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.

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.

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.

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.

Gastric Schwannoma, a benign neoplasm hiding as gastrointestinal stromal tumour.

Gastric Schwannomas are rare, submucosal mesenchymal tumours arising from the nerve plexus of the Gut wall. They have an incidence rate of 0.2% of all Gastric tumours. They are mostly asymptomatic, but can present with vague gastrointestinal symptoms and the fact that histopathologically, they are similar to Gastrointestinal stromal tumours, so they are most often misinterpreted as Gastrointestinal stromal tumours. We present a case, which was also hiding as GIST (Gastrointestinal stromal tumours) preoperatively, on the basis of Endoscopic and Radiological investigations. The diagnosis was then confirmed as Gastric Schwannoma, postoperatively by immunohistochemical analysis. Hence, it is important to diagnose correctly to make appropriate treatment decisions. The findings presented here can may be helpful for a clinician for diagnosing a Gastric Schwannoma.

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.

Retroperitoneal necrotizing fasciitis in a 42-year-old male patient: A case report.

Necrotizing fasciitis are rare but often fatal conditions. A retroperitoneal origin is very rare and limited to case studies; very few cases have been reported in the literature. We report a case of 42-year-old man who presented with complaints of severe constipation and paraumbilical abdominal pain for the past three days. On examination, the abdomen was tender and distended, giving features of bowel obstruction. CT scan suggested perforated appendix. Subsequent exploration revealed retroperitoneal necrotizing fasciitis extending down to right testicular tissue. After extensive debridement and drain placement, the patient was admitted to the ICU where with intensive monitoring and aggressive daily dressing the patient survived. Necrotizing fasciitis of other anatomical sites are easier to diagnose as compared to retroperitoneal origin. Focus should be placed while dealing with cases of acute abdomen as early diagnosis and prompt surgical intervention is needed for successful treatment.

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.

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).

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.

A Nonsynonymous/Synonymous Substitution Analysis of the B56 Gene Family Aids in Understanding B56 Isoform Diversity.

Gene duplication leads to the formation of gene families, wherein purifying or neutral selection maintains the original gene function, while diversifying selection confers new functions onto duplicated genes. The B56 gene family is highly conserved; it is encoded by one gene in protists and fungi, and five genes in vertebrates. B56 regulates protein phosphatase 2A (PP2A), an abundant heterotrimeric serine/threonine phosphatase that functions as a tumor suppressor and consists of a scaffolding "A" and catalytic "C" subunit heterodimer bound to a regulatory "B" subunit. Individual regulatory B56 subunits confer disparate functions onto PP2A in various cell-cell signaling pathways. B56 proteins share a conserved central core domain, but have divergent N- and C-termini which play a role in isoform specificity. We carried out a nonsynonymous/synonymous substitution analysis to better understand the divergence of vertebrate B56 genes. When five B56 paralogs from ten vertebrate species were analyzed, the gene family displayed purifying selection; stronger purifying selection was revealed when individual B56 isoforms were analyzed separately. The B56 core experienced stronger purifying selection than the N- and C-termini, which correlates with the presence of several contacts between the core and the AC heterodimer. Indeed, the majority of the contact points that we analyzed between B56 and the AC heterodimer experienced strong purifying selection. B56 subfamilies showed distinct patterns of selection in their N- and C-termini. The C-terminus of the B56-1 subfamily and the N-terminus of the B56-2 subfamily exhibited strong purifying selection, suggesting that these termini carry out subfamily-specific functions, while the opposite termini exhibited diversifying selection and likely carry out isoform-specific functions. We also found reduced synonymous substitutions at the N- and C-termini when grouping B56 genes by species but not by isoform, suggesting species-specific codon bias may have a role in regulating B56 gene expression.