Background

The structural and functional repair of sizeable subcutaneous cartilage defects remains a challenge in plastic and reconstructive surgery [1, 50,51], which was fraught with a couple of shortcomings [5, 52, 53]. Tissue-engineered cartilage grafts have emerged as a promising alternative to overcome these problems and satisfy the ever-increasing clinical need [54,55,56]. Currently, exosomes have been identified as the principal agent in mediating the therapeutic efficacy of the cell-based regenerative medicine approach [20, 21, 57, 58], and BMSC-Exos have been reported for promoting in situ cartilage defect repair [21, 26]. However, the therapeutic outcome for subcutaneous cartilage defect repair is still limited because of the lack of a suitable pro-chondrogenic environment [17, 40, 59]. Meanwhile, previous studies have also shown that chondrocytes could steer the chondrogenesis of stem cells in vitro and in vivo through paracrine effects [1, 7, 60]. In the present study, using a CPC-based cartilage tissue engineering approach, the potential of CC-Exos in promoting ectopia chondrogenesis and stabilizing cartilage regeneration in a subcutaneous environment was further investigated.

The current study demonstrated that CPC constructs supplied with CC-Exos could form homogeneous cartilage-like tissue with minimal hypertrophy in a subcutaneous environment, with no help from any chondrogenic factors. Furthermore, a series of in vitro experiments further confirmed that CC-Exos significantly promoted chondrogenesis-related factors at the mRNA and protein levels in CPCs, such as SOX-9 and COL II. Importantly, angiogenesis was inhibited by CC-Exos, which is known to be detrimental to cartilage regeneration leading to hypertrophic differentiation and subsequent calcification [44]. The observed contributions of CC-Exos to cartilage regeneration in vivo corroborate the in vitro findings and further support that CC-Exos alone could provide a preferable chondrogenic environment and help maintain the stability of cartilage tissue. Compared with BMSC-Exos where samples show more hypertrophic cartilage, the cartilage regeneration results achieved with the use of CC-Exos are significantly more favorable. Hence, the null hypothesis that there is no difference between CC-Exos and BMSC-Exos in cartilage regeneration results must be rejected.

To date, tissue engineering has offered promising solutions for clinical issues involving congenital and acquired cartilage defects [7, 61, 62]. However, the cartilage formation in subcutaneous environments is limited due to the lack of a proper chondrogenic niche [11, 12, 59]. Imitating the chondrogenic niche is a well-accepted approach to promote the ectopic chondrogenesis of progenitor cells [16, 18]. Exosomes have been studied extensively for their potential in participating in the maintenance of normal physiology via delivering various types of bioactive microRNAs, nucleic acids, proteins, and unique gene products [22, 63]. Recent studies have shown that chondrocytes and chondrocyte-related factors play key regulatory roles in the maintenance of the cartilage microenvironment and the ultimate cartilage phenotype of implanted stem cells [13,14,15]. In the present study, we further demonstrate that CC-Exos modulates CPC migration, proliferation, and cartilage matrix synthesis. Expression of SOX-9 and COL II by CPCs is upregulated in the presence of CC-Exos, which promotes chondrogenesis. This may be attributed to the TGF-β/SMAD signaling pathway, which is reported to play an essential role in chondrocyte differentiation and matrix maturation [41, 45, 64]. More investigations are needed to acquire the whole picture of the pathway involved in CC-Exos-induced chondrogenesis.

Additionally, reproducibly generating stable cartilage remains an unsolved challenge. Avoiding vessel ingrowth and hypertrophy is a critical factor in building stable cartilage [65, 66]. In the present study, compared to the positive control groups (BMSC-Exos), CC-Exos could maintain a stabilized phenotype of constructed cartilage at least within the investigated time frame, as evidenced by the presence of significantly less COL X-positive staining and minimal protein expression of COL X, IHH, and MMP 13 secreted by hypertrophic chondrocytes at 12 weeks. In addition, less CD31-positive microvessels are observed in the neo-cartilage of the CC-Exos group. However, after the addition of BMSC-Exos, expression of SDF-1 and VEGF is upregulated, which promotes cell homing and angiogenesis. This is beneficial for cartilage engineering during the early stage of implantation [40], which may account for the better neo-cartilage formation in the BMSC-Exos, as reported [21, 26]. However, it also has a disadvantage as evidenced by promoting associated ectopic cartilage hypertrophy. Recent studies have also shown that vascular invasion is one of the major mechanisms involved in hypertrophic cartilage differentiation [5, 44]. In vitro results also revealed that HUVEC migration and tube formation are reduced by CC-Exos when compared with BMSC-Exos. These results collaborate with data collected from an in vivo experiment, which shows CC-Exos have the ability to decrease angiogenesis in subcutaneous cartilage repair. Because CC-Exos can promote CPC migration, proliferation, and matrix synthesis in vitro, a more favorable prognosis is anticipated for long-term cartilage regeneration.

A novel method of imitating the chondrogenic niche is explored in the present work via the use of CC-Exos. After local injection of CC-Exos, the CPCs are rapidly directed to form neo-cartilage, when stimulated by chondroinductive mediators. Importantly, the engineered cartilage here can maintain the stabilized phenotype in non-chondrogenic niches, which is probably related to antiangiogenic factors secreted by CC-Exos that prevent neovascularization and hypertrophy. Because strategies that provide the conventional cartilage environment often require cell-based therapy [54, 55], the use of CC-Exos is advantageous from the perspectives of off-the-shelf and cell-free regenerative medicine approach for cartilage repair, and the ease of minimally invasive injection of CC-Exos concentrate.

Despite these encouraging results, the exact component(s) is yet to be elucidated. It is plausible that a myriad of components is present in the CC-Exos that can orchestrate cartilage regeneration including chondrogenesis and stability. However, the detailed mechanism of CC-Exos treatment to CPCs that caused the difference from that of BMSC-Exos is still unclear, and further investigation of RNA-seq is needed to dissect the components present in CC-Exos and to investigate their underlying mechanisms in cartilage repair. In addition, chondrogenesis is a complex process which is related to various signaling pathways, such as wnt, TGF-β, and hedgehog pathway. Here, we preliminarily demonstrated the induction of TGF-β and downstream SMAD2/3 expression after CC-Exos treatment. Further investigation is needed to acquire the entire picture of the pathway.

Conclusions

In summary, this study demonstrated that a novel exosome from chondrocytes could imitate the chondrogenic niche in a subcutaneous environment, which could facilitate chondrogenesis and maintenance of cartilage stability. This may contribute to its preferable chondroinductive niches coupled with its antiangiogenic properties. Thus, CC-Exos may represent a promising biologic-based therapeutic approach for the treatment of ectopic cartilage defects.