The Role Of Wnt Signal Pathway In The Pressure-regulated Chondrogenic Differentiation Of BMSCs/PRF Construct

Articular cartilage plays a key role in functional activities of joint for superiormechanical properties，including good flexibility and low friction. For human andmammal, joint injury is a common kind of injury. However, articular cartilage is difficultto auto-recover for lacking of direct blood supply and innervation. Hence, it is necessaryto seek a viable approach to treat this kind of injury. Although there are several ways exitalready, they all have some defects. Until recently, the development of tissue engineeredcartilage shed some light for clinical treatment. For tissue engineer, seed cells, scaffoldand growth factors are considered to be the key elements. Among these, bone marrowmesenchymal stem cells (BMSCs) has become the important seed cells for a readilyavailable source, easily passaged and multi-differentiation potential. Growth factors,including insulin-like growth factors (IGF-1), fibroblast growth factor (FGF) andtransforming growth factor-β (TGF-β), can be used to promote the proliferation andchondrogenic differentiation of stem cells. According to a series of studies conducted by the French scholar, platelet-rich fibrin (PRF) is an autologous biomaterial rich in multiplegrowth factors without any immune injection, which has been widely used in clinical toothimplantation. In our previous studies, we have observed that the PRF could slowly releasea pool of growth factors as the fibrin degrades and the α chain degranulates, and a type ofnewly formed transplant, consisting of cell sheet fragments of BMSCs and PRF, has beensuccessfully applied to repair and regenerate cartilage tissues. However, for constructingtissue-engineered cartilage, the effect of biochemistry and biomechanics are critical. It isreported that mechanical stimuli could promote chondrogenic differentiation of BMSCs,but its mechanism is still unclear. Our previous study have demonstrated that statichydraulic pressure of120KPa which was applied1h/d for consecutive4days couldsignificantly promote chondrogenic differentiation of BMSCs, while the mechanism ofthis kind of effect on BMSCs/PRF is still being explored by now. The Wnt signalingpathways are a group of signal transduction pathways made of proteins that pass signalsfrom outside of a cell through cell surface receptors to the inside of the cell. Two Wntsignaling pathways have been characterized mainly: the canonical Wnt pathway and thenoncanonical Wnt/calcium (Wnt/Ca2+) pathway, both of which are critical forchondrogenic differentiation. The canonical Wnt/β-catenin pathway takes part in theosteogenesis and mature of cartilage and inhibits the chondrogenic differentiation; whilenoncanonical Wnt/calcium pathway promotes chondrogenic differentiation via Wnt5a andpostpone the hypertrophic differentiation of chondrocytes. Nevertheless, the role of Wntsignaling pathways in chondrogenic differentiation of mechanical stimulated BMSCs isnot clear, and the relationship between the canonical Wnt/β-catenin and noncanonicalWnt/Ca2+pathway in the process of chondrogenic differentiation has not been reported yet.Hence, the present study aimed to investigate the effect of Wnt signaling pathways onchondrogenic differentiation of mechanical stimulated BMSCs alone or co-cultured withPRF using western blot and real time-PCR analyses, and explore the effect andrelationship of canonical and noncanonical Wnt signaling pathways in this process. The content of this item includes four parts summarized as following.1. Bone marrow was extracted by puncture method from4months old New Zealandrabbit to isolate the BMSC using density gradient centrifugation method. Then, thestemness of BMSCs (P3) were proved by cell surface marker characterizations and theirosteogenic/adipogenic differentiation potential. Then the BMSCs were cultured into cellsheet and combined with PRF granules, which was prepared with10ml blood taken fromauricle arterial by centrifuging at3000rpm/min for10minutes right away, to perform theconstruct of BMSCs/PRF.2. The cell sheet of BMSCs and the construct of BMSCs/PRF were both loadedappropriate pressure which we screened in previous studies, that was static hydraulicpressure of120KPa for60min, and then the expression of canonical and noncanonicalWnt signaling pathways related molecules was evaluated by Western Blot analyses. ForBMSCs without PRF，after being stimulated by static hydraulic pressure of120KPa, theexpression of p-GSK3β in canonical Wnt/β-catenin pathways was increased in15min andβ-catenin in30min, both of which showed highest expressional level in60min; while theexpression of key moleculars in noncanonical Wnt/Ca2+pathway increased in30min andreached highest in60min. For BMSCs/PRF, the expression of key moleculars incanonical pathways had no obvious change under such kind of pressure, while theexpression of noncanonical pathway related moleculars was unregulated significantly.These results indicated that both canonical Wnt/β-catenin and noncanonical Wnt/Ca2+pathway of BMSCs could be activated by static hydraulic pressure of120KPa, no matterco-cultured with or without PRF, and the expression of noncanonical pathway relatedmoleculars was higher in BMSCs/PRF.3. In this part, the role of canonical Wnt/β-catenin and noncanonical Wnt/Ca2+pathway for chondrogenic differentiation of BMSCs was detected. XAV939was chosen asinhibitor for canonical Wnt/β-catenin pathway at concentration of1μM、2μM、4μM,which was added in the medium16hours before pressure stimulation; and L-690,330waschosen as inhibitor for noncanonical Wnt/Ca2+pathway at concentration of20μM、50 μM、100μM, which was added in the medium5days before pressure stimulation. Thestatic hydraulic pressure of120KPa was applied for60min, and then the proteins andRNA were extracted for analyses. The specific proteins were quantified using WesternBlot to screen optimal concentration of inhibitors. The gene expression of PCNA wasdetected by real-time PCR analysis to evaluate the effect of different inhibitorconcentrations on cell proliferation. The results indicated that XAV939at a concentrationof2μM could significantly inhibit the canonical Wnt/β-catenin pathway and L-690,330ata concentration of50μM inhibit the noncanonical Wnt/Ca2+pathway, both of which hadno influence on cell proliferation. Based on the above results, the BMSCs cultured in vitrowere divided into four groups: Group I, BMSCs/PRF; Group II, BMSCs/PRF+statichydraulic pressure; Group III, BMSCs/PRF+inhibitors; and Group IV, BMSCs/PRF+static hydraulic pressure+inhibitor. Some specific markers, of chondrogenicdifferentiation in both canonical and noncanonical Wnt pathways, including Sox-9,Aggrecan and Col-II, were detected by Western blot and real-time PCR. We found that theexpression of Sox-9, Aggrecan and Col-II decreased after being inhibited by XAV939,which confirmed the promotion action of Wnt/β-catenin pathway in chondrogenicdifferentiation of BMSCs/PRF with pressure. Meanwhile, we found that the expression ofthese markers increased after being inhibited by L-690,330, which meant that the role ofWnt/Ca2+pathway in this process was negative regulation.4. The relationship between the canonical Wnt/β-catenin pathway and thenoncanonical Wnt/Ca2+pathway was studied in this section. We found that the specificmarkers of CaMKⅡ and PKC were significantly activated when BMSCs were inhibitedby XVA939at the concentration of2μM and stimulated by static hydraulic pressure of120KPa for60min. Similarly, the expression of β-catenin was dramatically increasedafter being inhibited by L-690,330. Based on these results, we concluded that there was aninhibitive effect between the canonical Wnt/β-catenin and the noncanonical Wnt/Ca2+pathway in the process of chondrogenic differentiation of mechanical stimulated BMSCswhen co-cultured with PRF. Conclusion: Both of the canonical Wnt/β-catenin and the noncanonical Wnt/Ca2+pathway could be activated in BMSCs by the static hydraulic pressure of120KPa for60min, no matter co-cultured with or without PRF. Although the activation effect of pressureon noncanonical Wnt/Ca2+pathway in BMSCs/PRF was obvious, PRF did not show sucheffect on the canonical Wnt/β-catenin pathway. The inhibitor of XAV939at aconcentration of2μM could significantly suppress the canonical Wnt/β-catenin pathwayand L-690,330at a concentration of50μM suppress the noncanonical Wnt/Ca2+pathway,both of which had no influence on cell proliferation. Using the method of reverse-blocking,we found that both of the canonical Wnt/β-catenin and the noncanonical Wnt/Ca2+pathway involved in the chondrogenic differentiation of mechanical stimulated BMSCs,whereas the former had a positive effect while the latter had a negative. Besides, theinhibitive effect between the canonical Wnt/β-catenin and the noncanonical Wnt/Ca2+pathway was proved in the present study.