Although autologous bone is considered as the most superior grafting material for bone regeneration [34
], other bone graft substitutes are increasingly preferred not only for the convenience of the surgeons but also for the good of the patients [35
]. In nature, bone healing occurs through both endochondral and intramembranous ossification [36
]. While intramembranous ossification is the fastest route of building bone matrix, it can only handle small and mechanically stable defects [36
]. Endochondral ossification, while it is slow and requires a lot more energy, is the predominant route of bone healing in fractures. During the process, mesenchymal progenitor cells differentiate into chondrocytes which deposit cartilaginous matrix called the soft callus. In animal models, the soft callus formation peaks at 7-9 days post trauma with a peak in collagen type 2 and proteoglycan such as aggrecan [38
]. The chondrocytes of the soft callus undergo maturation towards hypertrophy, becoming enlarged in size, and secreting alkaline phosphatase which mineralizes the extracellular matrix. The fate of the hypertrophic chondrocytes remains controversial. Classically, the hypertrophic chondrocytes undergo apoptosis and the osteoprogenitor cells invade the callus and replace the cartilage with bone. Alternatively, studies have shown that a proportion of hypertrophic chondrocytes do not undergo apoptosis and instead transdifferentiate into osteoblasts [12
]. In the classical model, the role of hypertrophic chondrocytes is less important after its accomplishment of synthesizing the necessary growth factors. This suggests that a hypertrophic cartilage matrix void of cells but high in endogenous growth factors can continue its route to be transformed into bone in vivo. To date, no clinical case using hypertrophic cartilage for bone regeneration has been reported. Despite abundant proof-of-principle studies, no available method can yet produce sufficient hypertrophic cartilage for clinical use. Here we demonstrated that by using the cell line ATDC5 cultured in micro-aggregates, it was feasible to produce hypertrophic cartilage in unlimited amounts. According to the FDA’s guideline for clinical issues concerning the use of xenotransplantation products in humans [40
], “cell lines from animals may be established and used in the production of xenotransplantation products”. Compared to primary cells, cell lines are easier to maintain and more consistent in quality. Since cell lines are carcinogenic, devitalization of the hypertrophic cartilage is necessary to reduce immunogenic concerns. Whether or not the devitalized MiTEC is still capable of inducing endochondral ossification in vivo needs further experiments. Bourgine et al. demonstrated that apoptosis-induced devitalized hypertrophic cartilage constructs could form bone in vivo while freeze-thawed devitalized constructs could not [18
]. They attributed this to the significant loss of glycosaminoglycans, mineral content, and ECM-bound cytokines during the freeze-thaw cycles. We also observed a loss of glycosaminoglycans, as indicated by Alcian Blue staining, in the SDS decellularized MiTEC, but not in the freeze-thaw devitalized MiTEC. Apparently, the bioactivity of the hypertrophic cartilage ECM may be lost when a sub-optimal devitalization/decellularization process is used. Hence, the balance of removing immunogenicity and retaining osteoinductivity needs to be fine-tuned, for which many devitalization/decellularization methods have been reported [41
]. Our model can potentially be adapted into a high throughput screening platforms in vivo and in vivo, allowing the devitalization/decellularization protocol to be empirically optimized.
Vascularization is a critical factor for the successful integration of large grafts. Hypertrophic cartilage has an intrinsic property of produce angiogenic growth factors, which play a crucial role in endochondral ossification and many studies have shown the positive effect of adding VEGF in bone regeneration [33
]. It has been shown that the lack of vascularization in the DCM constructs could be attributed to the loss of VEGF during the devitalization process [18
]. Vascularization in the case of endochondral bone formation is essential for the recruitment of the host cells in order to remodel the cartilage template and subsequent ossification. We showed that stimulating MiTEC with the hypoxia mimicking molecule phenanthroline boosted VEGF secretion to double its basal level, although the VEGF content which remained in the decellularized MiTEC has not been analyzed. Since vascularization is paramount to the success of this model, it may be necessary to further optimize it for instance by combining it with current vascularization strategies, i.e., VEGF impregnation [45
]. Similarly, pre-mineralization of MiTEC in vivo may be beneficial for bone formation in vivo. Calcium phosphate ceramics have a positive effect on osteogenesis [46
] and even soluble calcium ions have a positive effect on the osteogenic differentiation of hMSCs [47
]. The degree of mineralization is another aspect which can be optimized for bone formation in vivo.
The inductive potential of decellularized ECM on stem cells [48
] has been reported. It was hypothesized that the acellular ECM of a tissue possessed the instructive cues that drive the differentiation of stem cells into this particular tissue type [50
]. The instructive cues are the soluble factors and the macromolecules that regulate cell fate. To date, no study has demonstrated the inductive potential of hypertrophic cartilage matrix in vivo. In this paper, we show that devitalized/decellularized MiTEC, especially in combination with chondrogenic medium, enhanced the chondrogenic differentiation of hMSCs in vivo. From histological images it appeared that the MiTEC ECM remained intact and hMSCs were not able to enter the cartilage matrix. Thus, it’s arguable that the inductive potential of devitalized/decellularized MiTEC is caused by the soluble factors released from it, although the surface chemistry and topography of the tissue might also play a role. Interestingly, SDS decellularized MiTEC appeared to be more potent than the freeze-thaw devitalized aggregates. This may be similar to the biological activity of bone versus demineralized bone, where the removal of bone mineral makes the osteogenic molecules available for interaction with the cells [52
Unlike natural products harvested from cadavers where donor variation is inevitable, hypertrophic cartilage culture in vivo is a totally controllable process. There are many concerns over the variation in quality among allogenic bone grafts produced by different companies or even from different batches of product from the same company [53
]. Moreover, some products such as DBM and platelet gels containing “autologous growth factors” are not subjected to high level of regulatory scrutiny [55
]. In contrast, MiTEC can be cultured and tested for both safety and efficacy following stringent good manufacturing practice (GMP) protocols. Here, we demonstrated how MiTEC can be manipulated in a number of ways to modify its extracellular matrix composition. The well plate format of this system allows for high throughput screening to be used in drug discovery and, using a different cell source or cell sources, one can even switch this model to the production of a totally different type of tissue, for instance to produce liver tissue or to study tumor cell biology. In conclusion, we have shown a method to produce clinically relevant-sized hypertrophic cartilage for bone regeneration by endochondral ossification. We decellularized the hypertrophic tissue and showed that the remaining ECM had inductive potential on chondrogenic differentiation of hMSCs in vivo. The logical next phase of this work is the pre-clinical evaluation of MiTEC in orthotopic models.