Limb derived cells as a paradigm for engineering self‐assembling skeletal tissues

Abstract

Mimicking developmental events has been proposed as a strategy to engineer tissue constructs for regenerative medicine. However, this approach has not yet been investigated for skeletal tissues. Here, it is demonstrated that ectopic implantation of day-14.5 mouse embryonic long bone anlagen, dissociated into single cells and randomly incorporated in a bioengineered construct, gives rise to epiphyseal growth plate-like structures, bone and marrow, which share many morphological and molecular similarities to epiphyseal units that form after transplanting intact long bone anlage, demonstrating substantial robustness and autonomy of complex tissue self-assembly and the overall organogenesis process. In vitro studies confirm the self-aggregation and patterning capacity of anlage cells and demonstrate that the model can be used to evaluate the effects of large and small molecules on biological behaviour. These results reveal the preservation of self-organizing and self-patterning capacity of anlage cells even when disconnected from their developmental niche and subjected to system perturbations such as cellular dissociation. These inherent features make long bone anlage cells attractive as a model system for tissue engineering technologies aimed at creating constructs that have the potential to self-assemble and self-pattern complex architectural structures.

Uncoupling of in-vitro identity of embryonic limb derived skeletal progenitors and their in-vivo bone forming potential

“…Thus, the cells were able to maintain their developmental fidelity, suggesting that the bone formation is a cell intrinsic process. This has been demonstrated by previous work in our group 20 . However, when the cells were encapsulated in alginate, bone formation was very limited.…”

Developmentally Engineered Callus Organoid Bioassemblies Exhibit Predictive In Vivo Long Bone Healing

“…Ectopic samples were sectioned at 5 µm and tibias at 6 µm. Histology was performed according to previously reported methods of H&E, Alcian Blue, Masson's Trichrome, and Safranin O staining . Immuno‐histochemistry was performed on PFA‐fixed microtissues (Osterix), paraffin‐embedded PFA‐fixed microtissues (Indian Hedgehog), and paraffin‐embedded EDTA‐decalcified explants (human osteocalcin, CD31).…”

“…According to this strategy, cellular self‐assemblies and condensations of the appropriate length scale are key initiators for the formation of transient tissue structures capable of executing developmental programs with a high level of independence leading to organogenesis processes . These processes are regulated through the activation of tissue‐specific genes and pathways characterized by a high degree of autonomy resulting in tissues that are able to undergo a similar cascade of processes even ex vivo . This type of recapitulation of developmental events has previously been demonstrated with human adult stem cells, for example, for the formation of epithelial and liver organoids.…”

“…In the context of bone tissue engineering, fracture healing of long bones includes the formation of a cartilaginous “soft callus” that subsequently is transformed into bone, a process that resembles the well‐described and tightly synchronized process of endochondral ossification in the growth plate during development . The autonomy of the growth plate cartilage in embryonic cartilage anlagen was previously reported, and even when the cartilage anlage was decomposed into single cells and re‐implanted subcutaneously, re‐organization occurred and a growth plate‐like structure was formed . Furthermore, investigations inspired by “developmental engineering” demonstrated recapitulation of endochondral ossification in ectopic environments using embryonic stem cells or bone marrow mesenchymal stromal cells (BM‐MSCs) and orthotopically using rat or human BM‐MSCs.…”

The Third Era of Tissue Engineering: Reversing the Innovation Drivers

“…Qualitative examples of the DE approach have been successfully provided, for instance in bone tissue engineering, where cartilage intermediate templates have been shown to give rise to bone ossicles through endochondral ossification. The DE approach was also applied successfully using both adult [11,12] and embryonic [13,14] stem cell sources and both scaffold-free and scaffold-based approaches [15,16], demonstrating the robustness of this paradigm. However, to date, it has not yet been demonstrated that TE implants can guide regeneration in vivo, leading to an outcome where contaminating tissue structures are absent (contaminating tissues are tissues unrelated to the regenerative context and not contributing to TE product potency).…”