| Background: With the increase in sports injuries and social aging population, theincidence of bone and joint diseases is rising, which seriously affects the quality of humanlife.The causes associated with osteochondral defects are numerous and vary fromdegenerative diseases to trauma related injuries. With an aging population, the natural wearof thecartilaginous tissue often leads to osteoarthritis, a major cause ofosteochondraldefects. Articular cartilage and subchondral bone closely link with anatomical structure andinfluence on biological function each other, which together constitute a functional unit withinterdependent composite. Organizational structure, mechanical properties, metabolismand molecule transportation of different levels of this functional unit possess specificmaterial gradient and physiological differences,the osteochondral interface betweencartilage and bone plays a connecting role for barrier. Osteochondral defects containdamage to both the articular cartilage as well as the underlying subchondralbone.Both thecomposition and mechanical properties of different organizations which fully demonstratesthe importance of tissue interfaces. In order to repair an osteochondral defect the needs ofthe bone, cartilage and the bone-cartilage interface must be taken into account, because ofosteochondral interface is essential in preventing mechanical failure and maintainingnormal function of cartilage. Current tissue engineering (TE) strategies are hampered bythe difficulties inherent in designing a seamless interface between these two very differenttissues, which make the osteochondral integrated regeneration has been asignificant issue, both clinically and academically. Thus, the development of osteochondral tissueengineered interfaces would be a novel treatment for traumaticinjuries and agingassociated diseases that affect joints. This study included the following content:1.Toinvestigate the relationship between subchondral bone reconstruction and articularcartilage regeneration in process of spontaneous osteochondral repair;2.To develop anintegrated biphasic scaffold containing biomimetic interface;3.To evaluate characteristicsand biological behavior of the biphasic scaffoldsin vitro;4.To explore mechanism ofosteochondral defects repair and osteochondral interface regeneration using variousscheme of cell-scaffold constructsin vivo.Methods:1. The osteochondral defects (4mm in diameter and3mm in depth) werecreated in the trochlear groove on the unilateral femur of New Zealand white rabbits,which penetrated the subchondral bone without any treatment. The rabbits were sacrificedat1,4,12, and24weeks after operation, respectively. The specimens were obtained formacroscopic, histological, and immunohistochemical observations. According to theInternational Cartilage Repair Society Histological Scoring (ICRS), the effect of cartilagerepair was assessed. The histomorphometry parameters of subchondral bone were analyzedby micro-CT scan and reconstruction.2. Based on previousstudys, the articular cartilageextracellular matrix (ACECM) and hydroxyapatite (HAp) nanoparticles are employed andproportioned.By use of physical wet shatter and decellularization technology,"liquid-phasecosynthesis interdiffusion (LPCI)" technique, improved thermally induced phaseseparation (TIPS), physical and chemical crosslinking and freeze-drying technology tofabricate integrated biomimetic scaffolds including non-mineralized and mineralizedphasesas well as the continuous interfaces. Physical and chemicalcharacterization,biomechanical properties and permeability of scaffolds were determinated.3. The cells were seeded on scaffolds to construct cell-scaffold complexes as well as toobserve the cells adhesion, proliferation and growth distribution in the biphasic scaffoldsvia series of testing in vitro.In addition, cell-scaffold constructs were implantedsubcutaneously into mice in vivofor fluorescence imaging and histologicalexamination.4.Different scheme to seed cells intoACECM/HAp biphasic scaffolds formed cell-scaffold constructs and implanted into osteochondral defects of rabbit knee, specimenswere in batches harvested at4,12,24weeks time points after operations, performed grossobservation, histological staining and quantitative scoring, biochemical quantitativedetection, micro-CT scan and biomechanical evaluation.Results:1. Osteochondral defects could be repaired spontaneously in rabbit model. Withtime, defect was gradually filled with repair tissue, subchondral bone plate under the defectregion gradually migrated upward. Bone mineral density, bone volume fraction, mineralized tissue density, trabecular number, and trabecular thickness were increased, whiletrabecula spacing was decreased. Histological examination showed that fibrous repair waspredominant with rare hyal ine cartilage. With time, ICRS histological scores increasedgradually. Among the histomorphometry parameters of subchondral bone, the trabeculaspacing was negatively correlated with ICRS score (r=-0.584), and the otherhistomorphometry parameters were positively correlated with ICRS score (r values were0.6800.891). Subchondral bone remodeling and cartilage repair process were notcompletely synchronized.2. Utilizing ACECM and nano-HAp materials, combined use ofmodified technologies such as TIPS and LPCI successfully have prepared biphasicscaffolds containing mineralized and non-mineralized phaseswith a continuous interface,as well as good integrity of structure. Pore structure within upper layer of scaffolds presentmicrotube-like with good interconnectivity, which vertically arranged parallel orientation,while randomly arranged inlower layer of scaffold. There were relatively uniform porestructure but significant differences on pore size and porosity in the layers (P<0.05), thatshowed feature with “large and loose upper layer, small and dense lower layer”, pore sizevaried from128.2±20.3μm down to21.2±3.1μm.The upper porosity were92.6±6.2%,while porosity of the lower layer and contained from30±4.1%to44±3.2%due to contentof HAp increased.Scanning electron microscopy (SEM) showed that pore morphologychanged gradually from tubularto circular form, emerging a transitional structure similar tonatural osteochondral interface. Mechanical tests showed biphasic scaffold had goodresistance to compressive and shear strength,as well as the interface region providedphysical barrier with low permeability, which of7w/v%of HAp was appropriate ratio for scaffold concentration.3. CCK-8testing and SEM observationshowed that chondrocytesand BMSCs in upper layerpresent good adhesion and proliferation in vitro. ACECM/HApbiphasic scaffold was shown to promote cell distribution as guiding role and barrierfunction, cells in the upper layerwere arranged along pore distribution, no cells across theinterface into the lower layer via histological, SEM and confocal fluorescence microscopeobservation. No stratification found interfacial region, which can provide a suitablemicroenvironment for cells growth. Cells viability assessment in scaffolds and cell-scaffoldconstructs implantation experiment showed that upper layer of scaffolds retained maincomponent of ACECMwith chondrogenesis and lower layer with osteoconductive functionbased onits containing calcium and phosphorus, as well as interfacial region with lowpermeability for barrier effect, no cytotoxicity or good biocompatibility.4. Histologicalstaining tests showed BMSC-derived chondrogenic cells or chondrocytesladenACECM/HAp biphasic scaffolds repair in vivo is better than the control group(P<0.05).Particularlly, cartilage thickness of repair area was close to normal at24weekspost-operation. Matrix staining, cell morphology of neocartilage and surroundingtissue were normal, osteochondral interface or tidemarkhad regenerated, bone remodelingwas completed integrated with the surrounding bone tissue.Indentation mechanical testingshowed that neocartilage had good Young’s modulusat24weeks post-operation,there wasno differences with normal cartilage (P>0.05). High-quality subchondral bone formationwas observed by Micro-CT scan. Moreover, all the implants without failure during theobservation period, showing good initial stability for implantation.Conclusions:1.There is relevant correlation as well as independent process betweencartilage regeneration and subchondral bone reconstruction in the model of spontaneousosteochondral repair, and fast subchondral bone remodel ing may adversely affect articularcartilage repair.2. By selecting ACECM and nano HAp materials, adopting series ofoptimized techniques,using natural bone-cartilage tissue as a structure template, keepingthe "integration design" for layered scaffold, we have successfully constructed novelbiphasic scaffolds for osteochondral interface tissue engineering. The biphasicscaffoldspossessa non-mineralized upper layer with macroporous oriented structure, a mineralized lower layer with pyknotic cellular structure, and the interface between twolayers anchored integratedly, which had good resistance to compression and shear.Lowpermeability of the interfacial region provided regional characteristics of two layers,suggesting this biphasic scaffold meet the requirements of interfacial tissue engineeringbased on biomimetic property of components, structure, and function.3. ACECM/HApbiphasic scaffold have the ability to guide cellular distribution, may provide uniquemicroenvironment for suitable cells growth, proliferation and cartilage regeneration, aswell as good cell and tissue compatibility. Scaffolds with biomimetic properties ofbiochemical composition and structure,whichown unique barrier effect of interface, havebility to form a stable continuous tissue interface in vitro,that simulating the tissuecharacteristics of cartilage-bone interface, are suitable for osteochondral interfacetissueengineering.4. ACECM/HAp biphasic scaffold achieved a functional regeneration appliedto osteochondral defect repair. This integrated scaffold with low permeability can optimizethe formation of bone, cartilage and interface,which can be utilized as apromising materialfor osteochondral interface tissue engineering.Future application of interface tissueengineering strategies based on scaffolds, should pay attention to theproperties ofpermeable material and the inherent structure-function relationship of bone and cartilagefor a rapid translation into clinical application. |
PDF Full Text Request