionAscorbic acid factors has a promoting effect on chondrocytes secretion HYP(collagen synthesis),and can promote ariticular chondrocyte's differentiate into cartilage.Our finding indicate that the PCL-AA scaffolds have good biocompatibility and caciptity of healing articular cartilage defects.Adding ascorbic acid into PCL scaffords,which induced better cartilage formation not only in quantity but also in quality.The results are quite promising with respect to the use of PCL-AA scaffolds as aids of regeneration of articular cartilage using tissue engineering techniques.