Study On The Reparation Of Rabbit Condyle Cartilage Defects By Application Of BMSCs/PRF Construct Pre-modulated By Hydraulic Pressure

The temporomandibular joint (TMJ) is the only joint that retains relatively activeremodeling ability during one’s life. Condylar cartilage is an important part of the TMJwhich exhibits mechanical sensitivity and biomechanical reconstruction potential.Condylar cartilage defects or degenerative diseases could occur for a variety of reasons,such as inflammation, cancer, trauma, developmental abnormalities and so on, while theeffect of current therapies, including pridie drilling, autologous periosteal, perichondrealgraft, and autologous osteochondral, transplantation, are limited. With the developmentof modern medicine, tissue engineering shed some light on the reparation of articularcartilage defects. So far, bone marrow stem cells (BMSCs) are one of the most ideal seedcells for cartilage tissue engineering. Although a large number of studies have provedthat BMSCs were effective in the regeneration of articular hyaline cartilage in vivo afterbeing induced in vitro, there are still some problems in practical use of current method of cartilage tissue engineering. Firstly, seed cells are traditionally transplanted in the formof cell suspension, and the great mass of seed cells will be lost. Secondly, the currentscaffolds for cartilage tissue engineering show some drawbacks in biocompatibility andmechanical properties. Thirdly, the purified growth factors are unavailable for high price,while the exogenous growth factors might cause some other adverse reactions (i.e.immunologic rejection). Related studies have shown that both of the cell sheet and theplatelet-rich fibrin (PRF) could solve these problems effectively and have a goodprospect in application of articular cartilage defects reparation. However, it is stillunknown whether the PDLSCs/PRF construct could be used for reparation of articularcartilage defects. So far, tissue engineering cartilage is just similar to natural cartilage,but does not have the critical properties of natural cartilage. In order to obtain ideal effectof articular cartilage regeneration, we still have to mimic the microenvironment of seedcells in vitro and induce them in vivo. Studies have demonstrated that themicroenvironment of stem cells in vivo was composed of extracellular matrix, cytokinesand local mechanical stimulation, and stem cells were more sensitive to the mechanicalstimulation than the adult cells. In addition, biomechanical signals play a key role in theregulation of differentiation of stem cells. So, we wonder to ensure that whethermechanical stimulation is the critical factor for chondrocyte differentiation andmaintenance, and whether the regulation of mechanical environment could promote theeffect of BMSCs on reparation of cartilage defects significantly. Although several studieshave reported the biological effects and signaling mechanisms of single-layer cell sheetof BMSCs when stimulated by mechanical stimulation, these cells lack of the protectionof matrix, nutrition of cytokines and communication between mechanical stimulation andcytokines, and the extrinsic stimulation was very different from that in vivo. Therefore,the conclusion based on these studies could not illustrate the signal pathways andmolecular mechanisms of BMSCs on regeneration of condylar cartilage. In this study, weaimed to construct a cell transplant method consisting of cell sheet of BMSCs andplatelet-rich fibrin (PRF) to enhance the healing of condylar cartilage defects. To test itschondrogenesis potential, the complex was initially stimulated by pressure in vitro and then transplanted in the subcutis of nude mice combined with cartilage debris. Once theectopic chondrogenesis ability was identified, the complex was applied in the area ofcondylar cartilage defects to promote the healing. These overall results will shed somelight on the construction o of tissue engineering cartilage, and provide new opportunitiesfor the treatment of condylar cartilage defects. The content of this item includes threeparts summarized as following.1. Isolation and characterization of rabbit BMSCsObjective: To isolate Rabbit BMSCs by density gradient centrifugation combined withadherent cultivation and and characterize them by cell surface marker characterizationsand their osteogenic/adipogenic differentiation potential. Methods:3ml of bone marrowwas extracted by puncture method from New Zealand rabbit to isolate the BMSC usingdensity gradient centrifugation method. Then, the BMSCs (P3) were characterized by cellsurface marker characterizations and their osteogenic/adipogenic differentiation potential.Results: The isolated cells demonstrated typical growth curve and morphology withBMSCs. The detection of surface molecule expression revealed that the BMSCs werenegative for hematopoietic markers such as CD34and CD45but positive formesenchymal associated markers such as CD29and CD44. In addition, alizarinred-positive mineral deposits and Oil Red O-positive cells could be observed after beinginduced into osteogenic or adipogenic media, which provided strong evidence of theBMSCs multiple differentiation ability. Conclusion: The BMSCs were successfullyobtained from the bone marrow of rabbit by density gradient centrifugation method.2. Effect of the BMSCs/PRF constructs pre-loaded pressure on ectopicchondrogenesisObjective: To explore the ectopic chondrogenic effect of BMSCs/PRF construct regulatedby pressure in the chondrocyte microenvironment in vivo. Methods: The BMSC obtainedin part1were cultured into cell sheet and combined with PRF granules, which wasprepared with10ml blood taken from auricle arterial by centrifuging at3000rpm/min for 10minutes right away, to perform the construct of BMSCs/PRF. Then, the construct wasloaded appropriate pressure which we screened in previous studies, that was statichydraulic pressure of120KPa which was applied1h/d for consecutive4days. Inaccordance with the random number table, the45BALB/C nude mice aged4weeks wererandomly divided into3groups according to the adjuvant graft that was used combinedwith cartilage debris (n=15): Group A, adjuvant use of cell sheet fragments only; Group B,adjuvant use of the BMSCs/PRF construct; Group C, adjuvant use of the BMSCs/PRFconstruct loaded with120KPa static hydraulic pressure1h per day for consecutive4days.The animals were sacrificed2,4or8weeks post-surgery of subcutaneous transplantationand the section was stained with hematoxylin-eosin (HE) and toluidine blue. The meanoptical density value of representative images was calculated by the software Image-ProPlus6.0and statistically analyzed by SPSS13.0. Results: The histologic results showedthat chondrogenesis could be observed both in Group B and C at any time points, whichwas more favorable in Group C, presenting a large amount of chondrocytes. The meanoptical density values of representative images of Group B and C was increased in atime-dependent way, especially of Group C in the time point of4and8weeks incomparison to Group B (P<0.05), both of which were higher than that in Group A(P<0.05). Conclusion: The BMSCs/PRF construct pre-loaded static hydraulic pressureshowed much better ability of chondrogenesis in comparison to that did not loaded withpressure. Group A, the group of using BMSCs cell sheet only, showed unfavorable effecton chondrogenesis due to lack of growth factors provided by PRF and mechanicalstimulation.3. Effect of the BMSCs/PRF constructs pre-loaded pressure on the reparation ofcondyle cartilage defectsObjective: To explore the effect of BMSCs/PRF constructs pre-loaded pressure onreparation of condyle cartilage defects in rabbit. Methods: BMSCs/PRF construct,prepared using the stem cells labeled by BrdU, was pre-loaded with static hydraulicpressure of120KPa which was applied1h/d for consecutive4days. After establishing the model of bilateral condylar cartilage defects, the45New Zealand rabbits aged3-4months were randomly divided into5groups according to the adjuvant graft that wasused (n=9): Group A, without adjuvant graft; Group B, adjuvant use of the PRF granulesonly; Group C, adjuvant use of cell sheet fragments only; Group D, adjuvant use of theBMSCs/PRF construct; Group E, adjuvant use of the BMSCs/PRF construct pre-loadedwith pressure. The animals were sacrificed2,4or8weeks post-surgery and the sectionwas stained with hematoxylin-eosin (HE), toluidine blue and immunohistochemicalstaining of BrdU. The mean optical density value of representative images of toluidineblue staining was calculated by the software Image-Pro Plus6.0and statisticallyanalyzed by SPSS13.0. Results: General observation results revealed that, at every timepoint, the new-born tissues were the most favorable in Group E, especially in the groupof8weeks, presenting almost like normal condylar cartilage. The results of HE stainingalso demonstrated that the defects almost have been repaired in this group, whichmanifested as dense fibrous tissues on the surface layer and well arranged cartilage layers.The mean optical density values of representative images of all groups, except for GroupA, was increased in a time-dependent way, especially of Group E in the time point of8weeks (P<0.05). Regarding immunohistochemical staining of BrdU, manyimmunoreactive cells were found in the new born tissues, and the amount ofBrdU-positive cells was larger in Group D and E than that in Group C. Conclusion:BMSCs/PRF constructs pre-loaded with static hydraulic pressure of120KPa which wasapplied1h/d for consecutive4days could repair the condyle cartilage defects of therabbit TMJ successfully.Summary: The present study demonstrated that the newly developed transplant methodutilizing BMSCs/PRF, consisting of cell sheet fragments of BMSCs and PRF granules,could promote reparation of condyle cartilage defects. To mimic the mechanicalenvironment of condylar cartilage and explore the biological mechanism of thereparation process, the construct was loaded by appropriate pressure, static hydraulicpressure of120KPa which was applied1h/d for consecutive4days, which has been screened in previous studies. Multiple growth factors could be released slowly from awell-prepared PRF and affect BMSCs effectively, which will promote the proliferationand chondrogenic differentiation of BMSCs both in vitro and in vivo, especially in thecondition of cooperating with appropriate pressure. Static hydraulic pressure of120KPa,1h/d for consecutive4days could help to promote the reparation effects of BMSCs/PRFconstruct to the mandibular condylar cartilage defects of the rabbit.