Laser Micropatterning Promotes Rete Ridge Formation and Enhanced Engineered Skin Strength without Increased Inflammation.

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.

Cultured Epithelial Autograft Combined with Micropatterned Dermal Template Forms Rete Ridges In Vivo.

For patients with large, full-thickness burn wounds, sufficient donor sites for autografting are not available, and thus, alternate strategies must be used to close these wounds. Cultured epithelial autografts (CEAs) can aid in closing these wounds but are often associated with slow deposition of basement membrane proteins, leading to blistering and graft loss. Rete ridges and dermal papillae present at the dermal-epidermal junction (DEJ) play a key role in epidermal adhesion and skin homeostasis. Promoting the development of an interdigitated DEJ may enhance basement membrane protein deposition and provide enhanced physical interlock of the epidermis and dermis. To develop a dermal template with stable dermal papillae, an electrospun collagen scaffold was seeded with human dermal fibroblasts. Ridged topographies were patterned into the cell-seeded dermal template using laser ablation, creating wide and shallow (ActiveFX) or narrow and deep (DeepFX) wells. Micropatterned or flat (control) dermal templates were combined with CEAs immediately before grafting to full-thickness excisional wounds on immunodeficient mice. CEAs grafted in conjunction with ridged templates showed rete ridge formation at 2 weeks after grafting and led to increased epidermal thickness, proliferation, and stemness compared to templates with a flat DEJ. As this technology is further developed, the dermal papilla-containing dermal templates may be utilized in combination with CEAs to improve adhesion and clinical function. Impact statement Cultured epithelial autografts (CEAs) serve as an adjunct to conventional split-thickness autograft in patients with very large burns, but they are susceptible to blistering that can reduce engraftment. Blistering results, in part, from relatively slow basement membrane deposition after grafting. This study demonstrates that basement membrane deposition and rete ridge formation are enhanced by combination of CEAs with a micropatterned, cell-seeded dermal template. These findings may lead to improved treatment and increased survival in patients with very large burns.

Fractional CO2 laser micropatterning of cell-seeded electrospun collagen scaffolds enables rete ridge formation in 3D engineered skin.

Rete ridges are interdigitations of the epidermis and dermis of the skin that play multiple roles in homeostasis, including enhancing adhesion via increased contact area and acting as niches for epidermal stem cells. These structures, however, are generally absent from engineered skin (ES). To develop ES with rete ridges, human fibroblast-seeded dermal templates were treated with a fractional CO2 laser, creating consistently spaced wells at the surface. Constructs with and without laser treatment were seeded with keratinocytes, cultured for 10 days, and grafted onto athymic mice for four weeks. Rete-ridge like structures were observed in the laser-patterned (ridged) samples at the time of grafting and were maintained in vivo. Ridged grafts displayed improved barrier function over non-lasered (flat) grafts at the time of grafting and 4 weeks post-grafting. Presence of ridges in vivo corresponded with increased keratinocyte proliferation, epidermal area, and basement membrane length. These results suggest that this method can be utilized to develop engineered skin grafts with rete ridges, that the ridge pattern is stable for at least 4 weeks post-grafting, and that the presence of these ridges enhances epidermal proliferation and establishment of barrier function. STATEMENT OF SIGNIFICANCE: Rete ridges play a role in epidermal homeostasis, enhance epidermal-dermal adhesion and act as niches for epidermal stem cells. Despite their role in skin function, these structures are not directly engineered into synthetic skin. A new method to rapidly and reproducibly generate rete ridges in engineered skin was developed using fractional CO2 laser ablation. The resulting engineered rete ridges aided in the establishment of epidermal barrier function, basement membrane protein deposition and epidermal regeneration. This new model of engineered skin with rete ridges could be utilized as an in vitro system to study epidermal stem cells, a testbed for pharmaceutical evaluation or translated for clinical use in full-thickness wound repair.

Fractional CO2 laser ablation of porcine burn scars after grafting: Is deeper better?

INTRODUCTION: Fractional CO2 lasers have been used in clinical settings to improve scarring following burn injury. Though used with increasing frequency, the appropriate laser settings are not well defined and overall efficacy of this therapy has not been definitively established. As it has been proposed that for thick hypertrophic scars proportionally greater fluence and thus deeper ablation into the scar tissue would be most effective, the goal of this study was to examine the role of ablation depth on scar outcomes in a highly-controlled porcine model for burn scars-after grafting. METHODS: Properties of laser ablated wells were quantified on ex vivo pig skin as a function of laser energy (20, 70 or 150mJ). Full-thickness burn wounds were created on the dorsum of red Duroc pigs with the eschar excised and grafted with a split-thickness autograft meshed and expanded 1.5:1. After four weeks of healing, sites were treated with either 20, 70, or 150mJ pulse energy from a fractional CO2 laser at 5% density or left untreated as a control. Sites were treated every four weeks with three total sessions. Scar area, pigmentation, erythema, roughness, histology, and biomechanics were evaluated prior to each laser treatment at day 28, 56, and 83, as well as four weeks after the final laser treatment, day 112. Additional biopsies were collected at day 112 for gene expression analysis. RESULTS: The depth of the laser ablated wells increased with increasing pulse energy while the width of the wells was smaller in the 20mJ group and not significantly different in the 70 and 150mJ groups. Scar properties (area, color, biomechanics) were not significantly altered by laser therapy at any of the laser energies tested versus controls. Average scar roughness was improved by laser therapy in a dose dependent manner with scars treated with 150mJ of energy having the smoothest surface; however, these changes were not statistically significant. Assessment of matrix metalloproteinase 9 gene expression showed a slight upregulation in scars treated with 70 or 150mJ versus control scars and scars treated with 20mJ pulse energy. CONCLUSION: The current study demonstrated that the properties of the ablative well (depth and width) are not linearly correlated with laser pulse energy, with only a small increase in well depth at energies between 70 and 150mJ. Overall, the study suggests that there is little difference in outcomes as a function of laser energy. Fractional CO2 laser therapy did not result in any statistically significant benefit to scar properties assessed by quantitative, objective measures, thus highlighting the need for additional clinical investigation of laser therapy efficacy with non-treated controls and objective measures of outcome.

Early cessation of pressure garment therapy results in scar contraction and thickening.

Pressure garment therapy is often prescribed to improve scar properties following full-thickness burn injuries. Pressure garment therapy is generally recommended for long periods of time following injury (1-2 years), though it is plagued by extremely low patient compliance. The goal of this study was to examine the effects of early cessation of pressure garment therapy on scar properties. Full-thickness burn injuries were created along the dorsum of red Duroc pigs. The burn eschar was excised and wound sites autografted with split-thickness skin. Scars were treated with pressure garments within 1 week of injury and pressure was maintained for either 29 weeks (continuous pressure) or for 17 weeks followed by cessation of pressure for an additional 12 weeks (pressure released); scars receiving no treatment served as controls. Scars that underwent pressure garment therapy were significantly smoother and less contracted with decreased scar height compared to control scars at 17 weeks. These benefits were maintained in the continuous pressure group until week 29. In the pressure released group, grafts significantly contracted and became more raised, harder and rougher after the therapy was discontinued. Pressure cessation also resulted in large changes in collagen fiber orientation and increases in collagen fiber thickness. The results suggest that pressure garment therapy effectively improves scar properties following severe burn injury; however, early cessation of the therapy results in substantial loss of these improvements.

Structural, Chemical, and Mechanical Properties of Pressure Garments as a Function of Simulated Use and Repeated Laundering.

Pressure garments are widely employed for management of postburn scarring. Although pressure magnitude has been linked to efficacy, maintenance of uniform pressure delivery is challenging. An understanding of garment fabric properties is needed to optimize pressure delivery for the duration of garment use. To address this issue, compression vests were manufactured using two commonly used fabrics, Powernet or Dri-Tek Tricot, to achieve 10% reduction in circumference for a child-sized mannequin. Applied pressure was tracked on five anatomical sites over 23 hours, before laundering or after one and five laundering cycles. Load relaxation and fatigue of fabrics were tested before laundering or after one and five laundering cycles, and structural analysis via scanning electron microscopy was performed. Prior to laundering, pressure vests fabricated using Powernet or Dri-Tek Tricot generated a maximum pressure on the mannequin of 20 and 23 mm Hg, respectively. With both fabrics, pressure decreased during daily wear. Following five laundering cycles, Dri-Tek Tricot vests delivered a maximum of 7 vs 15 mm Hg pressure for Powernet at the same site. In cyclic tensile and load relaxation tests, exerted force correlated with fabric weave orientation with greatest force measured parallel to a fabric's long axis. The results demonstrate that Powernet exhibited the greatest applied force with the least garment fatigue. Fabric orientation with respect to the primary direction of tension was a critical factor in pressure generation and maintenance. This study suggests that fabrication of garments using Powernet with its long axis parallel to patient's body part circumference may enhance the magnitude and maintenance of pressure delivery.