Burns Induce Alterations in the Acyl Proteome of Mice and Humans.

ABSTRACT: Hypermetabolic reprogramming triggered by thermal injury causes substantial morbidity and mortality. Despite the therapeutic potential of targeting this response, the underlying mechanisms remain poorly understood. Interestingly, protein S-acylation is a reversible post-translational modification induced by metabolic alterations via DHHC acyltransferases. While this modification aids in the regulation of cellular functions, deregulated S-acylation contributes to various diseases by altering protein structure, stability, and localization. However, whether and how S-acylation may impact morbidity and mortality during post-burn hypermetabolism is unknown. In this study, we discovered that alterations in the acyl proteome play a key role in mediating adverse outcomes that occur after burn injury. Using a murine model, we show that burn injury induces profound changes in the expression of various DHHC isoforms in metabolic organs central to regulating post-burn hypermetabolism, the adipose tissue and liver. This was accompanied by increased levels of S-acylated proteins in several pathways involved in mediating the adverse hypermetabolic response, including ER stress, lipolysis, and browning. In fact, similar results were also observed in adipose tissue from severely burned patients, as reflected by increased S-acylation of ERK1/2, eIF2a, ATGL, FGF21, and UCP1 relative to non-burn controls. Importantly, pharmacologically targeting this post-translational modification using a non-selective DHHC inhibitor effectively attenuated burn-induced ER stress, lipolysis, and browning induction in an ex vivo explant model. Together, these findings suggest that S-acylation may facilitate the protein activation profile that drives burn-induced hypermetabolism and that targeting it could potentially be an effective strategy to restore metabolic function and improve outcomes after injury.

Fibrosis in burns: an overview of mechanisms and therapies.

Scar development remains a common occurrence and a major healthcare challenge affecting the lives of millions of patients annually. Severe injuries to the skin, such as burns can lead to pathological wound healing patterns, often characterized by dermal fibrosis or excessive scarring, and chronic inflammation. The two most common forms of fibrotic diseases following burn trauma are hypertrophic scars (HSCs) and keloids, which severely impact the patient's quality of life. Although the cellular and molecular mechanisms are similar, HSC and keloids have several distinct differences. In this review, we discuss the different forms of fibrosis that occur postburn injury, emphasizing how the extent of burn influences scar development. Moreover, we highlight how a systemic response induced by a burn injury drives wound fibrosis, including both the role of the inflammatory response, as well as the fate of fibroblast during skin healing. Finally, we list potential therapeutics aimed at alleviating pathological scar formation. An understanding of the mechanisms of postburn fibrosis will allow us to effectively move studies from bench to bedside.

Large animal models of thermal injury.

Burn injury results in a triad of inter-related adaptive responses: a systemic inflammatory response, a stress response, and a consequent hypermetabolic state which supports the former two. These pathological responses extend beyond the site of injury to affect distant organs and influence long-term outcomes in the patient. Animal models have proven valuable in advancing our understanding of mechanisms underlying the multifactorial manifestations of burn injury. While rodent models have been unprecedented in providing insights into signaling pathways, metabolic responses, protein turnover, cellular and molecular changes; small animal models do not replicate hypermetabolism, hyperinflammation, and wound healing after a burn injury as seen in humans. Herein, we provide a concise review of preferred large animal models utilized to understand burn pathophysiology based on organ systems and associated dysfunction. Additionally, we present a detailed protocol of contact burn injury in the Yorkshire pig model with a focus on preoperative care, anesthesia, analgesia, wound excision and grafting, dressing application, and frequency of dressing changes.

Small animal models of thermal injury.

Burns are a severe form of trauma that account for 1.1 million cases necessitating medical attention and 4500 mortalities annually in the United States alone. Importantly, the initial trauma is succeeded by extensive, prolonged physiological alterations that detrimentally impact multiple organ systems. Given the complexity of post-burn pathophysiology, in vitro experiments are insufficient to model thermal injuries. Therefore, compatible animal burn models are essential for studying burn-related phenomena. In this chapter, we discuss commonly employed small animal burn models and their comparability and applicability to human studies. In particular, we compare post-burn wound healing between the species as well as relevant hypermetabolic and inflammatory characteristics, providing a better understanding of the pros and cons of utilizing a small animal surrogate for human burns. We further provide an overview of the rodent scald burn model methodology as well as a comparison between elderly, aged and young animals, providing a guide for tailoring animal model choice based on the relevant research question.

Stem Cell Therapy for Burns: Story so Far.

Burn injuries affect approximately 11 million people annually, with fatalities amounting up to 180,000. Burn injuries constitute a global health issue associated with high morbidity and mortality. Recent years have seen advancements in regenerative medicine for burn wound healing encompassing stem cells and stem cell-derived products such as exosomes and conditioned media with promising results compared to current treatment approaches. Sources of stem cells used for treatment vary ranging from hair follicle stem cells, embryonic stem cells, umbilical cord stem cells, to mesenchymal stem cells, such as adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and even stem cells harvested from discarded burn tissue. Stem cells utilize various pathways for wound healing, such as PI3/AKT pathway, WNT-ß catenin pathway, TGF-ß pathway, Notch and Hedgehog signaling pathway. Due to the paracrine signaling mechanism of stem cells, exosomes and conditioned media derived from stem cells have also been utilized in burn wound therapy. As exosomes and conditioned media are cell-free therapy and contain various biomolecules that facilitate wound healing, they are gaining popularity as an alternative treatment strategy with significant improvement in outcomes. The treatment is provided either as direct injections or embedded in a natural/artificial scaffold. This paper reviews in detail the different sources of stem cells, stem cell-derived products, their efficacy in burn wound repair, associated signaling pathways and modes of delivery for wound healing.

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.

Burns in the Elderly: Potential Role of Stem Cells.

Burns in the elderly continue to be a challenge despite advances in burn wound care management. Elderly burn patients continue to have poor outcomes compared to the younger population. This is secondary to changes in the quality of the aged skin, leading to impaired wound healing, aggravated immunologic and inflammatory responses, and age-related comorbidities. Considering the fast-growing elderly population, it is imperative to understand the anatomic, physiologic, and molecular changes of the aging skin and the mechanisms involved in their wound healing process to prevent complications associated with burn wounds. Various studies have shown that stem cell-based therapies improve the rate and quality of wound healing and skin regeneration; however, the focus is on the younger population. In this paper, we start with an anatomical, physiological and molecular dissection of the elderly skin to understand why wound healing is delayed. We then review the potential use of stem cells in elderly burn wounds, as well as the mechanisms by which mesenchymal stem cell (MSCs)-based therapies may impact burn wound healing in the elderly. MSCs improve burn wound healing by stimulating and augmenting growth factor secretion and cell proliferation, and by modulating the impaired elderly immune response. MSCs can be used to expedite healing in superficial partial thickness burns and donor site wounds, improve graft take and prevent graft breakdown.

Non-invasive cell counting of adherent, suspended and encapsulated mammalian cells using optical density.

In situ measurement to determine mammalian cell number in a non-invasive, non-destructive and reagent-free manner is needed to enable continuous cell manufacturing. An analytical method is presented for non-invasive cell counting by conducting multiwavelength spectral analysis of mammalian cells achieving a minimal detectable cell count of 62,500 at 295 nm. Light absorbance was insensitive to culture volume, giving an absolute cell count rather than a concentration. The activation state of cells was also considered. The study was extended to quantification within polymeric microcapsules as an advanced substrate for mammalian cell growth in bioreactor formats and resulted in an offset directly correlating with the absorbance maxima of the polymer. These studies provide feasibility for optical density as a simple end point to indirectly quantify mammalian cell number for continuous monitoring of cell cultures.

Coencapsulation of ISCs and MSCs Enhances Viability and Function of both Cell Types for Improved Wound Healing.

INTRODUCTION: We previously demonstrated that insulin secreting cells (ISCs) accelerate healing of chronic wounds, and it is known that mesenchymal stem cells (MSCs) also accelerate wound healing. Here, we report that the combination of both cell types coencapsulated into a synthetic hydrogel dressing accelerates chronic wound healing 3 × faster than control and 2 × faster than each cell type delivered singly. Specifically, insulin released by ISCs activates the PI3/Akt pathway, which is vital to the function and survival of MSCs. MSCs in turn improve the viability and function of ISCs. MATERIALS AND METHODS: MSCs and/or rat islet tumor RIN-m cells were encapsulated into polyethylene glycol diacrylate hydrogel sheets and applied to 1 cm2 full thickness excisional wounds on the dorsa of genetically diabetic male mice (BKS.Cg-m +/+Leprdb/J) in accordance with protocols approved by the Rutgers IACUC. Encapsulated cell viability was assessed using a LIVE/DEAD® Viability/Cytotoxicity Kit. Akt phosphorylation, insulin, VEGF, and TGF-ß1 secretion were assessed by ELISA. Animals were sacrificed on postoperative days 14 and 28 and wound tissue was collected for histological and western blot analysis. RESULTS: ISC:MSC combination groups had the highest levels of every secreted product and phosphorylated Akt, and closed wounds in 14 days, ISC-only or MSC-only groups closed wounds in 28 days, control groups closed wounds in 40 days. Further, ISC:MSC groups healed without intermediate scab or scar. CONCLUSIONS: Combining MSCs with ISCs results in a more robust healing response than singly delivered cells, warranting further investigation of coencapsulation for MSC therapies.

Convergence of Cell Pharmacology and Drug Delivery.

Cellular therapy is enabling new approaches to tackle significant unmet needs in areas such as regenerative medicine and immunotherapy. The pharmacology of cell therapeutics becomes of critical importance to assure that these new drugs work reproducibly and effectively. Cell pharmacology can benefit from adapting principles of classical molecular drug pharmacokinetics (PK) and pharmacodynamics (PD) to quantitatively understand rate-limiting constraints of cell fate after administration. Future innovations focused on improvements in drug delivery using a PK/PD perspective can aid in designing a cell therapeutic product to overcome any pharmacological barriers for a given disease application. Herein, we present a perspective on the development of an ex vivo mesenchymal stromal therapeutic using a PK/PD framework and also present examples of general cell engineering techniques that implicitly influence the PK/PD curve by genetically modifying cells to regulate their in vivo duration, biodistribution, and activity. Stem Cells Translational Medicine 2019;8:874&879.

The effect of low-magnitude, high-frequency vibration on poly(ethylene glycol)-microencapsulated mesenchymal stem cells.

Low-magnitude, high-frequency vibration has stimulated osteogenesis in mesenchymal stem cells when these cells were cultured in certain types of three-dimensional environments. However, results of osteogenesis are conflicting with some reports showing no effect of vibration at all. A large number of vibration studies using three-dimensional scaffolds employ scaffolds derived from natural sources. Since these natural sources potentially have inherent biochemical and microarchitectural cues, we explored the effect of low-magnitude, high-frequency vibration at low, medium, and high accelerations when mesenchymal stem cells were encapsulated in poly(ethylene glycol) diacrylate microspheres. Low and medium accelerations enhanced osteogenesis in mesenchymal stem cells while high accelerations inhibited it. These studies demonstrate that the isolated effect of vibration alone induces osteogenesis.

Biomanufacturing for clinically advanced cell therapies.

The achievements of cell-based therapeutics have galvanized efforts to bring cell therapies to the market. To address the demands of the clinical and eventual commercial-scale production of cells, and with the increasing generation of large clinical datasets from chimeric antigen receptor T-cell immunotherapy, from transplants of engineered haematopoietic stem cells and from other promising cell therapies, an emphasis on biomanufacturing requirements becomes necessary. Robust infrastructure should address current limitations in cell harvesting, expansion, manipulation, purification, preservation and formulation, ultimately leading to successful therapy administration to patients at an acceptable cost. In this Review, we highlight case examples of cutting-edge bioprocessing technologies that improve biomanufacturing efficiency for cell therapies approaching clinical use.

Hydrogel Microencapsulated Insulin-Secreting Cells Increase Keratinocyte Migration, Epidermal Thickness, Collagen Fiber Density, and Wound Closure in a Diabetic Mouse Model of Wound Healing.

Wound healing is a hierarchical process of intracellular and intercellular signaling. Insulin is a potent chemoattractant and mitogen for cells involved in wound healing. Insulin's potential to promote keratinocyte growth and stimulate collagen synthesis in fibroblasts is well described. However, there currently lacks an appropriate delivery mechanism capable of consistently supplying a wound environment with insulin; current approaches require repeated applications of insulin, which increase the chances of infecting the wound. In this study, we present a novel cell-based therapy that delivers insulin to the wound area in a constant or glucose-dependent manner by encapsulating insulin-secreting cells in nonimmunogenic poly(ethylene glycol) diacrylate (PEGDA) hydrogel microspheres. We evaluated cell viability and insulin secretory characteristics of microencapsulated cells. Glucose stimulation studies verified free diffusion of glucose and insulin through the microspheres, while no statistical difference in insulin secretion was observed between cells in microspheres and cells in monolayers. Scratch assays demonstrated accelerated keratinocyte migration in vitro when treated with microencapsulated cells. In excisional wounds on the dorsa of diabetic mice, microencapsulated RIN-m cells accelerated wound closure by postoperative day 7; a statistically significant increase over AtT-20ins-treated and control groups. Histological results indicated significantly greater epidermal thickness in both microencapsulated RIN-m and AtT-20ins-treated wounds. The results suggest that microencapsulation enables insulin-secreting cells to persist long enough at the wound site for a therapeutic effect and thereby functions as an effective delivery vehicle to accelerate wound healing.