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.

Single-cell mapping of regenerative and fibrotic healing responses after musculoskeletal injury.

After injury, a cascade of events repairs the damaged tissue, including expansion and differentiation of the progenitor pool and redeposition of matrix. To guide future wound regeneration strategies, we compared single-cell sequencing of regenerative (third phalangeal element [P3]) and fibrotic (second phalangeal element [P2]) digit tip amputation (DTA) models as well as traumatic heterotopic ossification (HO; aberrant). Analyses point to a common initial response to injury, including expansion of progenitors, redeposition of matrix, and activation of transforming growth factor ß (TGF-ß) and WNT pathways. Surprisingly, fibrotic P2 DTA showed greater transcriptional similarity to HO than to regenerative P3 DTA, suggesting that gene expression more strongly correlates with healing outcome than with injury type or cell origin. Differential analysis and immunostaining revealed altered activation of inflammatory pathways, such as the complement pathway, in the progenitor cells. These data suggests that common pathways are activated in response to damage but are fine tuned within each injury. Modulating these pathways may shift the balance toward regenerative outcomes.

Low bone mass and impaired fracture healing in mouse models of Trisomy21 (Down syndrome).

Individuals with Down syndrome (DS), the result of trisomy of human chromosome Hsa21 (Ts21), present with an array of skeletal abnormalities typified by altered craniofacial features, short stature and low bone mineral density (BMD). While bone deficits progress with age in both sexes, low bone mass is more pronounced in DS men than women and osteopenia appears earlier. In the current study, the reproductive hormone status (FSH, LH, testosterone) of 17 DS patients (males, ages range 19-52 years) was measured. Although testosterone was consistently low, the hypothalamic-pituitary-gonadal axis was intact with corresponding rises in FSH and LH. To provide further insight into the heterogeneity of the bone mass in DS, the skeletal phenotypes of three of the most used murine DS models, Ts65Dn (Ts65), TC1, and Dp16(Yey1) (Dp16) were characterized and contrasted. Evaluation of the bone phenotype of both male and female 3-month-old Dp16 mice demonstrated sexual dimorphism, with low bone mass apparent in males, as it is in Ts65, but not in female Dp16. In contrast, male TC1 mice had no apparent bone phenotype. To determine whether low bone mass in DS impacted fracture healing, fractures of the middle phalanx (P2) digits were generated in both male and female Dp16 mice at 15 weeks of age, an age where the sexually dimorphic low BMD persisted. Fracture healing was assessed via in vivo microCT over (13 weeks) 93 days post fracture (DPF). At 93 DPF, 0 % of DS male (n = 12) or female (n = 8) fractures healed, compared to 50 % of the male (n = 28) or female (n = 8) WT littermate fractures. MicroCT revealed periosteal unbridged mineralized callus formation across the fracture gap in Dp16 mice, which was confirmed by subsequent histology. These studies provide the first direct evidence of significantly impaired fracture healing in the setting of DS.

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.

Hyaline cartilage differentiation of fibroblasts in regeneration and regenerative medicine.

Amputation injuries in mammals are typically non-regenerative; however, joint regeneration is stimulated by BMP9 treatment, indicating the presence of latent articular chondrocyte progenitor cells. BMP9 induces a battery of chondrogenic genes in vivo, and a similar response is observed in cultures of amputation wound cells. Extended cultures of BMP9-treated cells results in differentiation of hyaline cartilage, and single cell RNAseq analysis identified wound fibroblasts as BMP9 responsive. This culture model was used to identify a BMP9-responsive adult fibroblast cell line and a culture strategy was developed to engineer hyaline cartilage for engraftment into an acutely damaged joint. Transplanted hyaline cartilage survived engraftment and maintained a hyaline cartilage phenotype, but did not form mature articular cartilage. In addition, individual hypertrophic chondrocytes were identified in some samples, indicating that the acute joint injury site can promote osteogenic progression of engrafted hyaline cartilage. The findings identify fibroblasts as a cell source for engineering articular cartilage and establish a novel experimental strategy that bridges the gap between regeneration biology and regenerative medicine.

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).

Aging Delays Epimorphic Regeneration in Mice.

Epimorphic regeneration is a multitissue regeneration process where amputation does not lead to scarring, but blastema formation and patterned morphogenesis for which cell plasticity and concerted cell-cell interactions are pivotal. Tissue regeneration declines with aging, yet if and how aging impairs epimorphic regeneration is unknown. Here, we show for the first time that aging derails the spatiotemporal regulation of epimorphic regeneration in mammals, first, by exacerbating tissue histolysis and delaying wound closure, and second, by impairing blastema differentiation and skeletal regrowth. Surprisingly, aging did not limit stem cell availability in the blastema but reduced osteoblast-dependent bone formation. Our data suggest that aging delays regeneration not by stem cell exhaustion, but functional defects of differentiated cells that may be driven by an aged wound environment and alterations in the spatiotemporal regulation of regeneration events. Our findings emphasize the importance of accurate timing of signaling events for regeneration and highlight the need for carefully timed interventions in regenerative medicine.

Evolution of epimorphosis in mammals.

Mammalian epimorphic regeneration is rare and digit tip regeneration in mice is the best-studied model for a multi-tissue regenerative event that involves blastema formation. Digit tip regeneration parallels human fingertip regeneration, thus understanding the details of this response can provide insight into developing strategies to expand the potential of human regeneration. Following amputation, the digit stump undergoes a strong histolytic response involving osteoclast-mediated bone degradation that is spatially and temporally linked to the expansion of blastema osteoprogenitor cells. Blastemal differentiation occurs via direct intramembranous ossification. Although robust, digit regeneration is imperfect: The amputated cortical bone is replaced with woven bone and there is excessive bone regeneration restricted to the dorsal-ventral axis. Ontogenetic and phylogenetic analysis of digit regeneration in amphibians and mammals raise the possibility that mammalian blastema is a product of convergent evolution and we hypothesize that digit tip regeneration evolved from a nonregenerative precondition. A model is proposed in which the mammalian blastema evolved in part from an adaptation of two bone repair strategies (the bone remodeling cycle and fracture healing) both of which are conserved across tetrapod vertebrates. The view that epimorphic regeneration evolved in mammals from a nonregenerative precondition is supported by recent studies demonstrating that complex regenerative responses can be induced from a number of different nonregenerative amputation wounds by specific modification of the healing response.

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.

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification.

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (µCT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo µCT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

BMP9 stimulates joint regeneration at digit amputation wounds in mice.

A major goal of regenerative medicine is to stimulate tissue regeneration after traumatic injury. We previously discovered that treating digit amputation wounds with BMP2 in neonatal mice stimulates endochondral ossification to regenerate the stump bone. Here we show that treating the amputation wound with BMP9 stimulates regeneration of a synovial joint that forms an articulation with the stump bone. Regenerated structures include a skeletal element lined with articular cartilage and a synovial cavity, and we demonstrate that this response requires the Prg4 gene. Combining BMP2 and BMP9 treatments in sequence stimulates the regeneration of bone and joint. These studies provide evidence that treatment of growth factors can be used to engineer a regeneration response from a non-regenerating amputation wound.

Axonal regrowth is impaired during digit tip regeneration in mice.

Mice are intrinsically capable of regenerating the tips of their digits after amputation. Mouse digit tip regeneration is reported to be a peripheral nerve-dependent event. However, it is presently unknown what types of nerves and Schwann cells innervate the digit tip, and to what extent these cells regenerate in association with the regenerative response. Given the necessity of peripheral nerves for mammalian regeneration, we investigated the neuroanatomy of the unamputated, regenerating, and regenerated mouse digit tip. Using immunohistochemistry for ß-III-tubulin (ß3T) or neurofilament H (NFH), substance P (SP), tyrosine hydroxylase (TH), myelin protein zero (P0), and glial fibrillary acidic protein (GFAP), we identified peripheral nerve axons (sensory and sympathetic), and myelinating- and non-myelinating-Schwann cells. Our findings show that the digit tip is innervated by two digital nerves that each bifurcate into a bone marrow (BM) and connective tissue (CT) branch. The BM branches are composed of sympathetic axons that are ensheathed by non-myelinating-Schwann cells whereas the CT branches are composed of sensory and sympathetic axons and are ensheathed by myelinating- and non-myelinating-Schwann cells. The regenerated digit neuroanatomy differs from unamputated digit in several key ways. First, there is 7.5 fold decrease in CT branch axons in the regenerated digit compared to the unampuated digit. Second, there is a 5.6 fold decrease in myelinating-Schwann cells in the regenerated digit compared to the unamputated digit that is consistent with the decrease in CT branch axons. Importantly, we also find that the central portion of the regenerating digit blastema is aneural, with axons and Schwann cells restricted to peripheral and distal blastema regions. Finally, we show that even with impaired innervation, digits maintain the ability to regenerate after re-amputation. Taken together, these data indicate that nerve regeneration is impaired in the context of mouse digit tip 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.

Digit Tip Regeneration: Merging Regeneration Biology with Regenerative Medicine.

Regeneration Biology is the study of organisms with endogenous regenerative abilities, whereas Regenerative Medicine focuses on engineering solutions for human injuries that do not regenerate. While the two fields are fundamentally different in their approach, there is an obvious interface involving mammalian regeneration models. The fingertip is the only part of the human limb that is regeneration-competent and the regenerating mouse digit tip has emerged as a model to study a clinically relevant regenerative response. In this article, we discuss how studies of digit tip regeneration have identified critical components of the regenerative response, and how an understanding of endogenous regeneration can lead to expanding the regenerative capabilities of nonregenerative amputation wounds. Such studies demonstrate that regeneration-incompetent wounds can respond to treatment with individual morphogenetic agents by initiating a multi-tissue response that culminates in structural regeneration. In addition, the healing process of nonregenerative wounds are found to cycle through nonresponsive, responsive and nonresponsive phases, and we call the responsive phase the Regeneration Window. We also find the responsiveness of mature healed amputation wounds can be reactivated by reinjury, thus nonregenerated wounds retain a potential for regeneration. We propose that regeneration-incompetent injuries possess dormant regenerative potential that can be activated by targeted treatment with specific morphogenetic agents. We believe that future Regenerative Medicine-based-therapies should be designed to promote, not replace, regenerative responses. Stem Cells Translational Medicine 2018;7:262-270.

The periosteal requirement and temporal dynamics of BMP2-induced middle phalanx regeneration in the adult mouse.

Regeneration of mammalian limbs is restricted to amputation of the distal digit tip, the terminal phalanx (P3). The adjacent skeletal element, the middle phalanx (P2), has emerged as a model system to investigate regenerative failure and as a site to test approaches aimed at enhancing regeneration. We report that exogenous application of bone morphogenetic protein 2 (BMP2) stimulates the formation of a transient cartilaginous callus distal to the amputation plane that mediates the regeneration of the amputated P2 bone. BMP2 initiates a significant regeneration response during the periosteal-derived cartilaginous healing phase of P2 bone repair, yet fails to induce regeneration in the absence of periosteal tissue, or after boney callus formation. We provide evidence that a temporal component exists in the induced regeneration of P2 that we define as the "regeneration window." In this window, cells are transiently responsive to BMP2 after the amputation injury. Simple re-injury of the healed P2 stump acts to reinitiate endogenous bone repair, complete with periosteal chondrogenesis, thus reopening the "regeneration window" and thereby recreating a regeneration-permissive environment that is responsive to exogenous BMP2 treatment.

Macrophages are required to coordinate mouse digit tip regeneration.

In mammals, macrophages are known to play a major role in tissue regeneration. They contribute to inflammation, histolysis, re-epithelialization, revascularization and cell proliferation. Macrophages have been shown to be essential for regeneration in salamanders and fish, but their role has not been elucidated in mammalian epimorphic regeneration. Here, using the regenerating mouse digit tip as a mammalian model, we demonstrate that macrophages are essential for the regeneration process. Using cell-depletion strategies, we show that regeneration is completely inhibited; bone histolysis does not occur, wound re-epithelialization is inhibited and the blastema does not form. Although rescue of epidermal wound closure in the absence of macrophages promotes blastema accumulation, it does not rescue cell differentiation, indicating that macrophages play a key role in the redifferentiation of the blastema. We provide additional evidence that although bone degradation is a component, it is not essential to the overall regenerative process. These findings show that macrophages play an essential role in coordinating the epimorphic regenerative response in mammals.

Analogous cellular contribution and healing mechanisms following digit amputation and phalangeal fracture in mice.

Regeneration of amputated structures is severely limited in humans and mice, with complete regeneration restricted to the distal portion of the terminal phalanx (P3). Here, we investigate the dynamic tissue repair response of the second phalangeal element (P2) post amputation in the adult mouse, and show that the repair response of the amputated bone is similar to the proximal P2 bone fragment in fracture healing. The regeneration-incompetent P2 amputation response is characterized by periosteal endochondral ossification resulting in the deposition of new trabecular bone, corresponding to a significant increase in bone volume; however, this response is not associated with bone lengthening. We show that cells of the periosteum respond to amputation and fracture by contributing both chondrocytes and osteoblasts to the endochondral ossification response. Based on our studies, we suggest that the amputation response represents an attempt at regeneration that ultimately fails due to the lack of a distal organizing influence that is present in fracture healing.

Epidermal closure regulates histolysis during mammalian (Mus) digit regeneration.

Mammalian digit regeneration progresses through consistent stages: histolysis, inflammation, epidermal closure, blastema formation, and finally redifferentiation. What we do not yet know is how each stage can affect others. Questions of stage timing, tissue interactions, and microenvironmental states are becoming increasingly important as we look toward solutions for whole limb regeneration. This study focuses on the timing of epidermal closure which, in mammals, is delayed compared to more regenerative animals like the axolotl. We use a standard wound closure device, Dermabond (2-octyl cyanoacrylate), to induce earlier epidermal closure, and we evaluate the effect of fast epidermal closure on histolysis, blastema formation, and redifferentiation. We find that fast epidermal closure is reliant upon a hypoxic microenvironment. Additionally, early epidermal closure eliminates the histolysis stage and results in a regenerate that more closely replicates the amputated structure. We show that tools like Dermabond and oxygen are able to independently influence the various stages of regeneration enabling us to uncouple histolysis, wound closure, and other regenerative events. With this study, we start to understand how each stage of mammalian digit regeneration is controlled.

The mammalian blastema: regeneration at our fingertips.

In the mouse, digit tip regeneration progresses through a series of discrete stages that include inflammation, histolysis, epidermal closure, blastema formation, and redifferentiation. Recent studies reveal how each regenerative stage influences subsequent stages to establish a blastema that directs the successful regeneration of a complex mammalian structure. The focus of this review is on early events of healing and how an amputation wound transitions into a functional blastema. The stepwise formation of a mammalian blastema is proposed to provide a model for how specific targeted treatments can enhance regenerative performance in humans.