| ObjectiveCombines RGD peptides on surface of silk fibroin materials to implementmodification of silk ligament, to enhance the efficiency of adhesion and spreading ofBMSC and apply it to the anterior cruciate ligament reconstruction experiments on animals,provide a theoretical basis for the further preparation of tissue-engineered artificial silkligament research.MethodsPrepare a New Zealand white rabbit about a week age, extract its BMSCs, subcultureand detect cell types by parallel flow cytometry and immunofluorescence. Activatecarboxyl of silk fibroin by EDC/NHS, Make surface modification of silk ligament inGRGDSPC with PBS solution preparation.Put6silk ligaments modified by RGDsequence peptides in6orifice as experimental group and put6pure silk ligaments inanother6orifice as control group. Add the third generation cell suspension of BMSCsevenly on silk ligament and place them in the constant temperature incubator culture, after4h and12h, collect cell suspension respectively by digestion method and count them bycell count plate.Get fourth generation cells, compound with artificial silk ligament for7days toformate the composite materials of BMSC-silk ligament. Part36New Zealand whiterabbits in3groups stochasticly (the experimental group, control group and blank group),12in each group. Experimental group is disposed with the composite materials of BMSC-silk ligament modified by RGD peptides and control group with composite materialsunmodified by RGD peptides. Reconstruct anterior cruciate ligament in aseptic conditions.After4weeks,8weeks and16weeks, take inspection of observation, histology andbiomechanics respectively. Process experimental data by SPSS13.0. Result1. cultivation and identification of BMSCs.Separate and purificate BMSCs by adherent method, observe under invertedmicroscope, cells are adherent cell growth and irregular spindle. Take some cellssuspension to immunofluorescence test, the results shows CD34negative and CD44, CD50positive, it shows characteristics of stem cells.2. BMSC adhesion rate on artificial silk ligamentDeterminate cell adhesion rate by digestion method, the results showed:after4hours,adhesion rate of the experimental group is0.26±0.05and the control group is0.12±0.03;after12hours, adherence rate of the test group is0.46±0.04and the controlgroup is0.21±0.02. The adhesion rate of the RGD peptide modified silk ligament issignificantly higher than the unmodified silk ligament, via statistical analysis, the twogroups have statistical significance (P <0.05).3. Transplant observation4weeks later, in experimental group, loose connective tissue was relatively obvious,ligament was locally packaged by the synovial membrane, both ends of ligaments werestable in bone tunnel, biopsy showed much inflammatory cells, like neutrophils andlymphocytes, some elastic fibers appeared in the silk bundle. In control group, looseconnective tissue appeared but synovial membrane surrounding the ligament is not obvious,biopsy showed much inflammatory cells, no significant fiber ingrowth. In blank group, nosynovial membrane surrounding the ligament, biopsy showed much inflammatory cells, nosignificant fiber ingrowth.8weeks later, in experimental group, synovial membranegrowed obviously and surrounded the ligament, some dense connective tissue ingrowth,biopsy showed that inflammatory cells decreased significantly, the fiber structureconnected with the silk bundle tightly. In control group, a little loose connective tissuesurrowed the ligaments, synovial membrane surrounding was obvious, biopsy showed thatinflammatory cells decreased significantly, a little connective tissue surrounded theligaments, no vascular structures. In blank group, synovial coverage is not obvious, biopsyshowed a little inflammatory cells.16weeks later, in experimental group, connective tissuegrowed obviously and surrounded the ligament almost completely, small blood vesselswere visible on the surface, synovial membrane was abundant, articular cartilage was intactwithout any abrasion, biopsy showed silk bundle was closely combined with denseconnective tissue, fibrous connective tissue growed in. In control group, ligament waspartly covered by soft tissue, synovial thrived, biopsy showed surface was surrowed by some fiber structure, no obvious vascular structures.In blank group,the ligament almostcovered with synovial, biopsy showed a small amount of connective tissue surrounding thesilk bundle. Take mechanical proof of the implants and record the maximum tensilebreaking strength, the results showed that tensile strength of silk ligament complexmodified by RGD peptides was obviously higher than the unmodified silk ligamentcomplex or pure silk ligament, the difference was statistically significant (P <0.05).Conclusion1. Silk ligament modified with RGD peptides can promote the adhesion and spreadingefficiency of BMSCs obviously.2. Silk artificial ligaments modified with RGD peptides can form complex withBMSCs, the complex have sign to convert to normal ligament tissue to sign under the kneemicroenvironment.SignificanceIn recent years, ligament damage cases showed a trend of increase and thecorresponding reconstruction surgery increase. In the exploration about reconstructionmaterials, the silk is considered to be a promising reconstruction material, but the materialis still flawed for its poor cell adhesion. In our experiment, through modifying RGDpeptides on the surface of silk, to enhance the potency of BMSCs adhesion and spreading.Under the rabbit knee microenvironment, Silk artificial ligaments modified with RGDpeptides showed obvious advantage than the control group and the blank group, it providetheoretical and experimental basis for preparation and clinical application oftissue-engineered artificial ligament silk in the future. |
