Induced regeneration of articular cartilage - identification of a dormant regeneration program for a non-regenerative tissue.

A mouse organoid culture model was developed to regenerate articular cartilage by sequential treatment with BMP2 and BMP9 (or GDF2) that parallels induced joint regeneration at digit amputation wounds in vivo. BMP9-induced chondrogenesis was used to identify clonal cell lines for articular chondrocyte and hypertrophic chondrocyte progenitor cells from digit fibroblasts. A protocol that includes cell aggregation enhanced by BMP2 followed by BMP9-induced chondrogenesis resulted in the differentiation of organized layers of articular chondrocytes, similar to the organization of middle and deep zones of articular cartilage in situ, and retained a differentiated phenotype following transplantation. In addition, the differentiation of a non-chondrogenic connective tissue layer containing articular chondrocyte progenitor cells demonstrated that progenitor cell sequestration is coupled with articular cartilage differentiation at a clonal level. The studies identify a dormant endogenous regenerative program for a non-regenerative tissue in which fibroblast-derived progenitor cells can be induced to initiate morphogenetic and differentiative programs that include progenitor cell sequestration. The identification of dormant regenerative programs in non-regenerative tissues such as articular cartilage represents a novel strategy that integrates regeneration biology with regenerative medicine.

Microcomputed tomography staging of bone histolysis in the regenerating mouse digit.

Humans and mice have the ability to regenerate the distal digit tip, the terminal phalanx (P3) in response to amputation. What distinguishes P3 regeneration from regenerative failure is formation of the blastema, a proliferative structure that undergoes morphogenesis to regenerate the amputated tissues. P3 regeneration is characterised by the phases of inflammation, tissue histolysis and expansive bone degradation with simultaneous blastema formation, wound closure and finally blastemal differentiation to restore the amputated structures. While each regenerating digit faithfully progresses through all phases of regeneration, phase progression has traditionally been delineated by time, that is, days postamputation (DPA), yet there is widespread variability in the timing of the individual phases. To diminish variability between digits during tissue histolysis and blastema formation, we have established an in-vivo method using microcomputed tomography (micro CT) scanning to identify five distinct stages of the early regeneration response based on anatomical changes of the digit stump. We report that categorising the initial phases of digit regeneration by stage rather than time greatly diminishes the variability between digits with respect to changes in bone volume and length. Also, stages correlate with the levels of cell proliferation, osteoclast recruitment and osteoprogenitor cell recruitment. Importantly, micro CT staging provides a means to estimate open versus closed digit wounds. We demonstrate two spatially distinct and stage specific bone repair/regeneration responses that occur during P3 regeneration. Collectively, these studies showcase the utility of micro CT imaging to infer the composition of radiolucent soft tissues during P3 blastema formation. Specifically, the staging system identifies the onset of cell proliferation, osteoclastogenesis, osteoprogenitor recruitment, the spatial initiation of de novo bone formation and epidermal closure.

Digit specific denervation does not inhibit mouse digit tip regeneration.

It is long-established that innervation-dependent production of neurotrophic factors is required for blastema formation and epimorphic regeneration of appendages in fish and amphibians. The regenerating mouse digit tip and the human fingertip are mammalian models for epimorphic regeneration, and limb denervation in mice inhibits this response. A complicating issue of limb denervation studies in terrestrial vertebrates is that the experimental models also cause severe paralysis therefore impairing appendage use and diminishing mechanical loading of the denervated tissues. Thus, it is unclear whether the limb denervation impairs regeneration via loss of neurotrophic signaling or loss of mechanical load, or both. Herein, we developed a novel surgical procedure in which individual digits were specifically denervated without impairing ambulation and mechanical loading. We demonstrate that digit specific denervation does not inhibit but attenuates digit tip regeneration, in part due to a delay in wound healing. However, treating denervated digits with a wound dressing that enhances closure results in a partial rescue of the regeneration response. Contrary to the current understanding of mammalian epimorphic regeneration, these studies demonstrate that mouse digit tip regeneration is not peripheral nerve dependent, an observation that should inform continued mammalian regenerative medicine approaches.

Epimorphic regeneration of the mouse digit tip is finite.

BACKGROUND: Structural regeneration of amputated appendages by blastema-mediated, epimorphic regeneration is a process whose mechanisms are beginning to be employed for inducing regeneration. While epimorphic regeneration is classically studied in non-amniote vertebrates such as salamanders, mammals also possess a limited ability for epimorphic regeneration, best exemplified by the regeneration of the distal mouse digit tip. A fundamental, but still unresolved question is whether epimorphic regeneration and blastema formation is exhaustible, similar to the finite limits of stem-cell mediated tissue regeneration. METHODS: In this study, distal mouse digits were amputated, allowed to regenerate and then repeatedly amputated. To quantify the extent and patterning of the regenerated digit, the digit bone as the most prominent regenerating element in the mouse digit was followed by in vivo µCT. RESULTS: Analyses revealed that digit regeneration is indeed progressively attenuated, beginning after the second regeneration cycle, but that the pattern is faithfully restored until the end of the fourth regeneration cycle. Surprisingly, when unamputated digits in the vicinity of repeatedly amputated digits were themselves amputated, these new amputations also exhibited a similarly attenuated regeneration response, suggesting a systemic component to the amputation injury response. CONCLUSIONS: In sum, these data suggest that epimorphic regeneration in mammals is finite and due to the exhaustion of the proliferation and differentiation capacity of the blastema cell source.

Mouse Digit Tip Regeneration Is Mechanical Load Dependent.

Amputation of the mouse digit tip results in blastema-mediated regeneration. In this model, new bone regenerates de novo to lengthen the amputated stump bone, resulting in a functional replacement of the terminal phalangeal element along with associated non-skeletal tissues. Physiological examples of bone repair, such as distraction osteogenesis and fracture repair, are well known to require mechanical loading. However, the role of mechanical loading during mammalian digit tip regeneration is unknown. In this study, we demonstrate that reducing mechanical loading inhibits blastema formation by attenuating bone resorption and wound closure, resulting in the complete inhibition of digit regeneration. Mechanical unloading effects on wound healing and regeneration are completely reversible when mechanical loading is restored. Mechanical unloading after blastema formation results in a reduced rate of de novo bone formation, demonstrating mechanical load dependence of the bone regenerative response. Moreover, enhancing the wound-healing response of mechanically unloaded digits with the cyanoacrylate tissue adhesive Dermabond improves wound closure and partially rescues digit tip regeneration. Taken together, these results demonstrate that mammalian digit tip regeneration is mechanical load-dependent. Given that human fingertip regeneration shares many characteristics with the mouse digit tip, these results identify mechanical load as a previously unappreciated requirement for de novo bone regeneration in humans. © 2021 American Society for Bone and Mineral Research (ASBMR).

Proximal digit tip amputation initiates simultaneous blastema and transient fibrosis formation and results in partial regeneration.

Complete extremity regeneration in mammals is restricted to distal amputations of the digit tip, the terminal phalanx (P3). In mice, P3 regeneration is mediated via the formation of a blastema, a transient population of progenitor cells that form from the blending of periosteal and endosteal/marrow compartmentalized cells that undergo differentiation to restore the amputated structures. Compartmentalized blastema cells are formed independently, and periosteal compartment-derived cells are required for restoration of amputated skeletal length. P3 regenerative capacity is progressively attenuated at increasingly more proximal amputation levels, eventually resulting in regenerative failure. The continuum of regenerative capacity within the P3 wound milieu is a unique model to investigate mammalian blastema formation in response to distal amputation, as well as the healing response associated with regenerative failure at proximal amputation levels. We report that P3 proximal amputation healing, previously reported to result in regenerative failure, is not an example of complete regenerative failure, but instead is characterized by a limited bone regeneration response restricted to the endosteal/marrow compartment. The regeneration response is mediated by blastema formation within the endosteal/marrow compartment, and blastemal osteogenesis progresses through intramembranous ossification in a polarized proximal to distal sequence. Unlike bone regeneration following distal P3 amputation, osteogenesis within the periosteal compartment is not observed in response to proximal P3 amputation. We provide evidence that proximal P3 amputation initiates the formation of fibrotic tissue that isolates the endosteal/marrow compartment from the periosteal compartment and wound epidermis. While the fibrotic response is transient and later resolved, these studies demonstrate that blastema formation and fibrosis can occur in close proximity, with the regenerative response dominating the final outcome. Moreover, the results suggest that the attenuated proximal P3 regeneration response is associated with the absence of periosteal-compartment participation in blastema formation and bone regeneration.

Blastema formation and periosteal ossification in the regenerating adult mouse digit.

While mammals cannot regenerate amputated limbs, mice and humans have regenerative ability restricted to amputations transecting the digit tip, including the terminal phalanx (P3). In mice, the regeneration process is epimorphic and mediated by the formation of a blastema comprised of undifferentiated proliferating cells that differentiate to regenerate the amputated structures. Blastema formation distinguishes the regenerative response from a scar-forming healing response. The mouse digit tip serves as a preclinical model to investigate mammalian blastema formation and endogenous regenerative capabilities. We report that P3 blastema formation initiates prior to epidermal closure and concurrent with the bone histolytic response. In this early healing response, proliferation and cells entering the early stages of osteogenesis are localized to the periosteal and endosteal bone compartments. After the completion of stump bone histolysis, epidermal closure is completed and cells associated with the periosteal and endosteal compartments blend to form the blastema proper. Osteogenesis associated with the periosteum occurs as a polarized progressive wave of new bone formation that extends from the amputated stump and restores skeletal length. Bone patterning is restored along the proximal-distal and medial digit axes, but is imperfect in the dorsal-ventral axis with the regeneration of excessive new bone that accounts for the enhanced regenerated bone volume noted in previous studies. Periosteum depletion studies show that this compartment is required for the regeneration of new bone distal to the original amputation plane. These studies provide evidence that blastema formation initiates early in the healing response and that the periosteum is an essential tissue for successful epimorphic regeneration in mammals.