Oriented Scaffold For The Repair Of Articular Cartilage Defects In Rabbits

Articular cartilage provides near frictionless motion between articulatingsurfaces and protects the bones of synovial joints from being damaged whensubjected to impact and load bearing. Although articular cartilage is highlysusceptible to damage, it has limited intrinsic regeneration and self-repaircapacity due to its innate avascular nature and low cell-to-matrix ratio. Severalmethods to improve the cartilage repair, including subchondral abrasion,microfracture, transplantion of osteochondral plugs, and autologous chondrocyteimplantation, have had limited success. Tissue engineering has proved to be themost promising alternative therapy that combines cells, scaffolds andenvironmental factors for repair of articular cartilage defects. Nevertheless, thepoor mechanical properties of in vitro engineered cartilage limit its potential forclinical applications. Anatomically and functionally, articular cartilage consists offour zones: the superficial, transitional, deep and calcified zones and generallyexhibits columnar orientation of cells and anisotropic direction of collagen fibers that run vertically from the tidemark towards the joint surface. This alignedcollagen fiber network is believed to be a critical factor required for thebiomechanical properties of articular cartilage. So, the designed structure ofcartilage scaffolds should mimic native articular cartilage, which has an orientedstructure associated with its mechanical and physiological functions. Furthermore,the oriented architecture of this scaffold should possess superior biomechanicalproperties and protect cells from early critical compression prior to secretion ofabundant ECM. Therefore, scaffold with biomimetic oriented architecture is animportant requirement for tissue-engineered (TE) cartilage. There has been muchresearch about using oriented scaffolds to fabricate TE cartilage in vitro over thedecades, but the report of in vivo study was seldom. Therefore, in vivoinvestigation is necessary for evaluating the effects of the scaffold orientation onthe structure and function of three-dimensional (3D) cartilage formation.The purpose of this study was to fabricate an oriented cartilage ECM-derivedscaffold combined with chondrogenic-induced BMSCs for enhancement of thebiomechanical property of TE cartilage in vivo. The resultant constructs werecultured for2weeks and4weeks at subcutaneous sites in nude mice, after whichbiochemical, histological and biomechanical properties were evaluated. The invitro and in vivo experiments showed that the oriented cartilage ECM-derivedscaffold possessed sufficient mechanical strength to meet the requirements ofcartilage regeneration. The value of Young’s modulus of the oriented scaffold was2.5times higher than that of non-oriented scaffold in vitro (p<0.05). Seededchondrogenic-induced BMSCs successfully adhered and distributed onto orientedand non-oriented scaffolds in vitro. Furthermore, implantation of orientedscaffolds combined with differentiated BMSCs in the dorsa of nude mice formedsuperior biomechanically functional TE cartilage constructs after4weeks, andefficient cartilaginous matrix secretion was observed. Then, we investigated the potential application of the ECM derived oriented scaffolds for articular cartilagerepair. The results demonstrated that oriented scaffold is a potential matrix forcartilage regeneration and has good physical properties to allow for cartilageregeneration in a high-load-bearing defect site.Part I Fabrication of oriented scaffold by TIPS technologyThe structure of a cartilage scaffold is required to mimic native articularcartilage, which has an oriented structure associated with its mechanical function.In this study, bovine articular cartilage was physically shattered, thendecellularized sequentially with use of hypotonic buffer, TritonX-100, and anuclease solution and made into a suspension. An oriented cartilage extracellularmatrix (ECM)-derived scaffold was fabricated using a modified temperaturegradient-guided thermal-induced phase separation (TIPS) technique followed byfreeze-drying，which composed of microtubules arranged in parallel in verticalsection. The physical characteristics of oriented scaffold and non-orientedscaffold were detected. And the mechanical property of oriented and non-orientedscaffold was determined by measurement of Young’s modulus. There was nosignificant difference in pore size, porosity, density and water absorption betweenthe two groups. The mechanical property was higher than that of a typicalnon-oriented scaffold (P<0.05). This may be attributed to an increased capacity tosupport more compressive stress due to the greater thickness of the microtubulewalls. These results indicate that the oriented scaffold possess better mechanicalproperty and has the potential for application in cartilage tissue engineering.Part II Oriented scaffold for cartilage tissue engineering in vivoIt has been widely reported that orientation of scaffolds can promote cellmigration and thus probably contributes to improving tissue regeneration in vitro, but the report of in vivo study was seldom. Therefore, in vivo investigation isnecessary for evaluating the effects of the scaffold orientation on the structureand function of three-dimensional (3D) cartilage formation. Oriented andnon-oriented scaffolds were seeded with chondrogenic-induced bonemesenchymal stem cells (BMSCs) and cell-scaffold constructs were implantedsubcutaneously in the dorsa of nude mice. SEM shows that chondrogenic-inducedBMSCs adhered to the scaffolds and displayed a spherical morphology. Moreover,cells were aligned along the oriented microtubules in the oriented scaffold. Asignificant increase in proliferation of chondrogenic-induced BMSCs seeded onoriented and non-oriented scaffolds was measured by MTT assay, althoughproliferation in the oriented scaffold group was higher than in the non-orientedscaffold group from day3to day9(P<0.05). However, there was no significantdifference in proliferation between the two groups at day11and day13.At4weeks post-implantation, all samples stained positive for safranin O, toluidineblue, and collagen type II, but negative for collagen type I. Oriented-structureconstructs contained numerous parallel giant bundles of densely packed collagenfibers with chondrocyte-like cells aligned along the fibers. Total DNA,glycosaminoglycans (GAG) and collagen contents increased with time and thesevalues were similar in the two groups. Compared with the native articularcartilage, the Young’s modulus of the tissue-engineered (TE) cartilage reached42.9%,23.0%in oriented and non-oriented scaffolds respectively, at4weeks.This demonstrated that the oriented cartilage ECM-derived scaffold not onlysupported differentiated BMSC proliferation and cartilage-specific ECMsecretion, but also enhanced the biomechanical property of TE cartilage throughproviding an oriented structure, thus indicating the potential application of thisstrategy for cartilage tissue engineering. Part III Oriented scaffold for the repair of articular cartilage defects in rabbitsTo engineer healthy neo-cartilage, the constructs must have mechanicalproperties matching those of native cartilage as well as appropriate for theloading conditions of the joint. In this study, we investigated the potentialapplication of the ECM derived oriented scaffolds for articular cartilage repair.Rabbit BMSCs were isolated, proliferated and seeded onto oriented andnon-oriented scaffolds. These constructs were implanted in the defect (4mmdiameter,2mm depth) on medial femoral condyle of adult New Zealand Whiterabbits. At6,12and24weeks post-implantation, the gross and histologicalappearances, biochemical content, and mechanical properties of the regenerationprocess was evaluated. By24weeks time point, the specimens showed goodcartilage repair. Micro-Computed Tomography (Micro-CT) showed the thicknessof the neo-cartilage was close to normal articular cartilage. Cartilage-like tissuewas verified histologically by rounded cells within a hyaline-like matrix thatpositive stained for Safranin O, tuoluidine blue and collagen II. No differences inthe histological scores based on O’Driscoll histological grading were observedbetween two groups. No difference in DNA content, glycosaminoglycan (GAG)or collagen content between the oriented and non-oriented groups was found. TheYoung’s modulus of oriented was higher than higher than that of a typicalnon-oriented scaffold (P<0.05). The results demonstrated that ECM derivedoriented scaffold is a potential matrix for cartilage regeneration and has goodphysical properties to allow for cartilage regeneration in a high-load-bearingdefect site.