Aging Impairs the Cellular Interplay between Myeloid Cells and Mesenchymal Cells during Skin Healing in Mice.

Impaired wound healing is a major issue in the elderly population and is associated with substantial health and economic burden, which is exponentially increasing with the growing aging population. While the underlying pathobiology of disturbed skin healing by aging is linked to several genetic and epigenetic factors, little is known about the cell-cell interaction during the wound healing process in aged individuals, particularly the mesenchymal stem cell (MSCs)-macrophages axis. In this study, by using a thermal injury animal model in which we compared the wound healing process of adult and young mice, we found that the insufficient pool of MSCs in adult animals are deficient in migrating to the wound bed and instead are restricted to the wound edge. We identified a deficiency of a CD90-positive MSC subpopulation in the wounds of adult animals, which is positively correlated with the number of F4/80+ macrophages. In vitro, we found that CD90+ cells preferentially adhere to the myeloid cells forming doublet cells. Thus, our findings highlight that in adult mice subjected to a thermal injury, impaired wound healing is likely mediated by a disturbed cellular interplay between myeloid cells and mesenchymal cells.

Biological characteristics of stem cells derived from burned skin-a comparative study with umbilical cord stem cells.

INTRODUCTION: Burned human skin, which is routinely excised and discarded, contains viable mesenchymal stromal/stem cells (burn-derived mesenchymal stromal/stem cells; BD-MSCs). These cells show promising potential to enable and aid wound regeneration. However, little is known about their cell characteristics and biological function. OBJECTIVES: This study had two aims: first, to assess critical and cellular characteristics of BD-MSCs and, second, to compare those results with multipotent well-characterized MSCs from Wharton's jelly of human umbilical cords (umbilical cord mesenchymal stromal/stem cells, UC-MSCs). METHODS: BD- and UC-MSCs were compared using immunophenotyping, multi-lineage differentiation, seahorse analysis for glycolytic and mitochondrial function, immune surface markers, and cell secretion profile assays. RESULTS: When compared to UC-MSCs, BD-MSCs demonstrated a lower mesenchymal differentiation capacity and altered inflammatory cytokine secretomes at baseline and after stimulation with lipopolysaccharides. No significant differences were found in population doubling time, colony formation, cell proliferation cell cycle, production of reactive oxygen species, glycolytic and mitochondrial function, and in the expression of major histocompatibility complex I and II and toll-like receptor (TLR). IMPORTANCE, TRANSLATION: This study reveals valuable insights about MSCs obtained from burned skin and show comparable cellular characteristics with UC-MSCs, highlighting their potentials in cell therapy and skin regeneration.

Metformin alleviates muscle wasting post-thermal injury by increasing Pax7-positive muscle progenitor cells.

BACKGROUND: Profound skeletal muscle wasting and weakness is common after severe burn and persists for years after injury contributing to morbidity and mortality of burn patients. Currently, no ideal treatment exists to inhibit muscle catabolism. Metformin is an anti-diabetic agent that manages hyperglycemia but has also been shown to have a beneficial effect on stem cells after injury. We hypothesize that metformin administration will increase protein synthesis in the skeletal muscle by increasing the proliferation of muscle progenitor cells, thus mitigating muscle atrophy post-burn injury. METHODS: To determine whether metformin can attenuate muscle catabolism following burn injury, we utilized a 30% total burn surface area (TBSA) full-thickness scald burn in mice and compared burn injuries with and without metformin treatment. We examined the gastrocnemius muscle at 7 and 14 days post-burn injury. RESULTS: At 7 days, burn injury significantly reduced myofiber cross-sectional area (CSA) compared to sham, p < 0.05. Metformin treatment significantly attenuated muscle catabolism and preserved muscle CSA at the sham size. To investigate metformin's effect on satellite cells (muscle progenitors), we examined changes in Pax7, a transcription factor regulating the proliferation of muscle progenitors. Burned animals treated with metformin had a significant increase in Pax7 protein level and the number of Pax7-positive cells at 7 days post-burn, p < 0.05. Moreover, through BrdU proliferation assay, we show that metformin treatment increased the proliferation of satellite cells at 7 days post-burn injury, p < 0.05. CONCLUSION: In summary, metformin's various metabolic effects and its modulation of stem cells make it an attractive alternative to mitigate burn-induced muscle wasting while also managing hyperglycemia.

Electrospun Polyurethane-Gelatin Composite: A New Tissue-Engineered Scaffold for Application in Skin Regeneration and Repair of Complex Wounds.

Wound healing is vital for patients with complex wounds including burns. While the gold standard of skin transplantation ensures a surgical treatment to heal wounds, it has its limitations, for example, insufficient donor sites for patients with large burn wounds and creation of wounds and pain when harvesting the donor skin. Therefore, tissue-engineered skin is of paramount importance. The aim of this study is to investigate and characterize an elastomeric acellular scaffold that would demonstrate the ability to promote skin regeneration. A hybrid gelatin-based electrospun scaffold is fabricated via the use of biodegradable polycarbonate polyurethane (PU). It is hypothesized that the addition of PU would enable a tailored degradation rate and an enhanced mechanical strength of electrospun gelatin. Introducing 20% PU to gelatin scaffolds (Gel80-PU20) results in a significant increase in the degradation resistance, yield strength, and elongation of these scaffolds without altering the cell viability. In vivo studies using a mouse excisional wound biopsy grafted with the scaffolds reveals that the Gel80-PU20 scaffold enables greater cell infiltration than clinically established matrices, for example, Integra (dermal regeneration matrix, DRM), a benchmark scaffold. Immunostaining shows fewer macrophages and myofibroblastic cells on the Gel80-PU20 scaffold when compared with the DRM. The findings show that electrospun Gel80-PU20 scaffolds hold potential for generating tissue substitutes and overcoming some limitations of conventional wound care matrices.

Promotion of dermal regeneration using pullulan/gelatin porous skin substitute.

Tissue-engineered dermal substitutes represent a promising approach to improve wound healing and provide more sufficient regeneration, compared with current clinical standards on care of large wounds, early excision, and grafting of autografts. However, inadequate regenerative capacity, impaired regeneration/degradation profile, and high cost of current commercial tissue-engineered dermal regeneration templates hinder their utilization, and the development of an efficient and cost-effective tissue-engineered dermal substitute remains a challenge. Inspired from our previously reported data on a pullulan/gelatin scaffold, here we present a new generation of a porous pullulan/gelatin scaffold (PG2) served as a dermal substitute with enhanced chemical and structural characteristics. PG2 shows excellent biocompatibility (viability, migration, and proliferation), assessed by in vitro incorporation of human dermal fibroblasts in comparison with the Integra® dermal regeneration template (Control). When applied on a mouse full-thickness excisional wound, PG2 shows rapid scaffold degradation, more granulation tissue, more collagen deposition, and more cellularity in comparison with Control at 20 days post surgery. The faster degradation is likely due to the enhanced recruitment of inflammatory macrophages to the scaffold from the wound bed, and that leads to earlier maturation of granulation tissue with less myofibroblastic cells. Collectively, our data reveal PG2's characteristics as an applicable dermal substitute with excellent dermal regeneration, which may attenuate scar formation.

Stem cells derived from burned skin - The future of burn care.

BACKGROUND: Thermal injuries affect millions of adults and children worldwide and are associated with high morbidity and mortality. The key determinant for the survival of burns is rapid wound healing. Large wounds exceed intrinsic wound-healing capacities, and the currently available coverage materials are insufficient due to lack of cellularity, availability or immunological rejection. METHODS: Using the surgically debrided tissue, we isolated viable cells from burned skin. The isolated cells cultured in tissue culture dishes and characterized. FINDINGS: We report here that debrided burned skin, which is routinely excised from patients and otherwise considered medical waste and unconsciously discarded, contains viable, undamaged cells which show characteristics of mesenchymal skin stem cells. Those cells can be extracted, characterized, expanded, and incorporated into created epidermal-dermal substitutes to promote wound healing in immune-compromised mice and Yorkshire pigs without adverse side effects. INTERPRETATION: These findings are of paramount importance and provide an ideal cell source for autologous skin regeneration. Furthermore, this study highlights that skin contains progenitor cells resistant to thermal stress. FUND: Canadian Institutes of Health Research # 123336. CFI Leader's Opportunity Fund: Project # 25407 National Institutes of Health 2R01GM087285-05A1. EMHSeed: Fund: 500463, A generous donation from Toronto Hydro. Integra© Life Science Company provided the meshed bilayer Integra© for porcine experiments.

The response of muscle progenitor cells to cutaneous thermal injury.

BACKGROUND: Severe burn results in a systemic response that leads to significant muscle wasting. It is believed that this rapid loss in muscle mass occurs due to increased protein degradation combined with reduced protein synthesis. Alterations in the microenvironment of muscle progenitor cells may partially account for this pathology. The aim of this study was to ascertain the response of muscle progenitor cells following thermal injury in mice and to enlighten the cellular cascades that contribute to the muscle wasting. METHODS: C57BL/6 mice received a 20% total body surface area (TBSA) thermal injury. Gastrocnemius muscle was harvested at days 2, 7, and 14 following injury for protein and histological analysis. RESULTS: We observed a decrease in myofiber cross-sectional area at 2 days post-burn. This muscle atrophy was compensated for by an increase in myofiber cross-sectional area at 7 and 14 days post-burn. Myeloperoxidase (MPO)-positive cells (neutrophils) increased significantly at 2 days. Moreover, through Western blot analysis of two key mediators of the proteolytic pathway, we show there is an increase in Murf1 and NF-κB 2 days post-burn. MPO-positive cells were also positive for NF-κB, suggesting that neutrophils attain NF-κB activity in the muscle. Unlike inflammatory and proteolytic pathways, the number of Pax7-positive muscle progenitor cells decreased significantly 2 days post-burn. This was followed by a recovery in the number of Pax7-positive cells at 7 and 14 days, suggesting proliferation of muscle progenitors that accompanied regrowth. CONCLUSION: Our data show a biphasic response in the muscles of mice exposed to burn injury, with phenotypic characteristics of muscle atrophy at 2 days while compensation was observed later with a change in Pax7-positive muscle progenitor cells. Targeting muscle progenitors may be of therapeutic benefit in muscle wasting observed after burn injury.

Enhanced translation of mRNAs encoding proteins involved in mRNA translation during recovery from heat shock.

UNLABELLED: The mRNAs encoding poly (A) binding protein (PABP1), eukaryotic elongation factor 1A (eEF1A) and ribosomal protein S6 (RPS6) belong to the family of terminal oligo pyrimidine tract (TOP) containing mRNAs. Translation of the TOP mRNAs is regulated by growth signals and usually codes for proteins involved in mRNA translation. Previous studies from our laboratory showed that translation of PABP1 mRNA was preferentially enhanced during recovery of HeLa cells from heat shock. Presence of the 5' TOP cis element was required for the observed increase of PABP1 mRNA translation. In the studies reported here we showed that translation of two additional TOP mRNAs such as, eEF1A and RPS6 was similarly enhanced during recovery. In addition, we showed by in vivo cross-linking experiments that the cellular nucleic acid binding protein ZNF9 binds to all three TOP mRNAs examined in these studies as well as to the ß-actin mRNA that lacks a TOP cis element. Binding of ZNF9 to mRNAs was observed in both heat-shocked and non heat- shocked cells. However, depletion of ZNF9 by siRNA prevented the preferred stimulation of PABP1, eEF1A and RPS6 expression during recovery from heat shock. There was no detectable effect of ZNF9 depletion on the basal level of expression of either ß-actin or PABP1, eEF1A and RPS6 in HeLa cells following recovery from heat shock. CONCLUSION: Although the presence of ZNF9 was required for the translational stimulation of PABP1, eEF1A and RPS6 mRNAs, the mechanistic details of this process are still unclear. Since ZNF9 was shown to bind both TOP and non-TOP mRNAs, it is uncertain whether ZNF9 exerts its stimulatory effect on TOP mRNA translation following recovery from heat shock through the TOP cis-element. Perhaps additional factors or post-translational modification(s) of ZNF9 following heat shock are necessary for the preferred increase of TOP mRNA translation.