Developing cartilage constructed with the appropriate matrix composition and persistent chondrogenesis remains an enduring challenge in cartilage defects. Cartilage progenitor cell (CPC)-based tissue engineering has attracted recent attention because of its strong chondrogenic differentiation capacity. However, due to the lack of a suitable chondrogenic niche, the clinical application of CPC-regenerated cartilage in the subcutaneous environment remains a challenge. In this study, exosomes derived from chondrocytes (CC-Exos) were used to provide the CPC constructs with a cartilage signal in subcutaneous environments for efficient ectopic cartilage regeneration. Rabbit CPC-alginate constructs were prepared and implanted subcutaneously in nude mice. CC-Exos were injected into the constructs at the same dose (30 μg exosomes per 100 μL injection) after surgery and thereafter weekly for a period of 12 weeks. Exosomes derived from bone mesenchymal stem cells (BMSC-Exos) were used as the positive control. The mice in the negative control were administered with the same volume of PBS. At 4 and 12 weeks after implantation, the potential of CC-Exos and BMSC-Exos to promote chondrogenesis and stability of cartilage tissue in a subcutaneous environment were analyzed by histology, immunostaining, and protein analysis. The influences of BMSC-Exos and CC-Exos on chondrogenesis and angiogenic characteristics in vitro were assessed via coculturing with CPCs and human umbilical vein endothelial cells. The CC-Exos injection increased collagen deposition and minimized vascular ingrowth in engineered constructs, which efficiently and reproducibly developed into cartilage. The generated cartilage was phenotypically stable with minimal hypertrophy and vessel ingrowth up to 12 weeks, while the cartilage formed with BMSC-Exos was characterized by hypertrophic differentiation accompanied by vascular ingrowth. In vitro experiments indicated that CC-Exos stimulated CPCs proliferation and increased expression of chondrogenesis markers while inhibiting angiogenesis. These findings suggest that the novel CC-Exos provides the preferable niche in directing stable ectopic chondrogenesis of CPCs. The use of CC-Exos may represent an off-the-shelf and cell-free therapeutic approach for promoting cartilage regeneration in the subcutaneous environment.

Bone marrow-derived stem cells (BMSCs) and chondrocytes have been reported to present “dedifferentiation” and “phenotypic loss” during the chondrogenic differentiation process in cartilage tissue engineering, and cartilage progenitor cells (CPCs) are novel seeding cells for cartilage tissue engineering. In our previous study, cartilage progenitor cells from different subtypes of cartilage tissue were isolated and identified in vitro, but the study on in vivo chondrogenic characteristics of cartilage progenitor cells remained rarely. In the current study, we explored the feasibility of combining cartilage progenitor cells with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) to produce tissue-engineered cartilage and compared the proliferation ability and chondrogenic characteristics of cartilage progenitor cells with those of bone marrow-derived stem cells and chondrocytes. These three cells combined with PHBV were cultured in vitro for 1 week without chondrogenic induction and then transplanted subcutaneously into nude mice for 6 weeks. The cell-PHBV constructs were evaluated by gross observation, histological staining, glycosaminoglycan content measurement, biomechanical analysis and RT-PCR. The chondrocyte-PHBV constructs and CPC-PHBV constructs became an ivory-whitish cartilage-like tissue, while the BMSC-PHBV constructs became vascularized 6 weeks after the subcutaneous implantation. Histological examination showed that many typical cartilage structures were present in the chondrocyte group, some typical cartilage structures were observed in the CPC group, while no typical cartilage structures were observed in the BMSC group. Cartilage progenitor cells may undergo chondrogenesis without chondrogenic induction and are better at chondrogenesis than BMSCs but worse than chondrocytes in the application of cartilage tissue engineering.

With the help of biomaterials, cartilage stem/progenitor cells (CSPCs) derived from cartilage tissue present a promising choice for cartilage regeneration. In our previous study, we investigated whether CSPCs could be ideal seeding cells for cartilage tissue regeneration. Biomaterials are fabricated to accelerate tissue regeneration, providing a suitable environment for cell attachment, proliferation, and differentiation. Among the biomaterials used in cartilage regeneration medicine, alginate and collagen are classified as natural biomaterials and are characterized by high biocompatibility, bioactivity, and non-toxic degradation products. However, it is unclear which material would have a competitive advantage in CSPC-based cartilage regeneration in vivo. In the present study, we employed alginate and type Ⅰ collagen as substrates for CSPCs and chondrocytes, which was made control group, to explore a more suitable biomaterials for CSPCs to fabricate tissue-engineered cartilage, in vivo. Hematoxylin and eosin (HE) staining, Safranin O, immunohistochemical assay, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to evaluate the tissue-engineered cartilage in vivo. Compared with the alginate group, collagen enhanced the expression of cartilage-specific genes, such as ACAN, SOX9, and COLII, more markedly. Furthermore, the marker genes of expression, dedifferentiation, and hypertrophy, COLI and COLX, were downregulated in the collagen group. The results demonstrated that collagen as a substrate was superior to alginate in increasing the accumulation of cartilage-like ECM for CSPCs in vivo. In summary, compared with alginate, collagen hydrogel is an effective biomaterial for CSPC-based cartilage regeneration.

Repair of cartilage defects remains a challenge for surgeons, owing to its poor self‑repairing capacity. Cartilage tissue engineering, particularly marrow stem cell‑based cartilage regeneration, provides a promising option for the regeneration of damaged cartilage. Although producing tissue‑engineered cartilage from marrow stem cells appeared to be a feasible method, constructing certain sub‑types of cartilage, including elastic cartilage, remains difficult. Therefore, the present study explored the feasibility of constructing elastic cartilage by culturing bone marrow‑derived stem cells (BMSCs) in the supernatant of elastic cartilage cells to generate elastic cartilage. The elastic cartilage cells were obtained from the auricle cartilage of a newborn pig, and BMSCs were isolated from pig bone marrow aspirate. The supernatant of the chondrocytes was collected and then used to the culture BMSCs. At various time‑points, the differentiation of BMSCs was evaluated by gross view, histological examination and quantitative polymerase chain reaction. BMSCs changed from spindle‑shaped cells into polygonal cells with increasing culture time. The expression of collagen II and elastin was observed in the cells cultured in the supernatant of elastic chondrocytes, while no expression was observed in the control cells. Furthermore, the expression of collagen I and collagen X was downregulated in the cells cultured in the supernatant of elastic cartilage cells. The supernatant of elastic cartilage cells promoted the differentiation of BMSCs into elastic cartilage cells, which may be a promising method for constructing certain sub‑types of tissue‑engineered cartilage.

Hypertrophic scars have long posed a significant challenge in wound healing, primarily because of an imbalance between Type I (COL‐I) and Type III (COL‐III) collagen caused by excessive fibroblast activation. Existing treatments cannot directly regulate collagen formation to intervene in scar hyperplasia. This study is the first to elucidate the key mechanism through which human umbilical cord‐derived mesenchymal stem cell (hUC‐MSCs) exosomes expressing miR‐185‐5p achieve scarless healing through remodeling of the collagen‐type ratio. In this method, a hypoxic three‐dimensional (3D‐HO) suspension culture system that more closely mimics the in vivo microenvironment was established, and functional exosomes with enhanced physiological activity and higher yields were harvested. In vivo experiments demonstrated that 3D‐HO exosomes significantly improved the COL‐III/COL‐I regeneration ratio during mouse wound healing. Mechanistic studies revealed that the RhoA/YAP signaling axis plays a key regulatory role in the collagen regeneration ratio. Further molecular analysis revealed enrichment of miR‐185‐5p in 3D‐HO exosomes, which directly target RhoA to regulate fibroblasts. Both in vivo and in vitro functional interventions confirmed miR‐185‐5p in maintaining collagen type balance. In summary, this study demonstrates that microenvironment‐enhanced hUC‐MSCs exosomes remodel the composition of different types of collagen via the miR‐185‐5p–RhoA/YAP signaling axis, thereby driving scarless healing.