Heather Megan Powell

Scarring remains problematic post-burn injury even with pressure garment therapy, the current standard of care for scar suppression. Unfortunately, there is no consensus regarding the minimum pressure magnitude required for efficacy. Thus, to fill this knowledge gap, the relationship between the magnitude of applied pressure and scar outcomes was assessed using a porcine burn-excise-autograft model. Scars were treated with adjustable, low-fatigue pressure garments fit to 10, 20, or 30 mmHg; control scars were untreated (n = 16/group). Scar contraction, thickness, histological appearance, and biomechanics were assessed, and garment fatigue was measured. At all magnitudes of applied pressure, use of garments significantly reduced scar contraction and thickness, and improved scar pliability and elasticity, versus controls. Utilizing adjustable, low-fatigue garments, pressures of 10, 20, and 30 mmHg were reliably maintained throughout the study, demonstrating the efficacy of pressure garment therapy at pressures as low as 10 mmHg. While 30 mmHg pressure garments significantly reduced contraction compared to 10 or 20 mmHg, these garments required greater adjustment of the garment to reach the target pressure, which in practice could necessitate more frequent garment replacement. The results showed that although greatest improvement was observed at 30 mmHg, pressure as low as 10 mm Hg is effective at reducing post-burn scarring if that pressure is maintained throughout the garment’s use.

Engineered skin (ES) can serve as an advanced therapy for treatment of large full-thickness wounds, but delayed vascularization can cause ischemia, necrosis, and graft failure. To accelerate ES vascularization, this study assessed incorporation of polydopamine (PDA) microparticles loaded with different concentrations of basic fibroblast growth factor (bFGF) into collagen scaffolds, which were subsequently seeded with human fibroblasts to create dermal templates (DTs), and then keratinocytes to create ES. DTs and ES were evaluated in vitro and following grafting to full-thickness wounds in immunodeficient mice. In vitro, metabolic activity of DTs was enhanced with PDA+bFGF, though this increase was not observed following seeding with keratinocytes to generate ES. After grafting, ES with bFGF-loaded PDA microparticles displayed dose-dependent increases in CD31-positive vessel formation vs. PDA-only controls (p < 0.001 at day 7; p < 0.05 at day 14). Interestingly, ES containing PDA+bFGF microparticles exhibited an almost 3-fold increase in water loss through the skin and a less-organized basal keratinocyte layer at day 14 post-grafting vs. controls. This was associated with significantly increased inflammatory cell infiltrate vs. controls at day 7 in vivo (p < 0.001). The results demonstrate that PDA microparticles are a viable method for delivery of growth factors in ES. However, further investigation of bFGF concentrations, and/or investigation of alternative growth factors, will be required to promote vascularization while reducing inflammation and maintaining epidermal health.

To provide an optimal wound bed for epidermal regeneration, a viable dermis is needed. As the dermis is destroyed in full-thickness burns, dermal templates (DTs) are used to create a healthy dermis for grafting and other procedures. Neonatal foreskin has exclusively been used as the source of dermal fibroblasts in commercial DTs, as the tissue is readily available and because these cells are assumed to be more proliferative and capable of superior wound healing compared with adult fibroblasts. The goal of this study was to assess the function of adult fibroblasts compared with neonatal fibroblasts for DT construction and epidermal regeneration. Primary fibroblasts were isolated from neonatal or adult surgical discard tissue (n = 4 each). Expression of collagen type 1 A1 (COL1A1) and matrix metalloprotease 1 (MMP1), MMP3, and MMP9 was assessed for each cell strain, and proliferation was quantified in two-dimensional (2D) cultures and 3D DTs. Subsequently, DTs were constructed from each cell strain by inoculating fibroblasts onto electrospun collagen scaffolds. DT contraction, extracellular matrix remodeling, and cell viability were assessed over 7 days in culture, and the ability of the DTs to promote epidermal regeneration was assessed using primary adult keratinocytes. No differences in gene expression were observed in neonatal versus adult fibroblasts in 2D culture. Neonatal fibroblasts were significantly more proliferative at day 7 when cultured in 2D; however, fibroblast proliferation was independent of donor age in 3D culture. Neonatal DTs contracted significantly more than adult DTs (68.8% ± 6.2% vs. 91.7% ± 4.2% original wound area, respectively). Upon seeding with keratinocytes, a robust, stratified epidermis formed in all DT groups, with no statistically significant differences in dermal or epidermal thickness, basal keratinocyte proliferation, epidermal barrier function, or basement membrane deposition. Analysis of gene expression revealed modest differences in the expression of MMP1, COL1A1, and ACTA2 in neonatal versus adult engineered skin in vitro, which were not associated with any discernable histological differences. These results indicate that the fabrication of DTs with adult fibroblasts can promote epidermal regeneration equivalent to that of neonatal fibroblasts but with less in vitro contraction, which may enable the treatment of larger wound areas.

Background: Keloids are disfiguring, fibrotic scar-like lesions that are challenging to treat and commonly recur after therapy. A deeper understanding of the mechanisms driving keloid formation is necessary for the development of more effective therapies. Reduced vitamin D receptor (VDR) expression has been observed in keloids, implicating vitamin D signaling in keloid pathology. Vitamin D exhibits anti-proliferative and anti-inflammatory properties, suggesting it could have therapeutic utility in keloid disorder. The current study investigated vitamin D-regulated gene expression in keloid keratinocytes and the effects of inhibiting an enzyme involved in vitamin D metabolism on the phenotype of keloid-derived keratinocytes. Methods: Normal and keloid-derived primary keratinocytes were isolated from normal skin and keloid lesions, respectively, and were cultured in the absence or presence of vitamin D. In some experiments, inhibitors of the vitamin D metabolizing enzyme CYP24A1, ketoconazole or VID400 were added in the absence or presence of vitamin D. Cellular proliferation, migration and gene expression were measured. Results: We observed significant overexpression of CYP24A1 mRNA in keloid versus normal keratinocytes and increased CYP24A1 protein levels in keloids versus normal skin. CYP24A1 encodes 24 hydroxylase and is induced by vitamin D in a feedback loop that regulates vitamin D levels; thus, inhibition of CYP24A1 activity may locally increase active vitamin D levels. Ketoconazole, a non-specific cytochrome P-450 inhibitor, reduced proliferation of keloid and normal keratinocytes, but VID400, a specific CYP24A1 inhibitor, only significantly affected keloid keratinocyte proliferation. Neither inhibitor significantly reduced keratinocyte migration. The two inhibitors had different effects on vitamin D target gene expression in keratinocytes. Specifically, ketoconazole treatment reduced CYP24A1 expression in normal and keloid keratinocytes, whereas VID400 increased CYP24A1 expression. Both inhibitors decreased expression of profibrotic genes, including periostin and hyaluronan synthase 2, in keloid-derived cells. Combined treatment of keloid keratinocytes with vitamin D and ketoconazole or VID400 increased the effects of vitamin D treatment on target genes, although the effects were gene- and cell type-specific. Conclusions: The data suggest that reduction of vitamin D inactivation with CYP24A1 inhibitors may reduce profibrotic gene expression in keloid-derived cells. Therefore, CYP24A1 inhibitors may serve as adjunctive therapies to suppress keloid-associated gene expression changes.

Rete ridges play multiple important roles in native skin tissue function, including enhancing skin strength, but they are largely absent from engineered tissue models and skin substitutes. Laser micropatterning of fibroblast-containing dermal templates prior to seeding of keratinocytes was shown to facilitate rete ridge development in engineered skin (ES) both in vitro and in vivo. However, it is unknown whether rete ridge development results exclusively from the microarchitectural features formed by ablative processing or whether laser treatment causes an inflammatory response that contributes to rete ridge formation. In this study, laser-micropatterned and non-laser- treated ES grafts were developed and assessed during culture and for four weeks post grafting onto full-thickness wounds in immunodeficient mice. Decreases in inflammatory cytokine secretion were initially observed in vitro in laser-treated grafts compared to non-treated controls, although cytokine levels were similar in both groups five days after laser treatment. Post grafting, rete ridge-containing ES showed a significant increase in vascularization at week 2, and in collagen deposition and biomechanics at weeks 2 and 4, compared with controls. No differences in inflammatory cytokine expression after grafting were observed between groups. The results suggest that laser micropatterning of ES to create rete ridges improves the mechanical properties of healed skin grafts without increasing inflammation.

Segmental bone defects present complex clinical challenges. Nonunion, malunion, and infection are common sequalae of autogenous bone grafts, allografts, and synthetic bone implants due to poor incorporation with the patient’s bone. The current project explores the osteogenic properties of periosteum to facilitate graft incorporation. As tissue area is a natural limitation of autografting, mechanical strain was implemented to expand the periosteum. Freshly harvested, porcine periosteum was strained at 5 and 10% per day for 10 days with non-strained and free-floating samples serving as controls. Total tissue size, viability and histologic examination revealed that strain increased area to a maximum of 1.6-fold in the 10% daily strain. No change in tissue anatomy or viability via MTT or Ki67 staining and quantification was observed among groups. The osteogenic potential of the mechanical expanded periosteum was then examinedin vivo. Human cancellous allografts were wrapped with 10% per day strained, fresh, free-floating, or no porcine periosteum and implanted subcutaneously into female, athymic mice. Tissue was collected at 8- and 16-weeks. Gene expression analysis revealed a significant increase in alkaline phosphatase and osteocalcin in the fresh periosteum group at 8-weeks post implantation compared to all other groups. Values among all groups were similar at week 16. Additionally, histological assessment with H&E and Masson-Goldner Trichrome staining showed that all periosteal groups outperformed the non-periosteal allograft, with fresh periosteum demonstrating the highest levels of new tissue mineralization at the periosteum-bone interface. Overall, mechanical expansion of the periosteum can provide increased area for segmental healing via autograft strategies, though further studies are needed to explore culture methodology to optimize osteogenic potential.

Significance: The physical and psychological sequalae of burn injuries account for 10 million disability-adjusted life years lost annually. Hypertrophic scarring (HSc) following burn injury results in reduced mobility, contracture, pain, itching, and aesthetic changes for burn survivors. Despite the prevalence of scarring and the number of scar therapies available, none are highly effective at preventing HSc following burn injury. Recent Advances: Recent studies modulating the mechanical environment surrounding incisional and excisional wounds have shown off-loading of tension to be a powerful strategy to prevent scar formation. Preclinical studies applying force perpendicular to the surface of the skin or using a combination of pressure both circumferentially and perpendicularly have shown substantial reductions in scar thickness and contraction following burn injury. Critical Issues: Though pressure therapy is highly effective in preclinical studies, outcomes in clinical studies have been variable and may be a result of differing therapy protocols and garment material fatigue. A recent adult clinical study reported a significant reduction in pressure after one month of use and significant reduction between one and two months of use, resulting in below therapeutic doses of pressure applied after only one month of use Future Directions: To enhance efficacy of pressure garments, new low-fatigue materials must be developed for use in standard garments or garments must be redesigned to allow for adjustment to compensate for the loss of pressure with time. Additionally, measurements of applied pressure should be performed routinely during clinic visits to ensure that therapeutic doses of pressure are being delivered.