"Trooping the color”: restoring the original donor skin color by addition of melanocytes to bioengineered skin analogs

Purpose: Autologous skin substitutes to cover large skin defects are used since several years. Melanocytes, although essential for solar protection and pigmentation of skin, are not yet systematically added to such substitutes. In this experimental study, we reconstructed melanocyte-containing dermo-epidermal skin substitutes from donor skins of different skin pigmentation types and studied them in an animal model. Features pertinent to skin color were analyzed and compared in both skin substitutes and original donor skin. Methods: Keratinocytes, melanocytes, and fibroblast were isolated, cultured, and expanded from skin biopsies of light- and dark-pigmented patients. For each donor, melanocytes and keratinocytes were seeded in different ratios (1:1, 1:5, 1:10) onto collagen gels previously populated with autologous fibroblasts. Skin substitutes were then transplanted onto full-thickness wounds of immuno-incompetent rats. After 8weeks, macroscopic and microscopic analyses were conducted with regard to skin color and architecture. Results: Chromameter evaluation revealed that skin color of reconstructed light- and dark-pigmented skin was very similar to donor skin, independent of which melanocyte/keratinocyte ratio was added. Histological analyses of the skin analogs confirmed these findings. Conclusion: These data suggest that adding autologous melanocytes to bioengineered dermo-epidermal skin analogs can sustainably restore the patients' native skin colo

Rebuild, restore, reinnervate: do human tissue engineered dermo-epidermal skin analogs attract host nerve fibers for innervation?

Purpose: Tissue engineered skin substitutes are a promising tool to cover large skin defects, but little is known about reinnervation of transplants. In this experimental study, we analyzed the ingrowth of host peripheral nerve fibers into human tissue engineered dermo-epidermal skin substitutes in a rat model. Using varying cell types in the epidermal compartment, we wanted to assess the influence of epidermal cell types on reinnervation of the substitute. Methods: We isolated keratinocytes, melanocytes, fibroblasts, and eccrine sweat gland cells from human skin biopsies. After expansion, epidermal cells were seeded on human dermal fibroblast-containing collagen type I hydrogels as follows: (1) keratinocytes only, (2) keratinocytes with melanocytes, (3) sweat gland cells. These substitutes were transplanted into full-thickness skin wounds on the back of immuno-incompetent rats and were analyzed after 3 and 8weeks. Histological sections were examined with regard to myelinated and unmyelinated nerve fiber ingrowth using markers such as PGP9.5, NF-200, and NF-145. Results: After 3weeks, the skin substitutes of all three epidermal cell variants showed no neuronal ingrowth from the host into the transplant. After 8weeks, we could detect an innervation of all three types of skin substitutes. However, the nerve fibers were restricted to the dermal compartment and we could not find any unmyelinated fibers in the epidermis. Furthermore, there was no distinct difference between the constructs resulting from the different cell types used to generate an epidermis. Conclusion: Our human tissue engineered dermo-epidermal skin substitutes demonstrate a host-derived innervation of the dermal compartment as early as 8weeks after transplantation. Thus, our substitutes apparently have the capacity to attract nerve fibers from adjacent host tissues, which also grow into grafts and thereby potentially restore skin sensitivit

Premiere use of Integra™ artificial skin to close an extensive fetal skin defect during open in utero repair of myelomeningocele

Background: There are fetuses demonstrating very large myelomeningocele lesion which can not be covered with autochothonous skin. Material and Methods: We use Integra™ artifical skin for intrauterine coverage of the back lesion. A reverse latissimus dorsi flap was used postnataly to reinforce the repair site. Conclusion: Integra™ appears to be a suitable coverage for large soft tissue defects in utero. Moreover, a postnatal reverse latissimus dorsi flap appears to markedly strengthen tissue coverage over a spinal cord rescued in uter

Rebuild, restore, reinnervate: do human tissue engineered dermo-epidermal skin analogs attract host nerve fibers for innervation?

PURPOSE: Tissue engineered skin substitutes are a promising tool to cover large skin defects, but little is known about reinnervation of transplants. In this experimental study, we analyzed the ingrowth of host peripheral nerve fibers into human tissue engineered dermo-epidermal skin substitutes in a rat model. Using varying cell types in the epidermal compartment, we wanted to assess the influence of epidermal cell types on reinnervation of the substitute. METHODS: We isolated keratinocytes, melanocytes, fibroblasts, and eccrine sweat gland cells from human skin biopsies. After expansion, epidermal cells were seeded on human dermal fibroblast-containing collagen type I hydrogels as follows: (1) keratinocytes only, (2) keratinocytes with melanocytes, (3) sweat gland cells. These substitutes were transplanted into full-thickness skin wounds on the back of immuno-incompetent rats and were analyzed after 3 and 8 weeks. Histological sections were examined with regard to myelinated and unmyelinated nerve fiber ingrowth using markers such as PGP9.5, NF-200, and NF-145. RESULTS: After 3 weeks, the skin substitutes of all three epidermal cell variants showed no neuronal ingrowth from the host into the transplant. After 8 weeks, we could detect an innervation of all three types of skin substitutes. However, the nerve fibers were restricted to the dermal compartment and we could not find any unmyelinated fibers in the epidermis. Furthermore, there was no distinct difference between the constructs resulting from the different cell types used to generate an epidermis. CONCLUSION: Our human tissue engineered dermo-epidermal skin substitutes demonstrate a host-derived innervation of the dermal compartment as early as 8 weeks after transplantation. Thus, our substitutes apparently have the capacity to attract nerve fibers from adjacent host tissues, which also grow into grafts and thereby potentially restore skin sensitivity

Comparison of in vivo immune responses following transplantation of vascularized and non-vascularized human dermo-epidermal skin substitutes

PURPOSE: Autologous bio-engineered dermo-epidermal skin substitutes (DESS) represent an alternative therapeutic option for a definitive treatment of skin defects in human patients. Largely, the interaction of host immune cells with transplanted DESS is considered to be essential for the granulation tissue formation, graft take, and its functionality. The aim of this study was to compare the spatiotemporal distribution and density of host-derived monocytes/macrophages and granulocytes in vascularized (vascDESS) versus non-vascularized DESS (non-vascDESS) in a rat model. METHODS: Keratinocytes and the stromal vascular fraction (SVF) were derived from human skin or human adipose tissue, respectively. Human SVF containing both endothelial and mesenchymal/stromal progenitors was used to develop a vascularized collagen type I-based dermal component in vitro. The donor-matched, monolayer-expanded adipose stromal cells lacking endothelial cells were used as a negative control. Subsequently, human keratinocytes were seeded on top of hydrogels to build dermo-epidermal skin grafts. After transplantation onto full-thickness skin wounds on the back of immuno-incompetent rats, grafts were excised and analyzed after 1 and 3 weeks. The expression of distinct inflammatory cell markers specific for host-derived monocytes/macrophages (CD11b, CD68) or granulocytes (HIS48) was analyzed by immunofluorescence microscopy. RESULTS: All skin grafts were infiltrated by host-derived monocytes/macrophages (CD11b(+), CD68(+)) and granulocytes (HIS48(+)) between 1-3 week post-transplantation. When compared to non-vascDESS, the vascDESS showed an increased granulocyte infiltration at all time points analyzed with the majority of cells scattered throughout the whole dermal part. Whereas a moderate number of rat monocytes/macrophages (CD11b(+), CD68(+)) were found in vascDESS at 1 week, only a few cells were detected in non-vascDESS. We observed a time-dependent decrease of monocytes/macrophages in all transplants at 3 weeks. CONCLUSIONS: These results demonstrate a distinct spatiotemporal distribution of monocytes/macrophages as well as granulocytes in our transplants that closely resemble the one observed during physiological wound healing. The differences identified between vascDESS and non-vascDESS may indicate that human endothelial cells lining blood capillaries of vascDESS accelerate infiltration of monocytes and leukocytes

Characterization of M1 and M2 polarization of macrophages in vascularized human dermo-epidermal skin substitutes in vivo

AIMS AND OBJECTIVES: Vascularized bio-engineered human dermo-epidermal skin substitutes (vascDESS) hold promise for treating burn patients, including those with severe full-thickness wounds. We have previously shown that vascDESS promote wound healing by enhanced influx of macrophages and granulocytes. Immediately following transplantation, macrophages infiltrate the graft and differentiate into a pro-inflammatory (M1) or a pro-healing M2 phenotype. The aim of this study was to characterize the activation state of macrophages infiltrating skin transplants at distinct time points following transplantation. METHODS: Keratinocytes and the stromal vascular fraction (SVF) were derived from human skin or adipose tissue, respectively. Human SVF containing both endothelial and mesenchymal/stromal cells was used to generate vascularized dermal component in vitro, which was subsequently covered with human keratinocytes. Finally, vascDESS were transplanted on the back of immuno-incompetent rats, excised, and analyzed after 1 and 3 weeks using immunohistological techniques. RESULTS: A panel of markers of macrophage M1 (nitric oxide synthase: iNOS) and M2 (CD206) subclass was used. All skin grafts were infiltrated by both M1 and M2 rat macrophages between 1-3 weeks post-transplantation. CD68 (PG-M1) was used as a pan-macrophage marker. The number of CD68+CD206+ M2-polarized macrophages was higher in 3-week transplants as compared to early-stage transplants (1 week). In contrast, the number of CD68+iNOS+ M1 cells was markedly decreased in later stages in vivo. CONCLUSIONS: Macrophages exhibit a heterogeneous and temporally regulated polarization during skin wound healing. Our results suggest that the phenotype of macrophages changes during healing from a more pro-inflammatory (M1) profile in early stages after injury, to a less inflammatory, pro-healing (M2) phenotype in later phases in vivo

Fulminant wound infection with Clostridium perfringens and Bacillus cereus in a healthy five year old boy

Trauma related soft tissue infections with Clostridium perfringens and Bacillus cereus in children are rare. We report a case of a healthy five-year old boy with a posttraumatic fulminant localized necrotisizing facial soft tissue infection.A five-year old healthy boy was admitted after a sledging accident. He presented with a laceration of the right cheek. Within 12 h after rinsing and primarily closure under analgosedation he developed localized severe pain and fever. Immediate wound revision under general anesthesia revealed necrotic subcutaneous tissue with an abscess cavity. After surgical debridement the wound was initially left open. An empiric antibiotic therapy with amoxicillin/clavulanate was initiated. A facial bone fracture was excluded by computed tomography. C. perfringens., B. cereus, B. licheniformis and B. pumilus were recovered. A correct intravenous combination therapy was initiated followed by secondary wound closure. The local situation and general condition of the boy improved markedly and no further intervention was necessary. Keywords: Infection, Children, Clostridium perfringens, Bacillus cereus, Traum

In utero Plastic Surgery in Zurich: Successful Use of Distally Pedicled Random Pattern Transposition Flaps for Definitive Skin Closure during Open Fetal Spina Bifida Repair

&lt;b&gt;&lt;i&gt;Background:&lt;/i&gt;&lt;/b&gt; One of the intraoperative challenges of fetal spina bifida repair is skin closure when there is an extended skin defect. Thus, we examined whether distally pedicled random pattern transposition flaps (TFs) are a valid option to overcome this problem. &lt;b&gt;&lt;i&gt;Subjects and Methods:&lt;/i&gt;&lt;/b&gt; All patients undergoing in utero repair of spina bifida with application of a TF for back skin closure were analyzed focusing on intraoperative flap characteristics and postoperative flap performance. &lt;b&gt;&lt;i&gt;Results:&lt;/i&gt;&lt;/b&gt; In 30 (70%) of the 43 fetuses a primary skin closure was achieved, in 5 (12%) a skin substitute was used, and in 8 (18%) a TF was applied. Flap raising and insertion was uneventful and perfusion was sufficient in all 8 fetuses (100%). In 3 fetuses (37%) the donor sites were closed primarily, and in 5 (63%) a skin substitute was used for coverage. At birth, 7 flaps were viable and provided robust skin coverage over the center of the former lesion. Complications included a small skin defect with CSF leakage in 1 patient (13%). &lt;b&gt;&lt;i&gt;Conclusion:&lt;/i&gt;&lt;/b&gt; During open fetal spina bifida repair, TFs can be safely and efficaciously used to obtain solid and durable skin coverage over lesions too large to allow conventional primary skin closure.</jats:p

"Trooping the color": restoring the original donor skin color by addition of melanocytes to bioengineered skin analogs

PURPOSE: Autologous skin substitutes to cover large skin defects are used since several years. Melanocytes, although essential for solar protection and pigmentation of skin, are not yet systematically added to such substitutes. In this experimental study, we reconstructed melanocyte-containing dermo-epidermal skin substitutes from donor skins of different skin pigmentation types and studied them in an animal model. Features pertinent to skin color were analyzed and compared in both skin substitutes and original donor skin. METHODS: Keratinocytes, melanocytes, and fibroblast were isolated, cultured, and expanded from skin biopsies of light- and dark-pigmented patients. For each donor, melanocytes and keratinocytes were seeded in different ratios (1:1, 1:5, 1:10) onto collagen gels previously populated with autologous fibroblasts. Skin substitutes were then transplanted onto full-thickness wounds of immuno-incompetent rats. After 8 weeks, macroscopic and microscopic analyses were conducted with regard to skin color and architecture. RESULTS: Chromameter evaluation revealed that skin color of reconstructed light- and dark-pigmented skin was very similar to donor skin, independent of which melanocyte/keratinocyte ratio was added. Histological analyses of the skin analogs confirmed these findings. CONCLUSION: These data suggest that adding autologous melanocytes to bioengineered dermo-epidermal skin analogs can sustainably restore the patients' native skin color