Human Skin Grafts Research Articles

Three Dimensional Bioprinting of a Vascularized and Perfusable Skin Graft Using Human Keratinocytes, Fibroblasts, Pericytes, and Endothelial Cells

Multilayered skin substitutes comprising allogeneic cells have been tested for the treatment of nonhealing cutaneous ulcers. However, such nonnative skin grafts fail to permanently engraft because they lack dermal vascular networks important for integration with the host tissue. In this study, we describe the fabrication of an implantable multilayered vascularized bioengineered skin graft using 3D bioprinting. The graft is formed using one bioink containing human foreskin dermal fibroblasts (FBs), human endothelial cells (ECs) derived from cord blood human endothelial colony-forming cells (HECFCs), and human placental pericytes (PCs) suspended in rat tail type I collagen to form a dermis followed by printing with a second bioink containing human foreskin keratinocytes (KCs) to form an epidermis. In vitro, KCs replicate and mature to form a multilayered barrier, while the ECs and PCs self-assemble into interconnected microvascular networks. The PCs in the dermal bioink associate with EC-lined vascular structures and appear to improve KC maturation. When these 3D printed grafts are implanted on the dorsum of immunodeficient mice, the human EC-lined structures inosculate with mouse microvessels arising from the wound bed and become perfused within 4 weeks after implantation. The presence of PCs in the printed dermis enhances the invasion of the graft by host microvessels and the formation of an epidermal rete. Impact Statement Three Dimensional printing can be used to generate multilayered vascularized human skin grafts that can potentially overcome the limitations of graft survival observed in current avascular skin substitutes. Inclusion of human pericytes in the dermal bioink appears to improve both dermal and epidermal maturation.

Human CD8+ Tregs expressing a MHC-specific CAR display enhanced suppression of human skin rejection and GVHD in NSG mice

AbstractPolyclonal CD8+CD45RClow/− Tregs are potent regulatory cells able to control solid organ transplantation rejection and even induce tolerance. However, donor major histocompatibility complex (MHC)–specific Tregs are more potent than polyclonal Tregs in suppressing T-cell responses and preventing acute as well as chronic rejection in rodent models. The difficulty of identifying disease-relevant antigens able to stimulate Tregs has reduced the possibility of obtaining antigen-specific Tregs. To bypass this requirement and gain the advantage of antigen specificity, and thus improve the therapeutic potential of CD8+ Tregs, we stably introduced a chimeric antigen receptor (CAR) derived from a HLA-A*02 antigen-specific antibody (A2-CAR) in human CD8+ Tregs and developed a clinically compatible protocol of transduction and expansion. We demonstrated that A2-CAR CD8+ Tregs were not phenotypically altered by the process, were specifically activated, and did not exhibit cytotoxic activity toward HLA-A*02+ kidney endothelial cells (ECs). We showed that A2-CAR CD8+ Tregs were more potent suppressors of immune responses induced by HLA-A*02 mismatch than control-CAR CD8+ Tregs, both in vitro and in vivo, in models of human skin graft rejection and graft-versus-host disease (GVHD) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. We showed that integrity of human skin graft was preserved with A2-CAR CD8+ Tregs at least 100 days in vivo after administration, and that interaction between the A2-CAR CD8+ Tregs and HLA-A*02+ kidney ECs resulted in a fine-tuned and protolerogenic activation of the ECs without cytotoxicity. Together, our results demonstrated the relevance of the CAR engineering approach to develop antigen-specific CAR-CD8+ Tregs for clinical trials in transplantation, and potentially in other diseases.

595 New autologous engineered pigmented skin for human skin graft

595 New autologous engineered pigmented skin for human skin graft

Local Growth Hormone Therapy for Pressure Ulcer Healing on a Human Skin Mouse Model.

The growth hormone is involved in skin homeostasis and wound healing. We hypothesize whether it is possible to improve pressure ulcer (PU) healing by locally applying the recombinant human growth hormone (rhGH) in a human skin mouse model. Non-obese diabetic/severe combined immunodeficient mice (n = 10) were engrafted with a full-thickness human skin graft. After 60 days with stable grafts, human skin underwent three cycles of ischemia-reperfusion with a compression device to create a PU. Mice were classified into two groups: rhGH treatment group (n = 5) and control group (n = 5). In the rhGH group for local intradermal injections, each had 0.15 mg (0.5IU) applied to the PU edges, once per week for four weeks. Evaluation of the wound healing was conducted with photographic and visual assessments, and histological analysis was performed after complete wound healing. The results showed a healing rate twice as fast in the rhGH group compared to the control group (1.25 ± 0.33 mm2/day versus 0.61 ± 0.27 mm2/day; p-value < 0.05), with a faster healing rate during the first 30 days. The rhGH group showed thicker skin (1953 ± 457 µm versus 1060 ± 208 µm; p-value < 0.05) in the repaired area, with a significant decrease in collagen type I/III ratio at wound closure (62 days, range 60–70). Local administration of the rhGH accelerates PU healing in our model. The rhGH may have a clinical use in pressure ulcer treatment.

Bispecific T-Cell Engager -Armored Chimeric Antigen Receptor T Cells Overcome Antigen Escape From EGFRvIII-Targeted Therapy For Glioblastoma

Abstract INTRODUCTION Immune therapy with T cells engineered to express chimeric antigen receptors (CARs) represents a promising therapy for patients with glioblastoma (GBM). However, clinical responses have been limited due to heterogeneous target antigen expression and outgrowth of tumors lacking the antigen targeted by CAR T cells directed against a single target. In clinical studies with CART-EGFRvIII, EGFRvIII-targeted T cells successfully localized to the brain tumor microenvironment, but ultimately failed to prevent disease progression with post-treatment specimens demonstrating high levels of wild-type EGFR despite reduced expression of EGFRvIII. METHODS We developed a novel bicistronic CAR construct engineered for local delivery of bispecific T-cell engagers (BiTEs) that target residual tumor. Specifically, EGFRvIII-targeted CAR T cells were engineered to secrete BiTEs against wild-type EGFR, which is frequently amplified and overexpressed in GBM. RESULTS Human T cells were efficiently transduced with the dual CART.BiTE transgene. These modified cells secreted biologically active EGFR-specific BiTEs that not only redirected CAR T cells but also recruited and activated untransduced bystander T cells against wild-type EGFR. Recapitulating clinical data, EGFRvIII CAR T cells were unable to completely treat tumors with heterogenous EGFRvIII expression, leading to outgrowth of EGFRvIII-negative, EGFR-positive GBM. Conversely, CART.BiTE cells cured mice even in the setting of antigen-loss, against heterogeneous and well-established intracerebral tumors in mice. Unlike CAR T cells directly targeting EGFR, which caused toxicity in human skin grafts in vivo, secreted BiTE-EGFR was both locally effective and did not result in toxicity against grafted human skin. CONCLUSION This is the first instance in which CARs and BiTEs have been combined into a single platform of immune therapy. Our results demonstrate that CARs and BiTEs can be combined strategically to mitigate antigen heterogeneity in GBM and also provide a unique T-cell-based delivery method for BiTEs to tumors in the brain.

Varying Negative Pressure Wound Therapy Acute Effects on Human Split-Thickness Autografts.

Over 6.5 million people in the United States suffer from traumatic, burn, acute, and chronic wounds yearly. When reconstruction is required, split and full-thickness autografts are a first line of treatment intervention. Negative pressure wound therapy (NPWT) is gaining traction as an adjunct modality to improve graft survival, yet the specifics on what settings to apply topically over the graft is unsubstantiated and associated with morbidities. This study was performed in an effort to understand initial changes in wound and graft healing with a long-term goal of surface pressure optimization. Excess skin from elective procedures from six human subjects was trimmed to 0.012 inch in order represent a split-thickness autografts. These grafts were treated continuously with either -75 mm Hg (n = 4), -125 mm Hg (n = 4), or no pressure (n = 4) for 3 hours. Six skin grafts were treated with no sponge or pressure control (n = 6). RNAseq was performed on all treatment groups and compared with no pressure control. Significant gene expression changes with a subset focusing on inflammatory, cellular/extracellular matrix proliferation and angiogenic mediators and having greater than 2-fold were confirmed with immunohistochemistry staining. There are 95 significant gene transcription differences among all treatment groups. NPWT leads to significantly increased gene expression of FGFR1, ET-1, and 22 Keratin proteins. Between -75 and -125 mm Hg groups, there are 19 significant gene changes. Proinflammatory genes S100A8 and Tenacin C (TNC) demonstrate an 8.8- and 9.1-fold change, respectively, and is upregulated in -125 mm Hg group and downregulated in -75 mm Hg group. Fibrinogen genes fibrinogen gamma chain and fibrinogen alpha chain had respective log2-fold changes of -7.9 and -7.4 change between treatment groups and were downregulated in -125 mm Hg group and upregulated in -75 mm Hg group. There are varying effects of surface pressures on human split-thickness autografts during the imbibition time period. NPWT may improve cellular migration, proliferation, and angiogenesis over controls. Human skin grafts respond differently to -125 and -75 mm Hg within 3 hours of NPWT treatment. The results suggest -75 mm Hg leads to less inflammation and increased fibrinogen production compared with the -125 mm Hg group, at least initially. Reducing "time to heal" with NPWT is critical to successful outcomes and quality of life within young patients who often experience pain/discomfort when treated at the current standard pump settings. The results from this study and continued investigation may quickly translate to the clinical setting by finding the ideal pressure setting utilized in an effort to reduce NPWT length of treatment, improve patient comfort, satisfaction, and psychosocial well-being.

CAR-T cells secreting BiTEs circumvent antigen escape without detectable toxicity.

Chimeric antigen receptor (CAR)-T-cell therapy for solid tumors is limited due to heterogeneous target antigen expression and outgrowth of tumors lacking the antigen targeted by CAR-T cells directed against single antigens. Here, we developed a bicistronic construct to drive expression of a CAR specific for EGFRvIII, a glioblastoma-specific tumor antigen, and a bispecific T-cell engager (BiTE) against EGFR, an antigen frequently overexpressed in glioblastoma but also expressed in normal tissues. CART.BiTE cells secreted EGFR-specific BiTEs that redirect CAR-T cells and recruit untransduced bystander T cells against wild-type EGFR. EGFRvIII-specific CAR-T cells were unable to completely treat tumors with heterogenous EGFRvIII expression, leading to outgrowth of EGFRvIII-negative, EGFR-positive glioblastoma. However, CART.BiTE cells eliminated heterogenous tumors in mouse models of glioblastoma. BiTE-EGFR was locally effective but was not detected systemically after intracranial delivery of CART.BiTE cells. Unlike EGFR-specific CAR-T cells, CART.BiTE cells did not result in toxicity against human skin grafts in vivo.

Abstract LB-066: BiTE-armored CARs overcome antigen escape in EGFRvIII-targeted therapy for glioblastoma

Abstract Glioblastoma (GBM) is a devastating disease with an extremely poor prognosis. Immune therapy with T cells engineered to express chimeric antigen receptors (CARs), represents a promising alternative to conventional therapy, but responses have been limited to date due to heterogeneous target antigen expression and the emergence of immune escape following treatment with CAR T cells directed at a single target. This was observed in clinical studies with CART-EGFRvIII, wherein EGFRvIII-targeted T cells successfully localized to the brain tumor microenvironment, but ultimately failed to prevent disease progression—with post-treatment specimens demonstrating high levels of wild-type EGFR despite reduced expression of EGFRvIII. To help address this therapeutic barrier, we developed a novel CAR construct engineered for local delivery of bispecific T-cell engagers (BiTEs) that target residual tumor. Specifically, EGFRvIII-targeted CAR T cells were engineered to secrete BiTEs against wild-type EGFR, which is not expressed in normal brain but frequently amplified in GBM, recurrent disease and other cancers. Our results demonstrate that human T cells are efficiently transduced with the dual CART.BiTE transgene, and that modified cells successfully secrete biologically active EGFR-specific BiTEs that not only redirect CAR T cells but also recruit and activate untransduced bystander T cells against wild-type EGFR. Recapitulating clinical data, EGFRvIII CAR T cells were unable to completely treat tumors with heterogenous EGFRvIII expression, leading to outgrowth of EGFRvIII-negative, EGFR-positive recurrence. Conversely, CART.BiTE cells mediated potent antitumor efficacy in the setting of heterogeneous tumors and EGFRvIII antigen loss. In these models, infusion of CART.BiTE cells yielded extended survival and cured mice with even late-stage, established intracerebral tumors, including those derived from glioma cell lines and patient-derived glioma neurosphere cultures. Unlike CAR T cells directly targeting EGFR, which caused on-target toxicity against human skin grafts in vivo, secreted BiTE-EGFR was locally effective at exceedingly low concentrations that did not mediate immune responses against healthy human tissue. These results demonstrate that CARs and BiTEs can be combined strategically to mitigate the impact of antigen heterogeneity in GBM, and also provide a unique T-cell-based delivery method for BiTEs to tumors in the brain. Citation Format: Bryan D. Choi, Xiaoling Yu, Ana P. Castano, Amanda A. Bouffard, William T. Curry, Bob S. Carter, Marcela V. Maus. BiTE-armored CARs overcome antigen escape in EGFRvIII-targeted therapy for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-066.

Immune Reaction to Type XVII Collagen Induces Intramolecular and Intermolecular Epitope Spreading in Experimental Bullous Pemphigoid Models.

Bullous pemphigoid (BP), the most common autoimmune blistering disease, is induced by autoantibodies to type XVII collagen (COL17). Previous studies demonstrated that COL17 harbors several epitopes targeted by autoreactive T and B cells and that the target epitopes change sequentially during the disease course. To elucidate the details of the humoral immune response to COL17, we used an active BP mouse model in which BP is induced by the adoptive transfer of spleen cells from wild-type mice immunized with human COL17-expressing skin grafting to immunodeficient COL17-humanized (Rag-2−/−, mouse Col17−/−, human COL17+) mice. By immunoblot analysis, antibodies to the NC16A domain and other extracellular domains (ECDs) of COL17 were detected earlier than antibodies to intracellular domains (ICDs) in the active BP model. Time course analysis by enzyme-linked immunosorbent assay demonstrated a delayed peak of antibodies to ICD epitopes in active BP model. The blockade of CD40–CD40 ligand interaction soon after the adoptive transfer suppressed the production of antibodies to the non-collagenous 16A (NC16A) domain but not to an ICD epitope, suggesting the sequential activation from T and B cells against the ECD epitopes including the NC16A domain to those against ICD epitopes in vivo. Both wild-type mice immunized with a fragment of the NC16A domain and the recipients of those spleen cells produced IgG antibodies to ICD and ECD epitopes, showing intramolecular epitope spreading from the NC16A domain to other epitopes of COL17. Furthermore, we found that a portion of the active BP model mice show intermolecular epitope spreading from human COL17 to murine BP230. The appearance of antibodies to ICD epitopes of COL17 or of antibodies to murine BP230 did not correlate with the skin changes in the mice, suggesting that those antibodies have low pathogenicity. These results suggest that the immune response to the ECD epitopes of COL17, especially to the NC16A domain, triggers intramolecular, and intermolecular epitope spreading to ICD epitopes of COL17 and to murine BP230. These novel findings provide insight into the mechanism of epitope spreading in organ-specific, antibody-mediated autoimmune disorders.

Application of acellular human dermis and skin grafts for lower extremity reconstruction.

To use both acellular human dermis and skin grafting simultaneously for improved skin grafting without contracture. The study also aims to address the lack of research on the application of an acellular human dermis in diverse clinical cases. The study examined patients who had received acellular human dermis (CGDerm, CGBio, Seoul, Korea) and split-thickness skin grafting (STSG) simultaneously for lower limb, full-thickness skin defects between September 2012 and June 2014. The researchers performed chart reviews retrospectively and examined the patients based on the following factors: gender, age, injury mechanism, size, exposed structure, pre-coverage dressing method, coverage method, post-operational engraftment and total healing period, contracture development, elasticity, and infection development. A sample of 27 patients with a total of 30 wounds took part in the study. Of these wounds, 29 showed successful engraftment without infection or contracture. In one case, continued seroma was observed and, following new coverage of both the acellular human dermis and STSG, engraftment was successful. Human dermis can play an important role in securing the availability of surrounding tissue and in contracture prevention, both of which are key to lower limb reconstruction. Of the types available, acellular human dermis showed lower infection rates than other human dermis types, and its engraftment rate was higher than in STSG-only cases. These findings suggest that acellular human dermis use in STSG is effective and safe in lower limb reconstruction.

Allogeneic skin donors from a tissue bank in Southern Brazil: clinical and epidemiological profiles and microbial colonization of skin.

An allogeneic human skin graft is a temporary biologic dressing used in extensive burns that can be a providential treatment for affected patients. Skin quality depends directly on its microbial decontamination after processing in a tissue bank. Our objective was to describe the skin donor profiles in relation to the analysis of the microbial colonization of the donated skin. This clinical study includes epidemiological and microbiological data on skin donors from 2012 to 2014. The donor information database was compiled from the medical records of skin donors filed in the tissue bank. The donors were assessed regarding the microbial colonization of the skin at the time of processing in the tissue bank. We found a statistically significant association (P = 0.020) between lower average age of the donor and the presence of microbial colonization. We observed that Gram-negative bacteria (GNB) are associated with male gender (P = 0.015), source hospital A (P = 0.034), and over 7 days stay in an intensive care unit (ICU) (P = 0.001). We also observed that Staphylococcus aureus is associated with skin-harvesting hospital C (P = 0.034) and that Gram-positive bacilli (GPB) are associated with up to 7 days stay in an ICU (P = 0.009). We found significant associations between the type of microorganism colonizing the skin and the epidemiological and clinical profiles of the donors. This information is extremely important when determining the potential use of skin source and so optimizing the donation of allogeneic skin for transplantation.

Regulation of human T cell responses by dNP2-ctCTLA-4 inhibits human skin and microvessel graft rejection

Manipulation of human T cell functioning by delivery of macromolecules such as DNA, RNA, or protein is limited, unless the human T cells have been stimulated or electropermeabilized. To achieve successful adaptation and survival of a grafted organ, the alloreactive T cells that induce graft rejection must be regulated. Corticosteroids, calcineurin inhibitors, and mTOR inhibitors, which are systemic immunosuppressants, are currently used for transplantation, with significant side effects. In this study, we demonstrated that a cell-permeable peptide (CPP), dNP2, could efficiently deliver proteins into human CD4 and CD8 T cells. We confirmed regulatory functioning of the cytoplasmic domain of CTLA-4 conjugated with dNP2 (dNP2-ctCTLA-4) in human T cell activation, proliferation, and chemokine receptor expression. We utilized a human skin allograft system in SCID/beige mice to examine whether dNP2-ctCTLA-4 could inhibit allograft rejection by controlling T cell responses. The grafted skin tissue inflammation, allogeneic T cell infiltration, and blood cytokine level was markedly reduced by dNP2-ctCTLA-4, resulting in successful transplantation. In addition, it also inhibited T cell alloresponses against microvessels formed form Bcl-2-transduced human umbilical vein endothelial cells implanted into Balb/c Rag1−/−/IL-2Rγ−/- double knockout (DKO) mice, assessed as reduced T cell infiltration and granzyme B expression. These results collectively suggest that dNP2 peptide conjugation offers a valuable tool for delivering macromolecules like proteins into human T cells, and dNP2-ctCTLA-4 is a novel agent that shows potential in controlling human T cell responses to allow successful adaptation of grafted tissues.

Murine type VII collagen distorts outcome in human skin graft mouse model for dystrophic epidermolysis bullosa

Human skin graft mouse models are widely used to investigate and develop therapeutic strategies for the severe generalized form of recessive dystrophic epidermolysis bullosa (RDEB), which is caused by biallelic null mutations in COL7A1 and the complete absence of type VII collagen (C7). Most therapeutic approaches are focused on reintroducing C7. Therefore, C7 and anchoring fibrils are widely used as readouts in therapeutic research with skin graft models. In this study, we investigated the expression pattern of human and murine C7 in a grafting model, in which human skin is reconstituted out of in vitro cultured keratinocytes and fibroblasts. The model revealed that murine C7 was deposited in both human healthy control and RDEB skin grafts. Moreover, we found that murine C7 is able to form anchoring fibrils in human grafts. Therefore, we advocate the use of human‐specific antibodies when assessing the reintroduction of C7 using RDEB skin graft mouse models.

Identification of functionally unique CD4 T cells that are the circulating counterparts of epidermal resident memory T cells

Abstract As a barrier organ, the skin contains specialized T cell populations that combat infection and also help maintain tissue homeostasis and promote wound repair. Circulating skin-tropic T cells can also be identified in the blood based on their expression of the cutaneous lymphocyte antigen (CLA), but the developmental and functional relationships between these circulating CLA+ T cells and tissue-resident T cells in the skin are not fully understood. Using 33-parameter Cytometry by time-of-flight (CyTOF) analysis we identified a novel population of skin-tropic CD4+ T cells in human blood that expresses the CD103 integrin and phenotypically and functionally resembles epidermal resident memory T cells (TRM) in the skin. RNA sequencing identified a set of signature genes shared by circulating CD103+CLAhi T cells in the blood and epidermal TRM cells in the skin, and suggested that CD103+CLAhiT cells contribute to normal skin function and response to tissue damage. Finally, using humanized NSG mice carrying full-thickness human skin grafts we demonstrated that, despite their identification as TRM, CD103+CLAhi T cells in the skin are capable of exiting the tissue and re-entering circulation. Thus, CD103+CLAhi T cells in the blood represent a circulating T cell population of cutaneous TRM, and this provides novel opportunities for the study and therapeutic manipulation of TRM cells in the contexts of cutaneous infection, inflammation, and tissue-repair.

749 Murine type VII collagen distorts outcome in human skin graft mouse models for dystrophic epidermolysis bullosa

749 Murine type VII collagen distorts outcome in human skin graft mouse models for dystrophic epidermolysis bullosa

Deletion of a Pathogenic Mutation-Containing Exon of COL7A1 Allows Clonal Gene Editing Correction of RDEB Patient Epidermal Stem Cells

Recessive dystrophic epidermolysis bullosa is a severe skin fragility disease caused by loss of functional type VII collagen at the dermal-epidermal junction. A frameshift mutation in exon 80 of COL7A1 gene, c.6527insC, is highly prevalent in the Spanish patient population. We have implemented gene-editing strategies for COL7A1 frame restoration by NHEJ-induced indels in epidermal stem cells from patients carrying this mutation. TALEN nucleases designed to cut within the COL7A1 exon 80 sequence were delivered to primary patient keratinocyte cultures by non-integrating viral vectors. After genotyping a large collection of vector-transduced patient keratinocyte clones with high proliferative potential, we identified a significant percentage of clones with COL7A1 reading frame recovery and Collagen VII protein expression. Skin equivalents generated with cells from a clone lacking exon 80 entirely were able to regenerate phenotypically normal human skin upon their grafting onto immunodeficient mice. These patient-derived human skin grafts showed Collagen VII deposition at the basement membrane zone, formation of anchoring fibrils, and structural integrity when analyzed 12 weeks after grafting. Our data provide a proof-of-principle for recessive dystrophic epidermolysis bullosa treatment through ex vivo gene editing based on removal of pathogenic mutation-containing, functionally expendable COL7A1 exons in patient epidermal stem cells.

Surface-enhanced Raman scattering nanosensors for in vivo detection of nucleic acid targets in a large animal model

Although nanotechnology has led to important advances in in vitro diagnostics, the development of nanosensors for in vivo detection remains very challenging. Here, we demonstrated the proof-of-principle of in vivo detection of nucleic acid targets using a promising type of surface-enhanced Raman scattering (SERS) nanosensor implanted in the skin of a large animal model (pig). The in vivo nanosensor used in this study involves the “inverse molecular sentinel” detection scheme using plasmonics-active nanostars, which have tunable absorption bands in the near infrared region of the “tissue optical window”, rendering them efficient as an optical sensing platform for in vivo optical detection. Ex vivo measurements were also performed using human skin grafts to demonstrate the detection of SERS nanosensors through tissue. In this study, a new core–shell nanorattle probe with Raman reporters trapped between the core and shell was utilized as an internal standard system for self-calibration. These results illustrate the usefulness and translational potential of the SERS nanosensor for in vivo biosensing.

Simultaneous silencing of TGF-β1 and COX-2 reduces human skin hypertrophic scar through activation of fibroblast apoptosis.

Excessive skin scars due to elective operations or trauma represent a challenging clinical problem. Pathophysiology of hypertrophic scars entails a prolonged inflammatory and proliferative phase of wound healing. Over expression of TGF-β1 and COX-2 play key regulatory roles of the aberrant fibrogenic responses and proinflammatory mediators. When we silenced TGF-β1 and COX-2 expression simultaneously in primary human fibroblasts, a marked increase in the apoptotic cell population occurred in contrast to those only treated with either TGF-β1 or COX-2 siRNA alone. Furthermore, using human hypertrophic scar and skin graft implant models in mice, we observed significant size reductions of the implanted tissues following intra-scar administration of TGF-β1/COX-2 specific siRNA combination packaged with Histidine Lysine Polymer (HKP). Gene expression analyses of those treated tissues revealed silencing of the target gene along with down regulations of pro-fibrotic factors such as α-SMA, hydroxyproline acid, Collagen 1 and Collagen 3. Using TUNEL assay detection, we found that the human fibroblasts in the implanted tissues treated with the TGF-β1/COX-2siRNAs combination exhibited significant apoptotic activity. Therefore we conclude that a synergistic effect of the TGF-β1/COX-2siRNAs combination contributed to the size reductions of the hypertrophic scar implants, through activation of fibroblast apoptosis and re-balancing between scar tissue deposition and degradation.

Maintenance of human skin tissue and cellularity after engraftment in immunodeficient mouse model

A lack of an appropriate animal model to study human skin in vivo remains a major problem. Human skin explant can be conserved only few days in culture medium. For these reasons, it is necessary to developed humanized mice model with human skin graft for studying of skin innate and adaptive responses. Humanized mice model with human skin engraftment have been Non-obese diabetic-SCID gamma chain null mice (NSG, NOD.Cg-PrkdcscidIL2rg tmlWjl/Sz) were engrafted with fresh human skin obtained from healthy volunteers undergoing plastic surgery of breast, abdomen or face lifting. Eighty-two mice were engrafted with 16 different human skin with standard operating procedure. A failure of human skin engraftment was found in 21 (25.6%) of the engrafted mice, due to partial or total human skin necrosis. We observed very good or good engrafted skin behavior in 61 humanized mice (78.6%), with preservation of the structures of the human skin compared to non-engrafted human skin and conservation of the major skin resident immune Antigen Presenting Cells 1 to 3-week post-graft. Here, we described the standard operating procedure, and performance of human skin engraftment in NOD-Scid IL2rgamma(null) mouse model with the maintenance of skin epidermal tissue and cells

500-Gray γ-Irradiation May Increase Adhesion Strength of Lyophilized Cadaveric Split-Thickness Skin Graft to Wound Bed.

Human cadaveric skin grafts are considered as the "gold standard" for temporary wound coverage because they provide a more conductive environment for natural wound healing. Lyophilization, packing, and terminal sterilization with gamma-ray can facilitate the application of cadaveric split-thickness skin grafts, but may alter the adhesion properties of the grafts. In a pilot study, we found that 500 Gy γ-irradiation seemed not to reduce the adherence between the grafts and wound beds. We conducted this experiment to compare the adherences of lyophilized, 500-Gy γ-irradiated skin grafts to that of lyophilized, nonirradiated grafts. Pairs of wounds were created over the backs of Sprague- Dawley rats. Pairs of "lyophilized, 500-Gy γ-irradiated" and "lyophilized, nonirradiated" cadaveric split-thickness skin grafts were fixed to the wound beds. Adhesion strength between the grafts and the wound beds was measured and compared. On post-skin-graft day 7 and day 10, the adhesion strength of γ-irradiated grafts was greater than that of the nonirradiated grafts. Because lyophilized cadaveric skin grafts can be vascularized and the collagen of its dermal component can be remodeled after grafting, the superior adhesion strength of 500-Gy γ-irradiated grafts can be explained by the collagen changes from irradiation.