Model to Inhibit Contraction in Third-Degree Burns Employing Split-Thickness Skin Graft and Administered Bone Marrow-Derived Stem Cells.

Third-degree burns typically result in pronounced scarring and contraction in superficial and deep tissues. Established techniques such as debridement and grafting provide benefit in the acute phase of burn therapy, nevertheless, scar and contraction remain a challenge in deep burns management. Our ambition is to evaluate the effectiveness of novel cell-based therapies, which can be implemented into the standard of care debridement and grafting procedures. Twenty-seven third-degree burn wounds were created on the dorsal area of Red Duroc pig. After 72 h, burns are surgically debrided using a Weck knife. Split-thickness skin grafts (STSGs) were then taken after debridement and placed on burn scars combined with bone marrow stem cells (BM-MSCs). Biopsy samples were taken on days 17, 21, and 45 posttreatment for evaluation. Histological analysis revealed that untreated control scars at 17 days are more raised than burns treated with STSGs alone and/or STSGs with BM-MSCs. Wounds treated with skin grafts plus BM-MSCs appeared thinner and longer, indicative of reduced contraction. qPCR revealed some elevation of α-SMA expression at day 21 and Collagen Iα2 in cells derived from wounds treated with skin grafts alone compared to wounds treated with STSGs + BM-MSCs. We observed a reduction level of TGFß-1 expression at days 17, 21, and 45 in cells derived from wounds treated compared to controls. These results, where the combined use of stem cells and skin grafts stimulate healing and reduce contraction following third-degree burn injury, have a potential as a novel therapy in the clinic.

The effect of mesenchymal stem cells improves the healing of burn wounds: a phase 1 dose-escalation clinical trial.

Background: Stem cell therapy holds promise to improve healing and stimulate tissue regeneration after burn injury. Preclinical evidence has supported this; however, clinical studies are lacking. We examined the application of bone marrow-derived mesenchymal stem cells (BM-MSC) to deep second-degree burn injuries using a two-dose escalation protocol. Methods: Ten individuals aged 18 years or older with deep second-degree burn wounds were enrolled. The first five patients were administered 2.5 × 10³ BM-MSC/cm2 to their wounds. After safety of the initial dose level was assessed, a second group of five patients was treated with a higher concentration of 5 × 10³ allogeneic BM-MSC/cm2. Safety was assessed clinically and by evaluating cytokine levels in mixed recipient lymphocyte/donor BM-MSC reactions (INFγ, IL-10 and TNFα). At each visit, we performed wound measurements and assessed wounds using a Patient and Observer Scar Assessment Scale (POSAS). Results: All patients responded well to treatment, with 100% closure of wounds and minimal clinical evidence of fibrosis. No adverse reactions or evidence of rejection were observed for both dose levels. Patients receiving the first dose concentration had a wound closure rate of 3.64 cm2/day. Patients receiving the second dose concentration demonstrated a wound closure rate of 10.47 cm2/day. The difference in healing rates between the two groups was not found to be statistically significant (P = 0.17). Conclusion: BM-MSC appear beneficial in optimising wound healing in patients with deep second-degree burn wounds. Adverse outcomes were not observed when administering multiple doses of allogeneic BM-MSC. Lay Summary: Thermal injuries are a significant source of morbidity and mortality, constituting 5%-20% of all injuries and 4% of all deaths. Despite overall improvements in the management of acutely burned patients, morbidities associated with deeper burn injuries remain commonplace. Burn patients are too often left with significant tissue loss, scarring and contractions leading to physical loss of function and long-lasting psychological and emotional impacts.In previous studies, we have demonstrated the safety and efficacy of administering bone marrow-derived mesenchymal stem cells (BM-MSC) to chronic wounds with substantial improvement in healing and evidence of tissue regeneration. In this report, we have examined the application of BM-MSC to deep second-degree burn injuries in patients.The aim of the present phase I/II clinical trial was to examine the safety and efficacy of administering allogeneic BM-MSC to deep second-degree burns. We utilised two different dose levels at concentrations 2.5 × 103 and 5 × 103 cells/cm2. Patients with deep second-degree burn wounds up to 20% of the total body surface area were eligible for treatment. Allogeneic BM-MSC were applied to burn wounds topically or by injection under transparent film dressing <7 days after injury. Patients were followed for at least six months after treatment.Using two dose levels allowed us to gain preliminary information as to whether different amounts of BM-MSC administered to burn wounds will result in significant differences in safety/ clinical response. Once the safety and dose-response analysis were completed, we evaluated the efficacy of allogeneic stem cell therapy in the treatment of deep second-degree burn wounds.In this study, we examined the role of allogeneic BM-MSC treatment in patients with deep second-degree burn injuries, in a dose-dependent manner. No significant related adverse events were reported. Safety was evaluated both clinically and by laboratory-based methods. Efficacy was assessed clinically through evidence of re-pigmentation, hair follicle restoration and regenerative change. While these findings are encouraging, more studies will be needed to better establish the benefit of BM-MSC in the treatment of burn injuries.

Proteomic analysis of bone marrow-derived mesenchymal stem cell extracellular vesicles from healthy donors: implications for proliferation, angiogenesis, Wnt signaling, and the basement membrane.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) have shown therapeutic potential in various in vitro and in vivo studies in cutaneous wound healing. Furthermore, there are ubiquitous studies highlighting the pro-regenerative effects of BM-MSC extracellular vesicles (BM-MSC EVs). The similarities and differences in BM-MSC EV cargo among potential healthy donors are not well understood. Variation in EV protein cargo is important to understand, as it may be useful in identifying potential therapeutic applications in clinical trials. We hypothesized that the donors would share both important similarities and differences in cargo relating to cell proliferation, angiogenesis, Wnt signaling, and basement membrane formation-processes shown to be critical for effective cutaneous wound healing. METHODS: We harvested BM-MSC EVs from four healthy human donors who underwent strict screening for whole bone marrow donation and further Good Manufacturing Practices-grade cell culture expansion for candidate usage in clinical trials. BM-MSC EV protein cargo was determined via mass spectrometry and Proteome Discoverer software. Corresponding proteomic networks were analyzed via the UniProt Consortium and STRING consortium databases. RESULTS: More than 3000 proteins were identified in each of the donors, sharing > 600 proteins among all donors. Despite inter-donor variation in protein identities, there were striking similarities in numbers of proteins per biological functional category. In terms of biologic function, the proteins were most associated with transport of ions and proteins, transcription, and the cell cycle, relating to cell proliferation. The donors shared essential cargo relating to angiogenesis, Wnt signaling, and basement membrane formation-essential processes in modulating cutaneous wound repair. CONCLUSIONS: Healthy donors of BM-MSC EVs contain important similarities and differences among protein cargo that may play important roles in their pro-regenerative functions. Further studies are needed to correlate proteomic signatures to functional outcomes in cutaneous repair.

Combined Systemic and Intratumoral Administration of Human Papillomavirus Vaccine to Treat Multiple Cutaneous Basaloid Squamous Cell Carcinomas.

Importance: Squamous cell carcinoma (SCC) is the second most common form of skin cancer, and its incidence is increasing. When surgical management is not an option, finding a safe and efficacious treatment is a challenge. Mounting evidence suggests that the human papillomavirus (HPV) is involved in the pathogenesis of some SCCs. Objective: To assess whether the 9-valent HPV vaccine could be an effective treatment strategy for cutaneous SCC. Design, Setting, and Participants: A woman in her 90s with multiple, inoperable cutaneous basaloid SCCs was successfully treated at a university-based outpatient dermatology clinic with a combination of systemic and intratumoral delivery of the 9-valent HPV vaccine from March 17, 2016, through February 27, 2017, and then followed up through May 21, 2018. Main Outcomes and Measures: Reduction in tumor size and number after a combination of systemic and intratumoral administration of the HPV vaccine. Results: All tumors resolved 11 months after the first intratumoral injection of the vaccine. The patient remained free of tumors at the end of follow-up. Conclusions and Relevance: This is the first report, to our knowledge, of complete regression of a cutaneous malignant tumor after combined systemic and direct intratumoral injection of the 9-valent HPV vaccine. This report suggests that the HPV vaccine may have therapeutic utility for SCCs in patients who are poor surgical candidates, have multiple lesions, or defer surgery.

Assessment of Ablative Fractional CO2 Laser and Er:YAG Laser to Treat Hypertrophic Scars in a Red Duroc Pig Model.

Hypertrophic scarring is a fibroproliferative process that occurs following a third-degree dermal burn injury, producing significant morbidity due to persistent pain, itching, cosmetic disfigurement, and loss of function due to contractures. Ablative fractional lasers have emerged clinically as a fundamental or standard therapeutic modality for hypertrophic burn scars. Yet the examination of their histopathological and biochemical mechanisms of tissue remodeling and comparison among different laser types has been lacking. In addition, deficiency of a relevant animal model limits our ability to gain a better understanding of hypertrophic scar pathophysiology. To evaluate the effect of ablative fractional lasers on hypertrophic third-degree burn scars, we have developed an in vivo Red Duroc porcine model. Third-degree burn wounds were created on the backs of animals, and burn scars were allowed to develop for 70 days before treatment. Scars received treatment with either CO2 or erbium: yttrium aluminum garnet (YAG) ablative fractional lasers. Here, we describe the effect of both lasers on hypertrophic third-degree burn scars in Red Duroc pigs. In this report, we found that Er:YAG has improved outcomes versus fractional CO2. Molecular changes noted in the areas of dermal remodeling indicated that matrix metalloproteinase 2, matrix metalloproteinase 9, and Decorin may play a role in this dermal remodeling and account for the enhanced effect of the Er:YAG laser. We have demonstrated that ablative fractional laser treatment of burn scars can lead to favorable clinical, histological, and molecular changes. This study provides support that hypertrophic third-degree burn scars can be modified by fractional laser treatment.

Dual mechanism of type VII collagen transfer by bone marrow mesenchymal stem cell extracellular vesicles to recessive dystrophic epidermolysis bullosa fibroblasts.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe blistering disease resulting from a lack of type VII collagen production. Recent clinical trials have shown efficacy of bone marrow-derived mesenchymal stem cells (BM-MSCs) in the treatment of epidermolysis bullosa, including improved basement membrane restructuring and cutaneous wound healing. The mechanism as to how type VII collagen is transferred from donor stem cell to recipient RDEB cells has not been defined. Here, we submit the model that BM-MSC-derived extracellular vesicles serve at least two roles: 1) to help transport type VII collagen within the extracellular space; and 2) to feed RDEB fibroblasts with messenger RNA that codes for type VII collagen, resulting in COL7A1 translation and synthesis of type VII collagen alpha chain proteins by RDEB fibroblasts. Utilizing a chemoselective ligation detection method, we found RDEB cells that were treated simultaneously with BM-MSC EVs and an l-methionine analog, l-homopropargylglycine (HPG), synthesized collagen VII alpha chain protein that contained the alkyne group of HPG to react (i.e. undergo the Click-iT® reaction) with azide-modified Alexa 594, suggesting de novo synthesis of type VII collagen by RDEB fibroblasts. Thus, our results support a model in which BM-MSC EVs help increase type VII collagen levels available to recipient cells by 1) donating BM-MSC type VII collagen protein and 2) inducing RDEB fibroblasts to make their own type VII collagen protein. These findings allow us to hypothesize that the secretome of BM-MSCs could have therapeutic value in the treatment of RDEB-related skin disorders.

Bone Marrow Mesenchymal Stem Cell-Derived CD63+ Exosomes Transport Wnt3a Exteriorly and Enhance Dermal Fibroblast Proliferation, Migration, and Angiogenesis In Vitro.

Wnts are secreted glycoproteins that regulate stem cell self-renewal, differentiation, and cell-to-cell communication during embryonic development and in adult tissues. Bone marrow mesenchymal stem cells (BM-MSCs) have been shown to stimulate dermis repair and regeneration; however, it is unclear how BM-MSCs may modulate downstream Wnt signaling. While recent reports implicate that Wnt ligands and Wnt messenger RNAs (such as Wnt4) exist within the interior compartment of exosomes, it has been debated whether or not Wnts exist on the exterior surface of exosomes to travel in the extracellular space. To help answer this question, we utilized flow cytometry of magnetic beads coated with anti-CD63 antibodies and found, for the first time, that Wnt3a protein is detectable exteriorly on CD63+ exosomes derived from BM-MSCs over-secreting Wnt3a into serum-free conditioned media (Wnt3a CM). Our data suggest that CD63+ exosomes significantly help transport exterior Wnt3a signal to recipient cells to promote fibroblast and endothelial functions. During purification of exosomes, we unexpectedly found that use of ultracentrifugation alone significantly decreased the ability to detect exteriorly bound Wnt3a on CD63+ exosomes, however, polyethylene glycol (PEG)-mediated exosome-enrichment before exosome-purification (with ultracentrifugation into a sucrose cushion) resulted in exosomes more likely to retain exterior Wnt3a detectability and downstream Wnt/beta-catenin activity. Our findings indicate the important role that purification methods may have on stem cell-derived Wnt-exosome activity in downstream assays. The ability for BM-MSC Wnt3a CM and exosomes to stimulate dermal fibroblast proliferation and migration, and endothelial angiogenesis in vitro, was significantly decreased after CD63+-exosome depletion or knockdown of Wnt coreceptor LRP6 in recipient cells, suggesting both are required for optimal Wnt-exosome activity in our system. Thus, BM-MSC-derived CD63+ exosomes are a significant carrier of exterior Wnt3a within high Wnt environments, resulting in downstream fibroblast proliferation, migration, and angiogenesis in vitro.