Background: Although increasing studies have demonstrated that chondrogenic progenitor cells (CPCs) remain present in human osteoarthritic cartilage, the biological alterations of the CPCs from the less diseased lateral tibial condyle and the more diseased medial condyle of same patient remain to be investigated. Methods: CPCs were isolated from paired grade 1-2 and grade 3-4 osteoarthritic cartilage by virtue of cell migratory capacities. The cell morphology, immunophenotype, self-renewal, multi-differentiation, and cell migration of these CPCs were evaluated. Additionally, the distributions of CD105 + /CD271 + cells in OA osteochondral specimen were determined. Furthermore, a high-throughput mRNA sequencing was performed. Results: Migratory CPCs (mCPCs) robustly outgrew from mildly collagenases-digested osteoarthritic cartilages. The mCPCs from grade 3-4 cartilages (mCPCs, grades 3-4) harbored morphological characteristics, cell proliferation, and colony formation capacity that were similar to those of the mCPCs from the grade 1-2 OA cartilages (mCPCs, grades 1-2). However, the mCPCs (grades 3-4) highly expressed CD271. In addition, the mCPCs (grades 3-4) showed enhanced osteo-adipogenic activities and decreased chondrogenic capacity. Furthermore, the mCPCs (grades 3-4) exhibited stronger cell migration in response to osteoarthritis synovial fluids. More CD105 + /CD271 + cells resided in grade 3-4 articular cartilages. Moreover, the results of mRNA sequencing showed that mCPCs (grades 3-4) expressed higher migratory molecules. Conclusions: Our data suggest that more mCPCs (grades 3-4) migrate to injured articular cartilages but with enhanced osteo-adipogenic and decreased chondrogenic capacity, which might explain the pathological changes of mCPCs during the progression of OA from early to late stages. Thus, these dysfunctional mCPCs might be optional cell targets for OA therapies.

The reconstruction of irradiated bone defects after settlement of skeletal tumors remains a significant challenge in clinical applications. In this study, we explored radiation-induced skeletal stem cell (SSC) stemness impairments and rescuing effects of ferulic acid (FA) on SSCs in vitro and in vivo. The immunophenotype, cell renewal, cell proliferation, and differentiation of SSCs in vitro after irradiation were investigated. Mechanistically, the changes in tissue regeneration-associated gene expression and MAPK pathway activation in irradiated SSCs were evaluated. The regenerative capacity of SSCs in the presence of FA in an irradiated bone defect mouse model was also investigated. We found that irradiation reduced CD140a-and CD105-positive cells in skeletal tissues and mouse-derived SSCs. Additionally, irradiation suppressed cell proliferation, colony formation, and osteogenic differentiation of SSCs. The RNA-Seq results showed that tissue regeneration-associated gene expression decreased, and the Western blotting results demonstrated the suppression of phosphorylated p38/MAPK and ERK/MAPK in irradiated SSCs. Notably, FA significantly rescued the radiation-induced impairment of SSCs by activating the p38/MAPK and ERK/MAPK pathways. Moreover, the results of imaging and pathological analyses demonstrated that FA enhanced the bone repair effects of SSCs in an irradiated bone defect mouse model substantially. Importantly, inhibition of the p38/MAPK and ERK/MAPK pathways in SSCs by specific chemical inhibitors partially abolished the promotive effect of FA on SSC-mediated bone regeneration. In summary, our findings reveal a novel function of FA in repairing irradiated bone defects by maintaining SSC stemness and suggest that the p38/MAPK and ERK/MAPK pathways contribute to SSC-mediated tissue regeneration postradiation.

Background Although increasing evidence has demonstrated that human dental pulp stem cells (hDPSCs) are efficacious for the clinical treatment of skeletal disorders, the underlying mechanisms remain incompletely understood. Osteoarthritis (OA) is one of the most common degenerative disorders in joints and is characterized by gradual and irreversible cartilaginous tissue damage. Notably, immune factors were newly identified to be closely related to OA development. In this study, we explored the modulatory effects of clinical-grade hDPSCs on osteoarthritic macrophages and their protective effects on cartilaginous tissues in OA joints. Methods The cell morphology, immunophenotype, and inflammatory factor expression of osteoarthritic macrophages were explored by phase contrast microscope, transmission electron microscopy, immunostaining, flow cytometry, quantitative polymerase chain reaction, and enzyme linked immunosorbent assay, respectively. Additionally, the factors and signaling pathways that suppressed macrophage activation by hDPSCs were determined by enzyme-linked immunosorbent assay and western-blotting. Furthermore, hDPSCs were administered to a rabbit knee OA model via intra-articular injection. Macrophage activation in vivo and cartilaginous tissue damage were also evaluated by pathological analysis. Results We found that hDPSCs markedly inhibited osteoarthritic macrophage activation in vitro. The cell morphology, immunophenotype, and inflammatory factor expression of osteoarthritic macrophages changed into less inflammatory status in the presence of hDPSCs. Mechanistically, we observed that hDPSC-derived hepatocyte growth factor and transforming growth factor β1 mediated the suppressive effects on osteoarthritic macrophages. Moreover, phosphorylation of MAPK pathway proteins contributed to osteoarthritic macrophage activation, and hDPSCs suppressed their activation by partially inactivating those pathways. Most importantly, injected hDPSCs inhibited macrophage activation in osteochondral tissues in a rabbit knee OA model in vivo. Further histological analysis showed that hDPSCs alleviated cartilaginous damage to knee joints. Conclusions In summary, our findings reveal a novel function for hDPSCs in suppressing osteoarthritic macrophages and suggest that macrophages are efficient cellular targets of hDPSCs for alleviation of cartilaginous damage in OA. Graphical abstract hDPSCs treat OA via an osteoarthritic macrophages-dependent mechanisms. hDPSCs suppress the activation of osteoarthritic macrophages in vitro and in vivo and alleviate cartilaginous lesions in OA models.

Background Radial extracorporeal shockwave (r-ESW), an innovative and noninvasive technique, is gaining increasing attention in regenerative medicine due to its mechanobiological effects. Subchondral bone stem/progenitor cells (SCB-SPCs), originating from the pivotal zone of the osteochondral unit, have been shown to have multipotency and self-renewal properties. However, thus far, little information is available regarding the influences of r-ESW on the biological properties of SCB-SPCs and their therapeutic effects in tissue regeneration. Methods SCB-SPCs were isolated from human knee plateau osteochondral specimens and treated with gradient doses of r-ESW in a suspension stimulation system. The optimized parameters for SCB-SPC self-renewal were screened out by colony-forming unit fibroblast assay (CFU-F). Then, the effects of r-ESW on the proliferation, apoptosis, and multipotency of SCB-SPCs were evaluated. Moreover, the repair efficiency of radial shockwave-preconditioned SCB-SPCs was evaluated in vivo via an osteochondral defect model. Potential mechanisms were explored by western blotting, confocal laser scanning, and high-throughput sequencing. Results The CFU-F data indicate that r-ESW could augment the self-renewal of SCB-SPCs in a dose-dependent manner. The CCK-8 and flow cytometry results showed that the optimized shockwave markedly promoted SCB-SPC proliferation but had no significant influence on cell apoptosis. Radial shockwave exerted no significant influence on osteogenic capacity but strongly suppressed adipogenic ability in the current study. For chondrogenic potentiality, the treated SCB-SPCs were mildly enhanced, while the change was not significant. Importantly, the macroscopic scores and further histological analysis strongly demonstrated that the in vivo therapeutic effects of SCB-SPCs were markedly improved post r-ESW treatment. Further analysis showed that the cartilage-related markers collagen II and proteoglycan were expressed at higher levels compared to their counterpart group. Mechanistic studies suggested that r-ESW treatment strongly increased the expression of YAP and promoted YAP nuclear translocation in SCB-SPCs. More importantly, self-renewal was partially blocked by the YAP-specific inhibitor verteporfin. Moreover, the high-throughput sequencing data indicated that other self-renewal-associated pathways may also be involved in this process. Conclusion We found that r-ESW is capable of promoting the self-renewal of SCB-SPCs in vitro by targeting YAP activity and strengthening its repair efficiency in vivo, indicating promising application prospects.