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910 Cellular versus acellular matrix devices in treatment of diabetic foot ulcers: preliminary results of a comparative efficacy randomized controlled trial
Tissue Regeneration and Wound Healing | ABSTRACTS 909
Developing a 3D microfluidic model of immune-competent human skin using autologous cells H Abaci1, H Wobma1, Y Doucet2, JU Shin1, J Jackow2, Z Guo2, K Yeager1, G VunjakNovakovic2 and AM Christiano2 1 Columbia University Medical Center, New York, NY and 2 Columbia University, New York, NY Most studies of human skin toxicity and immune reactivity are carried out using animal models, however, these are often poorly representative of human physiology. Current 2D in vitro models using mature human skin cells lack the structural complexity and heterogeneous cell types necessary to provide meaningful functional readouts. In this study, we i) generated iPSC-derived skin constructs with autologous dendritic cells and T cells; and ii) designed a microfluidic platform which can recapitulate the infiltration of circulating lymphocytes into the skin through endothelium where they can interact with antigen presenting cells. We first created a polycarbonate-based platform with 96-well transwell inserts for micro skin tissues that were connected to each other with microfluidic channels. We next aimed to build a skin construct containing immune cells from autologous sources. This was made feasible by using peripheral blood mononuclear cell (PBMC)-derived iPSCs as the source for deriving keratinocytes and fibroblasts, and then differentiating CD14+ cells from the same blood donor into Langerhans cells (LCs). We successfully generated iPSC-derived skin constructs, incorporated LCs into the epidermis, and validated the LC migratory function in response to various chemical stimuli. To test whether we could mimic a psoriatic response, naı¨ve CD4+ T cells were isolated from PBMCs of the same blood donor and differentiated into specific Th cell populations. Upon activation, these cells infiltrated the epidermis of the autologous skin constructs. Our immune-competent skin model containing autologous immune cells represents an innovative in vitro platform for drug testing in immune-mediated skin diseases.
911
910
Cellular versus acellular matrix devices in treatment of diabetic foot ulcers: preliminary results of a comparative efficacy randomized controlled trial C Tchanque-Fossuo1, S Dahle2, H Lev-Tov3, C Li4 and R Isseroff5 1 University of California Davis, Sacramento, CA, 2 UC Davis/VA Northern California, Sacramento Medical Center, Mather, CA, 3 Department of Dermatology and Cutaneous Surgery, Miami, FL, 4 University of California Davis Department of Public Health Sciences, Division of Biostatistics, Davis, CA and 5 UC Davis/Dermatology Service VANCHS, Sacramento, CA Diabetic foot ulcers (DFU) heal with difficulty: standard of care (SOC) heals less than 30% at 20 weeks. Dermagraft, a bioengineered matrix containing live cells, has shown to increase healing over SOC, albeit with great cost and with modest improvement. We hypothesize there is no difference in healing using Dermagraft compared to the less expensive, noncellular matrix, Oasis. We conducted a prospective randomized, single-blind, controlled trial with three arms: SOC control, Dermagraft, or Oasis in patients with DFU. The primary outcome is percentage of subjects with complete wound closure by week 12 and a secondary outcome is the percentage with complete wound closure by week 28. During the 2-week screening phase, subjects with a 40% change in ulcer size were excluded. Patients were randomized into: SOC (n¼19), Dermagraft (n¼18) and Oasis (n¼18) for an active treatment phase (8 weeks) followed by maintenance phase (5 weeks SOC), study endpoint visit (week 12), and 4 monthly follow-up visits. There was equal distribution of demographical data among the groups and no difference in ulcer laterality or ulcer position among the groups. Although significant reduction in wound area from week 1 to 28 was noted in all three groups (p < 0.05), there was no significant difference in percent of subjects with complete wound closure by week 12 (55.5% Dermagraft, 72.2% Oasis, 57.9% SOC, p¼0.61) and week 28 (72.2% Dermagraft, 88.9% Oasis, 73.7% SOC, p¼0.50). This exploratory analysis suggests that the outcomes of treatment with either cellular Dermagraft or acellular Oasis matrix are comparable. Our study is still actively enrolling patients to meet the target, and we have yet to finalize the trial to make definitive conclusions.
912
Transient receptor potential (TRP) channels regulate cell migration of keratinocyte in vitro S Hiroyasu and JCR Jones Washington State University, Pullman, WA During re-epithelialization of skin wounds, basal keratinocytes proximal to the wound edge migrate over the wound bed in a directed manner. Our focus is to define the underlying mechanisms regulating such keratinocyte migration. Several reports indicate that cell migration is controlled by the modulation of Ca2+ concentration at the leading front (lamellipodium) of a moving cell. This modulation is determined by certain Ca2+ channels on the cell membrane including transient receptor potential (TRP) channels. To begin to analyze the function of Ca2+ in migrating keratinocytes, we first observed Ca2+ localization using Fluo-4 AM in live cells. During single cell migration, we detected high Ca2+ transients (flickers) in the lamellipodium which alternated from one lateral edge (flank) to the other. These flickers are temporally and spatially associated with attachment/detachment of the flanks as a single keratinocyte ‘walks’ over its substrate. Following a challenge with the Ca2+ chelator ethylene glycol-bis (b-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), both the Ca2+ concentration in the lamellipodium and cell motility are decreased. To screen the Ca2+ channels to identify which is responsible for the change in cell motility, we challenged the cells with the broad TRP channel inhibitors ruthenium red and 2-aminoethoxydiphenylborane (2-APB). Both inhibitors suppress single cell motility and collective cell migration. These results support the hypothesis that TRP channels support keratinocyte migration and are a potential target for improving wound healing in vivo.
Regulation of the dynamics of a6b4 integrin and vimentin in squamous cell carcinoma ZT Colburn and JCR Jones Washington State University, Pullman, WA The expression of the intermediate filament protein vimentin is a hallmark of squamous cell carcinoma. Moreover, vimentin expression is not only correlated with reduced survival but also with enhanced metastasis. Our goal was to test the possibility that vimentin promotes metastasis of squamous cell carcinoma by enhancing cancer cell motility. Thus, we investigated an association between vimentin filaments and motility-related, cell-matrix adhesion structures in tumor cells. Though vimentin filaments rarely associate with focal adhesions, they frequently interact with a6b4 integrin-rich hemidesmosome protein complexes organized into punctate structures at the cell-matrix interface. Interestingly, while most a6b4 integrin puncta are associated with keratin filaments, only a subset of puncta near the leading edge of front-rear polarized cells exhibit interaction with vimentin filaments. We evaluated a6b4 integrin-vimentin filament localization in motile cells by immunostaining immediately following time-lapse microscopy. These assays revealed that the lamellipodial localization of a6b4 integrin and the extension of vimentin filaments into lamellipodia are correlated with enhanced directed migration. To evaluate the function of these vimentin-associated matrix adhesions, we first generated b4 integrin knockdown cell lines. These cells exhibit reduced directed migration. Furthermore, the extension of vimentin filaments into their lamellipodia is inhibited. Structured-illumination (super-resolution) microscopy revealed that association of vimentin with a6b4 integrin reduces a6b4 integrin mobility and inhibits integrin turnover. Correspondingly, a6b4 integrin-vimentin association significantly restricts vimentin dynamics. Thus, our data indicate that sites of matrix interaction rich in a6b4 integrin and vimentin play important roles in the regulation of directed epithelial cell motility in cancer.
913
914
Midkine is upregulated in keloids A Tran1, I Dougherty1 and D Glass2 1 UT Southwestern Medical Center, Dallas, TX and 2 University of Texas Southwestern Medical Center, Dallas, TX Midkine is a 13 kDa heparin-binding growth factor involved in many important biological pathways including embryogenesis, tumorigenesis, and wound healing. It has been reported to stimulate collagen production (both type I and type III) and glycosaminoglycan synthesis in primary dermal fibroblasts. However, the role of midkine in the development of keloids (exuberant scarring of the skin) has not been extensively studied. To investigate whether midkine plays a role in keloid formation, we studied the expression of midkine in keloid and adjacent normal tissues from keloid patients. Results from both microarray analysis and from real-time PCR showed that midkine was strongly upregulated in keloid tissue. Immunohistochemistry further confirmed the increased expression of midkine in keloids, particularly in the dermis where dense hyalinized collagen bundles are concentrated. These findings suggest that midkine could potentially play a role in the pathogenesis of keloids, possibly by the modulation of extracellular matrix components.
Fibroblast heterogeneity in human skin: CD26 identifies a population of fibroblasts that upregulate collagen production in response to wounding CA Worthen1, TM Johnson1, JJ Voorhees1, J Orringer2 and GJ Fisher1 1 Department of Dermatology, University of Michigan, Ann Arbor, MI and 2 Dermatology, University of Michigan, Ann Arbor, MI Dermal fibroblasts are a heterogenous population of cells that are responsible for the deposition of extracellular matrix (ECM). After wounding, fibroblasts must quickly produce collagen and other ECM proteins to support overlying reepithelization and close the wound. Fibroblast sub-populations have been identified as collagen-producing cells that also express one or more markers such as CD34, CD90, and/or PDFG-a. Recently, a population of resident cells that express CD26 (dipeptidyl peptidase) have been reported to be responsible for the majority of ECM deposition during wound healing in mouse skin. We have investigated the properties of CD26-positive dermal cells in normal and wounded human skin. FACS analysis of dermal cells, which were freshly released from normal human skin samples by collagenase digestion, revealed a substantial population of CD26 positive cells. The majority of these cells (70%) were also CD90 positive, identifying them as resident fibroblasts. These CD26+/ CD90+ cells also expressed CD34. Gene expression analysis revealed similar levels of type I collagen expression in CD26+/CD90+ and CD26-/CD90+ dermal cells. In wounded human skin (3 weeks after CO2 laser-ablation), the number of CD26+/CD90+ cells increased 2.3fold (p¼0.026) and collagen expression in this population of cells increased 11.3-fold (p¼0.0007). In contrast, the number of CD26-/CD90+ cells did not increase, and collagen expression was significantly 3-fold lower than in the CD26+/CD90+ (p¼0.003) cells. Identification of CD26+/CD90+ dermal cells as a major source of new collagen deposition in would healing may facilitate development of novel strategies to improve wound healing in the elderly and treat non-healing wounds.
www.jidonline.org S157
Developing a 3D microfluidic model of immune-competent human skin using autologous cells H Abaci1, H Wobma1, Y Doucet2, JU Shin1, J Jackow2, Z Guo2, K Yeager1, G VunjakNovakovic2 and AM Christiano2 1 Columbia University Medical Center, New York, NY and 2 Columbia University, New York, NY Most studies of human skin toxicity and immune reactivity are carried out using animal models, however, these are often poorly representative of human physiology. Current 2D in vitro models using mature human skin cells lack the structural complexity and heterogeneous cell types necessary to provide meaningful functional readouts. In this study, we i) generated iPSC-derived skin constructs with autologous dendritic cells and T cells; and ii) designed a microfluidic platform which can recapitulate the infiltration of circulating lymphocytes into the skin through endothelium where they can interact with antigen presenting cells. We first created a polycarbonate-based platform with 96-well transwell inserts for micro skin tissues that were connected to each other with microfluidic channels. We next aimed to build a skin construct containing immune cells from autologous sources. This was made feasible by using peripheral blood mononuclear cell (PBMC)-derived iPSCs as the source for deriving keratinocytes and fibroblasts, and then differentiating CD14+ cells from the same blood donor into Langerhans cells (LCs). We successfully generated iPSC-derived skin constructs, incorporated LCs into the epidermis, and validated the LC migratory function in response to various chemical stimuli. To test whether we could mimic a psoriatic response, naı¨ve CD4+ T cells were isolated from PBMCs of the same blood donor and differentiated into specific Th cell populations. Upon activation, these cells infiltrated the epidermis of the autologous skin constructs. Our immune-competent skin model containing autologous immune cells represents an innovative in vitro platform for drug testing in immune-mediated skin diseases.
911
910
Cellular versus acellular matrix devices in treatment of diabetic foot ulcers: preliminary results of a comparative efficacy randomized controlled trial C Tchanque-Fossuo1, S Dahle2, H Lev-Tov3, C Li4 and R Isseroff5 1 University of California Davis, Sacramento, CA, 2 UC Davis/VA Northern California, Sacramento Medical Center, Mather, CA, 3 Department of Dermatology and Cutaneous Surgery, Miami, FL, 4 University of California Davis Department of Public Health Sciences, Division of Biostatistics, Davis, CA and 5 UC Davis/Dermatology Service VANCHS, Sacramento, CA Diabetic foot ulcers (DFU) heal with difficulty: standard of care (SOC) heals less than 30% at 20 weeks. Dermagraft, a bioengineered matrix containing live cells, has shown to increase healing over SOC, albeit with great cost and with modest improvement. We hypothesize there is no difference in healing using Dermagraft compared to the less expensive, noncellular matrix, Oasis. We conducted a prospective randomized, single-blind, controlled trial with three arms: SOC control, Dermagraft, or Oasis in patients with DFU. The primary outcome is percentage of subjects with complete wound closure by week 12 and a secondary outcome is the percentage with complete wound closure by week 28. During the 2-week screening phase, subjects with a 40% change in ulcer size were excluded. Patients were randomized into: SOC (n¼19), Dermagraft (n¼18) and Oasis (n¼18) for an active treatment phase (8 weeks) followed by maintenance phase (5 weeks SOC), study endpoint visit (week 12), and 4 monthly follow-up visits. There was equal distribution of demographical data among the groups and no difference in ulcer laterality or ulcer position among the groups. Although significant reduction in wound area from week 1 to 28 was noted in all three groups (p < 0.05), there was no significant difference in percent of subjects with complete wound closure by week 12 (55.5% Dermagraft, 72.2% Oasis, 57.9% SOC, p¼0.61) and week 28 (72.2% Dermagraft, 88.9% Oasis, 73.7% SOC, p¼0.50). This exploratory analysis suggests that the outcomes of treatment with either cellular Dermagraft or acellular Oasis matrix are comparable. Our study is still actively enrolling patients to meet the target, and we have yet to finalize the trial to make definitive conclusions.
912
Transient receptor potential (TRP) channels regulate cell migration of keratinocyte in vitro S Hiroyasu and JCR Jones Washington State University, Pullman, WA During re-epithelialization of skin wounds, basal keratinocytes proximal to the wound edge migrate over the wound bed in a directed manner. Our focus is to define the underlying mechanisms regulating such keratinocyte migration. Several reports indicate that cell migration is controlled by the modulation of Ca2+ concentration at the leading front (lamellipodium) of a moving cell. This modulation is determined by certain Ca2+ channels on the cell membrane including transient receptor potential (TRP) channels. To begin to analyze the function of Ca2+ in migrating keratinocytes, we first observed Ca2+ localization using Fluo-4 AM in live cells. During single cell migration, we detected high Ca2+ transients (flickers) in the lamellipodium which alternated from one lateral edge (flank) to the other. These flickers are temporally and spatially associated with attachment/detachment of the flanks as a single keratinocyte ‘walks’ over its substrate. Following a challenge with the Ca2+ chelator ethylene glycol-bis (b-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), both the Ca2+ concentration in the lamellipodium and cell motility are decreased. To screen the Ca2+ channels to identify which is responsible for the change in cell motility, we challenged the cells with the broad TRP channel inhibitors ruthenium red and 2-aminoethoxydiphenylborane (2-APB). Both inhibitors suppress single cell motility and collective cell migration. These results support the hypothesis that TRP channels support keratinocyte migration and are a potential target for improving wound healing in vivo.
Regulation of the dynamics of a6b4 integrin and vimentin in squamous cell carcinoma ZT Colburn and JCR Jones Washington State University, Pullman, WA The expression of the intermediate filament protein vimentin is a hallmark of squamous cell carcinoma. Moreover, vimentin expression is not only correlated with reduced survival but also with enhanced metastasis. Our goal was to test the possibility that vimentin promotes metastasis of squamous cell carcinoma by enhancing cancer cell motility. Thus, we investigated an association between vimentin filaments and motility-related, cell-matrix adhesion structures in tumor cells. Though vimentin filaments rarely associate with focal adhesions, they frequently interact with a6b4 integrin-rich hemidesmosome protein complexes organized into punctate structures at the cell-matrix interface. Interestingly, while most a6b4 integrin puncta are associated with keratin filaments, only a subset of puncta near the leading edge of front-rear polarized cells exhibit interaction with vimentin filaments. We evaluated a6b4 integrin-vimentin filament localization in motile cells by immunostaining immediately following time-lapse microscopy. These assays revealed that the lamellipodial localization of a6b4 integrin and the extension of vimentin filaments into lamellipodia are correlated with enhanced directed migration. To evaluate the function of these vimentin-associated matrix adhesions, we first generated b4 integrin knockdown cell lines. These cells exhibit reduced directed migration. Furthermore, the extension of vimentin filaments into their lamellipodia is inhibited. Structured-illumination (super-resolution) microscopy revealed that association of vimentin with a6b4 integrin reduces a6b4 integrin mobility and inhibits integrin turnover. Correspondingly, a6b4 integrin-vimentin association significantly restricts vimentin dynamics. Thus, our data indicate that sites of matrix interaction rich in a6b4 integrin and vimentin play important roles in the regulation of directed epithelial cell motility in cancer.
913
914
Midkine is upregulated in keloids A Tran1, I Dougherty1 and D Glass2 1 UT Southwestern Medical Center, Dallas, TX and 2 University of Texas Southwestern Medical Center, Dallas, TX Midkine is a 13 kDa heparin-binding growth factor involved in many important biological pathways including embryogenesis, tumorigenesis, and wound healing. It has been reported to stimulate collagen production (both type I and type III) and glycosaminoglycan synthesis in primary dermal fibroblasts. However, the role of midkine in the development of keloids (exuberant scarring of the skin) has not been extensively studied. To investigate whether midkine plays a role in keloid formation, we studied the expression of midkine in keloid and adjacent normal tissues from keloid patients. Results from both microarray analysis and from real-time PCR showed that midkine was strongly upregulated in keloid tissue. Immunohistochemistry further confirmed the increased expression of midkine in keloids, particularly in the dermis where dense hyalinized collagen bundles are concentrated. These findings suggest that midkine could potentially play a role in the pathogenesis of keloids, possibly by the modulation of extracellular matrix components.
Fibroblast heterogeneity in human skin: CD26 identifies a population of fibroblasts that upregulate collagen production in response to wounding CA Worthen1, TM Johnson1, JJ Voorhees1, J Orringer2 and GJ Fisher1 1 Department of Dermatology, University of Michigan, Ann Arbor, MI and 2 Dermatology, University of Michigan, Ann Arbor, MI Dermal fibroblasts are a heterogenous population of cells that are responsible for the deposition of extracellular matrix (ECM). After wounding, fibroblasts must quickly produce collagen and other ECM proteins to support overlying reepithelization and close the wound. Fibroblast sub-populations have been identified as collagen-producing cells that also express one or more markers such as CD34, CD90, and/or PDFG-a. Recently, a population of resident cells that express CD26 (dipeptidyl peptidase) have been reported to be responsible for the majority of ECM deposition during wound healing in mouse skin. We have investigated the properties of CD26-positive dermal cells in normal and wounded human skin. FACS analysis of dermal cells, which were freshly released from normal human skin samples by collagenase digestion, revealed a substantial population of CD26 positive cells. The majority of these cells (70%) were also CD90 positive, identifying them as resident fibroblasts. These CD26+/ CD90+ cells also expressed CD34. Gene expression analysis revealed similar levels of type I collagen expression in CD26+/CD90+ and CD26-/CD90+ dermal cells. In wounded human skin (3 weeks after CO2 laser-ablation), the number of CD26+/CD90+ cells increased 2.3fold (p¼0.026) and collagen expression in this population of cells increased 11.3-fold (p¼0.0007). In contrast, the number of CD26-/CD90+ cells did not increase, and collagen expression was significantly 3-fold lower than in the CD26+/CD90+ (p¼0.003) cells. Identification of CD26+/CD90+ dermal cells as a major source of new collagen deposition in would healing may facilitate development of novel strategies to improve wound healing in the elderly and treat non-healing wounds.
www.jidonline.org S157