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.

Biological Consequences of Vanadium Effects on Formation of Reactive Oxygen Species and Lipid Peroxidation.

Lipid peroxidation (LPO), a process that affects human health, can be induced by exposure to vanadium salts and compounds. LPO is often exacerbated by oxidation stress, with some forms of vanadium providing protective effects. The LPO reaction involves the oxidation of the alkene bonds, primarily in polyunsaturated fatty acids, in a chain reaction to form radical and reactive oxygen species (ROS). LPO reactions typically affect cellular membranes through direct effects on membrane structure and function as well as impacting other cellular functions due to increases in ROS. Although LPO effects on mitochondrial function have been studied in detail, other cellular components and organelles are affected. Because vanadium salts and complexes can induce ROS formation both directly and indirectly, the study of LPO arising from increased ROS should include investigations of both processes. This is made more challenging by the range of vanadium species that exist under physiological conditions and the diverse effects of these species. Thus, complex vanadium chemistry requires speciation studies of vanadium to evaluate the direct and indirect effects of the various species that are present during vanadium exposure. Undoubtedly, speciation is important in assessing how vanadium exerts effects in biological systems and is likely the underlying cause for some of the beneficial effects reported in cancerous, diabetic, neurodegenerative conditions and other diseased tissues impacted by LPO processes. Speciation of vanadium, together with investigations of ROS and LPO, should be considered in future biological studies evaluating vanadium effects on the formation of ROS and on LPO in cells, tissues, and organisms as discussed in this review.

Effect of Targeted Cytokine Inhibition on Progression of Post-Traumatic Osteoarthritis Following Intra-Articular Fracture.

Intra-articular fractures (IAF) result in significant and prolonged inflammation, increasing the chances of developing post-traumatic osteoarthritis (PTOA). Interleukin-one beta (IL-1ß) and Tumor Necrosis Factor-alpha (TNF-α) are key inflammatory factors shown to be involved in osteochondral degradation following IAF. As such, use of targeted biologics such as Infliximab (INX), a TNF-α inhibitor, and Anakinra (ANR), an interleukin-one (IL-1) receptor antagonist (IL1RA), may protect against PTOA by damping the inflammatory response to IAF and reducing osteochondral degradation. To test this hypothesis, IAFs were induced in the hindlimb knee joints of rats treated with INX at 10 mg/kg/day, ANR at 100 g/kg/day, or saline (vehicle control) by subcutaneous infusion for a period of two weeks and healing was evaluated at 8-weeks post injury. Serum and synovial fluid (SF) were analyzed for soluble factors. In-vivo microcomputed tomography (µCT) scans assessed bone mineral density and bone morphometry measurements. Cationic CA4+ agent assessed articular cartilage composition via ex vivo µCT. Scoring according to the Osteoarthritis Research Society International (OARSI) guidelines was performed on stained histologic tibia sections at the 56-day endpoint on a 0-6 scale. Systemically, ANR reduced many pro-inflammatory cytokines and reduced osteochondral degradation markers Cross Linked C-Telopeptide Of Type II (CTXII, p < 0.05) and tartrate-resistant acid phosphatase (TRAP, p < 0.05). ANR treatment resulted in increased chemokines; macrophage-chemotractant protein-1 (MCP-1), MPC-3, macrophage inhibitory protein 2 (MIP2) with a concomitant decrease in proinflammatory interleukin-17A (IL17A) at 14 days post-injury within the SF. Microcomputed tomography (µCT) at 56 days post-injury revealed ANR Treatment decreased epiphyseal degree of anisotropy (DA) (p < 0.05) relative to saline. No differences were found with OARSI scoring but contrast-enhanced µCT revealed a reduction in glycosaminoglycan content with ANR treatment. These findings suggest targeted cytokine inhibition, specifically IL-1 signaling, as a monotherapy has minimal utility for improving IAF healing outcomes but may have utility for promoting a more permissive inflammatory environment that would allow more potent disease modifying osteoarthritis drugs to mitigate the progression of PTOA after IAF.

Effects of Adjunct Antifibrotic Treatment within a Regenerative Rehabilitation Paradigm for Volumetric Muscle Loss.

The use of a rehabilitation approach that promotes regeneration has the potential to improve the efficacy of pro-regenerative therapies and maximize functional outcomes in the treatment of volumetric muscle loss (VML). An adjunct antifibrotic treatment could further enhance functional gains by reducing fibrotic scarring. This study aimed to evaluate the potential synergistic effects of losartan, an antifibrotic pharmaceutical, paired with a voluntary wheel running rehabilitation strategy to enhance a minced muscle graft (MMG) pro-regenerative therapy in a rodent model of VML. The animals were randomly assigned into four groups: (1) antifibrotic with rehabilitation, (2) antifibrotic without rehabilitation, (3) vehicle treatment with rehabilitation, and (4) vehicle treatment without rehabilitation. At 56 days, the neuromuscular function was assessed, and muscles were collected for histological and molecular analysis. Surprisingly, we found that the losartan treatment decreased muscle function in MMG-treated VML injuries by 56 days, while the voluntary wheel running elicited no effect. Histologic and molecular analysis revealed that losartan treatment did not reduce fibrosis. These findings suggest that losartan treatment as an adjunct therapy to a regenerative rehabilitation strategy negatively impacts muscular function and fails to promote myogenesis following VML injury. There still remains a clinical need to develop a regenerative rehabilitation treatment strategy for traumatic skeletal muscle injuries. Future studies should consider optimizing the timing and duration of adjunct antifibrotic treatments to maximize functional outcomes in VML injuries.

Retrospective characterization of a rat model of volumetric muscle loss.

Volumetric muscle loss (VML) is a pervasive injury within contemporary combat and a primary driver of disability among injured Service members. As such, VML has been a topic of investigation over the past decade as the field has sought to understand the pathology of these injuries and to develop treatment strategies which restore the form and function of the involved musculature. To date, much of this work has been performed in disparate animal models that vary significantly in terms of the species utilized, the muscle (or muscle group) affected, and the volume of muscle lost. Moreover, variation exists in the reporting of anatomical and functional outcomes within these models. When taken together, the ability to successfully assess comparative efficacy of promising therapies is currently limited. As such, greater scrutiny on the characterization of these VML models is needed to better assess the quality of evidence supporting further translation of putative therapies. Thus, the objective of this study was to retrospectively characterize anatomical and functional outcomes associated with one such VML model - the 6 mm biopsy punch model of the rat tibialis anterior muscle. Through these efforts, it was shown that this model is highly reproducible and consistent across a large number of experiments. As such, the data presented herein represent a reasonable benchmark for the expected performance of this model with utility for drawing inferences across studies and identifying therapies which have shown promise within the preclinical domain, and thus are ready for further translation towards the clinic.

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.

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.

Effect of genetic background on the dystrophic phenotype in mdx mice.

Genetic background significantly affects phenotype in multiple mouse models of human diseases, including muscular dystrophy. This phenotypic variability is partly attributed to genetic modifiers that regulate the disease process. Studies have demonstrated that introduction of the γ-sarcoglycan-null allele onto the DBA/2J background confers a more severe muscular dystrophy phenotype than the original strain, demonstrating the presence of genetic modifier loci in the DBA/2J background. To characterize the phenotype of dystrophin deficiency on the DBA/2J background, we created and phenotyped DBA/2J-congenic Dmdmdx mice (D2-mdx) and compared them with the original, C57BL/10ScSn-Dmdmdx (B10-mdx) model. These strains were compared with their respective control strains at multiple time points between 6 and 52 weeks of age. Skeletal and cardiac muscle function, inflammation, regeneration, histology and biochemistry were characterized. We found that D2-mdx mice showed significantly reduced skeletal muscle function as early as 7 weeks and reduced cardiac function by 28 weeks, suggesting that the disease phenotype is more severe than in B10-mdx mice. In addition, D2-mdx mice showed fewer central myonuclei and increased calcifications in the skeletal muscle, heart and diaphragm at 7 weeks, suggesting that their pathology is different from the B10-mdx mice. The new D2-mdx model with an earlier onset and more pronounced dystrophy phenotype may be useful for evaluating therapies that target cardiac and skeletal muscle function in dystrophin-deficient mice. Our data align the D2-mdx with Duchenne muscular dystrophy patients with the LTBP4 genetic modifier, making it one of the few instances of cross-species genetic modifiers of monogenic traits.

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.

Cl alloying improves thermal stability and increases luminescence in iodine-rich inorganic perovskites.

The inorganic perovskite CsPbI3 shows promising photophysical properties for a range of potential optoelectronic applications but is metastable at room temperature. To address this, Br can be alloyed into the X-site to create compositions such as CsPbI2Br that are stable at room temperature but have bandgaps >1.9 eV - severely limiting solar applications. Herein, in an effort to achieve phase stable films with bandgaps <1.85 eV, we investigate alloying chlorine into iodine-rich triple-halide CsPb(I0.8Br0.2-x Cl x )3 with 0 < x < 0.1. We show that partial substitution of iodine with bromine and chlorine provides a path to maintain broadband terrestrial absorption while improving upon the perovskite phase stability due to chlorine's smaller size and larger ionization potential than bromine. At moderate Cl loading up to ≈5%, X-ray diffraction reveals an increasingly smaller orthorhombic unit cell, suggesting chlorine incorporation into the lattice. Most notably, this Cl incorporation is accompanied by a significant enhancement over Cl-free controls in the duration of black-phase stability of up to 7× at elevated temperatures. Additionally, we observe up to 5× increased steady state photoluminescence intensity (PL), along with a small blue-shift. In contrast, at high loading (≈10%), Cl accumulates in a second phase that is visible at grain boundaries via synchrotron fluorescence microscopy and negatively impacts the perovskite phase stability. Thus, replacing small fractions of bromine for chlorine in the iodine-rich inorganic perovskite lattice results in distinct improvement thermal stability and optoelectronic quality while minimally impacting the bandgap.

Two-dimensional perovskite templates for durable, efficient formamidinium perovskite solar cells.

We present a design strategy for fabricating ultrastable phase-pure films of formamidinium lead iodide (FAPbI3) by lattice templating using specific two-dimensional (2D) perovskites with FA as the cage cation. When a pure FAPbI3 precursor solution is brought in contact with the 2D perovskite, the black phase forms preferentially at 100°C, much lower than the standard FAPbI3 annealing temperature of 150°C. X-ray diffraction and optical spectroscopy suggest that the resulting FAPbI3 film compresses slightly to acquire the (011) interplanar distances of the 2D perovskite seed. The 2D-templated bulk FAPbI3 films exhibited an efficiency of 24.1% in a p-i-n architecture with 0.5-square centimeter active area and an exceptional durability, retaining 97% of their initial efficiency after 1000 hours under 85°C and maximum power point tracking.

Strong-bonding hole-transport layers reduce ultraviolet degradation of perovskite solar cells.

The light-emitting diodes (LEDs) used in indoor testing of perovskite solar cells do not expose them to the levels of ultraviolet (UV) radiation that they would receive in actual outdoor use. We report degradation mechanisms of p-i-n-structured perovskite solar cells under unfiltered sunlight and with LEDs. Weak chemical bonding between perovskites and polymer hole-transporting materials (HTMs) and transparent conducting oxides (TCOs) dominate the accelerated A-site cation migration, rather than direct degradation of HTMs. An aromatic phosphonic acid, [2-(9-ethyl-9H-carbazol-3-yl)ethyl]phosphonic acid (EtCz3EPA), enhanced bonding at the perovskite/HTM/TCO region with a phosphonic acid group bonded to TCOs and a nitrogen group interacting with lead in perovskites. A hybrid HTM of EtCz3EPA with strong hole-extraction polymers retained high efficiency and improved the UV stability of perovskite devices, and a champion perovskite minimodule-independently measured by the Perovskite PV Accelerator for Commercializing Technologies (PACT) center-retained operational efficiency of >16% after 29 weeks of outdoor testing.

Development and characterization of an intra-articular fracture mediated model of post-traumatic osteoarthritis.

PURPOSE: This study aimed to develop and characterize a closed intra-articular fracture (IAF) mediated post-traumatic osteoarthritis (PTOA) model in rats to serve as a testbed for putative disease modifying interventions. METHODS: Male rats were subject to a 0 Joule (J), 1 J, 3 J, or 5 J blunt-force impact to the lateral aspect of the knee and allowed to heal for 14 and 56 days. Micro-CT was performed at time of injury and at the specified endpoints to assess bone morphometry and bone mineral density measurements. Cytokines and osteochondral degradation markers were assayed from serum and synovial fluid via immunoassays. Histopathological analyses were performed on decalcified tissues and assessed for evidence of osteochondral degradation. RESULTS: High-energy (5 J) blunt impacts consistently induced IAF to the proximal tibia, distal femur, or both while lower energy (1 J and 3 J) impacts did not. CCL2 was found to be elevated in the synovial fluid of rats with IAF at both 14- and 56-days post-injury while COMP and NTX-1 were upregulated chronically relative to sham controls. Histological analysis showed increased immune cell infiltration, increased osteoclasts and osteochondral degradation with IAF relative to sham. CONCLUSION: Based on results from the current study, our data indicates that a 5 J blunt-forced impact adequately and consistently induces hallmark osteoarthritic changes to the articular surface and subchondral bone at 56 days after IAF. Marked development of PTOA pathobiology suggest this model will provide a robust testbed for screening putative disease modifying interventions that might be translated to the clinic for militarily relevant, high-energy joint injuries.

Evaluating the potential use of functional fibrosis to facilitate improved outcomes following volumetric muscle loss injury.

Volumetric muscle loss (VML) was defined as the frank loss of skeletal muscle tissue with associated chronic functional deficits. Significant effort has been dedicated to developing approaches for treating VML injuries, most of which have focused on stimulating regeneration of the affected musculature via a variety of approaches (e.g., biomaterials). VML injury induces a prolonged inflammatory response which causes fibrotic tissue deposition and is thought to inhibit de novo myofiber regeneration despite observed improvements in functional outcomes (i.e., functional fibrosis; FF). Recent approaches have sought to attenuate inflammation and/or fibrosis as a means to create a permissive environment for regenerative therapies. However, there are currently no clinically available interventions capable of facilitating full restoration of form and function following VML injury; thus, an unmet clinical need exists for a near-term interventional strategy to treat affected patients. FF could serve as an alternative approach to facilitate improved functional outcomes following VML injuries. We sought to investigate whether intentionally exploiting the concept of FF (i.e., induction of a supraphysiological fibrotic response via the delivery of a polypropylene mesh combined with TGFß) would enhance the function of the VML affected musculature. We found that FF treatment induces enhanced fibrotic tissue deposition within the VML defect as evidenced by histological and molecular analysis. FF-treated animals exhibit improved in vivo muscle function compared to untreated control animals at 8 weeks post-injury, thus substantiating the concept that FF could serve as an efficacious approach for facilitating improved functional outcomes following VML injury. STATEMENT OF SIGNIFICANCE: VML injuries result in long-term functional impairments and reduced quality of life for affected individuals, namely combat injured US Service members, and no clinical interventions can restore the form and function of the injured limb. Extensive efforts have been aimed at developing therapeutics to address this critical gap; unfortunately, most interventions facilitate only modest regeneration. Interestingly, improved muscle function has been observed in VML studies following treatment with a therapeutic, despite a lack of myogenic tissue formation; a phenomenon termed Functional Fibrosis (FF). Herein we exploited the concept of FF to enhance the function of VML affected musculature. This finding is significant in that the commercially available interventions used to induce FF can be translated into the clinic near-term, thus improving the standard of care for VML injuries.

The impact of bilateral injuries on the pathophysiology and functional outcomes of volumetric muscle loss.

Volumetric muscle loss (VML)-defined as the irrecoverable loss of skeletal muscle tissue with associated persistent functional deficits-is among the most common and highly debilitating combat-related extremity injuries. This is particularly true in cases of severe polytrauma wherein multiple extremities may be involved as a result of high energy wounding mechanisms. As such, significant investment and effort has been made toward developing a clinically viable intervention capable of restoring the form and function of the affected musculature. While these investigations conducted to date have varied with respect to the species, breed, and sex of the chosen pre-clinical in-vivo model system, the majority of these studies have been performed in unilateral injury models, an aspect which may not fully exemplify the clinical representation of the multiply injured patient. Furthermore, while various components of the basal pathophysiology of VML (e.g., fibrosis and inflammation) have been investigated, relatively little effort has focused on how the pathophysiology and efficacy of pro-regenerative technologies is altered when there are multiple VML injuries. Thus, the purpose of this study was two-fold: (1) to investigate if/how the pathophysiology of unilateral VML injuries differs from bilateral VML injuries and (2) to interrogate the effect of bilateral VML injuries on the efficacy of a well-characterized regenerative therapy, minced muscle autograft (MMG). In contrast to our hypothesis, we show that bilateral VML injuries exhibit a similar systemic inflammatory response and improved muscle functional recovery, compared to unilateral injured animals. Furthermore, MMG treatment was found to only be effective at promoting an increase in functional outcomes in unilateral VML injuries. The findings presented herein add to the growing knowledge base of the pathophysiology of VML, and, importantly, reiterate the importance of comprehensively characterizing preclinical models which are utilized for early-stage screening of putative therapies as they can directly influence the translational research pipeline.

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).

Evolution of Stronger SARS-CoV-2 Variants as Revealed Through the Lens of Molecular Dynamics Simulations.

Using molecular dynamics simulations, the protein-protein interactions of the receptor-binding domain of the wild-type and seven variants of the severe acute respiratory syndrome coronavirus 2 spike protein and the peptidase domain of human angiotensin-converting enzyme 2 were investigated. These variants are alpha, beta, gamma, delta, eta, kappa, and omicron. Using 100 ns simulation data, the residue interaction networks at the protein-protein interface were identified. Also, the impact of mutations on essential protein dynamics, backbone flexibility, and interaction energy of the simulated protein-protein complexes were studied. The protein-protein interface for the wild-type, delta, and omicron variants contained several stronger interactions, while the alpha, beta, gamma, eta, and kappa variants exhibited an opposite scenario as evident from the analysis of the inter-residue interaction distances and pair-wise interaction energies. The study reveals that two distinct residue networks at the central and right contact regions forge stronger binding affinity between the protein partners. The study provides a molecular-level insight into how enhanced transmissibility and infectivity by delta and omicron variants are most likely tied to a handful of interacting residues at the binding interface, which could potentially be utilized for future antibody constructs and structure-based antiviral drug design.

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.