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Photodynamic therapy in skin ulcer closure
P3801
P3803
Development of a good manufacturing process facility for cell therapy of chronic nonhealing wounds Tatyana Yufit, MD, Roger Williams Medical Center, Providence, RI, United States; Polly Carson, Roger Williams Medical Center, Providence, RI, United States; Taehee Kwak, MD, Roger Williams Medical Center, Providence, RI, United States; Vincent Falanga, MD, Roger Williams Medical Center, Providence, RI, United States With increasing emphasis on translational bedside research, the need for appropriate regulatory oversight and approval has become essential. The requirements of the US Food and Drug Administration (FDA) for investigational new drug (IND) studies that are investigator-initiated have become increasingly stringent. In this report, we outline the steps that are required for developing an IND submission to the FDA and discuss the preparation and manufacturing issues facing investigators new to this type of research. At this time, the FDA is treating academic investigators (research) the same way they deal with the pharmaceutical industry. A substantial obstacle is the development of good manufacturing practice (GMP) facilities. This requires the establishment of standard operating procedures (SOPs) for each piece of equipment, procedures, quality assurance, personnel training, and documentation. The GMP facility needs to be approved and is subject to inspection and auditing at any time. There is a strongly intertwined relationship between the GMP, the clinical research team, the institutional review board (IRB), and FDA regulations. For the purpose of illustrating these challenges and how to overcome them, we will take the example of culturing autologous bone marrowederived mesenchymal stem cells for topical application to nonhealing human wounds. In this work, we have shown that cells delivered at a concentration of [1.5 million per cm2 of wound surface lead to accelerated healing of previously nonhealing wounds. The cells are delivered in a modified fibrin spray construct. All components of treatment, including cells, media, and fibrin are subject to SOP and regulatory issues. The development of the GMP facility for this work and the subsequent IND submission to the FDA is a formidable task with which academic investigators are not yet familiar. We will discuss how this can be accomplished.
Effects of aloe vera extract on fibroblasts and keratinocytes Jie Li, MD, University of Miami Miller School of Medicine, Miami, FL, United States; Eric Teplicki, University of Miami Miller School of Medicine, Miami, FL, United States; Qianli Ma, University of Miami Miller School of Medicine, Miami, FL, United States; Ran Huo, University of Miami Miller School of Medicine, Miami, FL, United States
Commercial support: None identified.
Background: Aloe vera (AV) extract has been incorporated in a vast number of topical preparations for wound healing and skin care; however, its effects on fibroblasts and keratinocytes, two integral cells in wound healing, remain unclear. Objective: To determine the in vitro effects of an AV extract on fibroblast and keratinocyte proliferation and migration. Methods: To assess proliferation, normal human fibroblasts and keratinocytes were treated with normal medium, or medium containing a preservative (normally mixed in with AV; contains sodium benzoate, potassium sorbate, and phosphoric acid) or AV/preservative. Media were changed every 24 hours. Cell numbers and viability were quantified with a cell counter. To assess cell migration, cross-shaped wounds were made on confluent cultures of treated and nontreated cells and photographed. Percentages of wound gap area covered by migrating cells were determined. Results: In fibroblast proliferation, fibroblast cultures with 1:50 dilutions of preservative, compared to normal media, showed 46% and 74% decreases in the number of viable fibroblast cells at days 3 and 5, respectively. When treated with AV/preservative at the same conditions, 31% and 32% increases in fibroblasts were observed. No significant effects of preservative or AV/preservative on fibroblast migration were found. For keratinocyte proliferation, 1:100 dilutions of preservative lead to 92% and 96% decreases in viable cells at days 3 and 5, respectively, while AV/preservative lead to 28 and 13% decreases (P \.01). In keratinocyte migration, no significant difference in migration was found by 1:100 preservative at 24 hours, while the corresponding AV/preservative group showed a 45% increase in migration. Conclusions: Surprisingly, the preservative normally mixed with AV led to significant inhibition of fibroblast and keratinocyte proliferation. However, AV was able to reverse these effects, and the AV/preservative group actually significantly increased fibroblast proliferation and keratinocyte migration over normal medium control. These in vitro data suggest that AV may have a beneficial role in wound healing, and that its effects may be even more pronounced in the presence of a less inhibitory preservative. Commercial support: None identified.
P3802
P3804
Photodynamic therapy in skin ulcer closure Marcello Monti, MD, Universita` degli Studi di Milano, Istituto Clinico Humanitas, Italy, Rozzano, Milano, Italy; Stefania Motta, Universita` degli Studi di Milano, Istituto Clinico Humanitas, Rozzano, Milano, Italy
Assessment of the effects on wound healing and gene expression of a bioelectric dressing using a porcine wound model and real time reverse transcriptase-polymerase chain reaction Stephen Davis, MD, University of Miami Miller School of Medicine, Miami, FL, United States; Joel Gil, University of Miami Miller School of Medicine, Miami, FL, United States; Jose Valdes, University of Miami Miller School Medicine, Miami, FL, United States; Robert Perez, University of Miami Miller School of Medicine, Miami, FL, United States; Yan Rivas, University of Miami Miller School of Medicine, Miami, FL, United States
Wound healing is the process of restoring integrity to traumatized tissue and involves the biologic principles of tissue response to injury with all of its associated inflammatory, immune, biochemical, cell biology, and signal transduction mechanisms. Photodynamic therapy with 5-aminolevulinic acid (ALA-PDT) is widely used in dermatology to destroy malignant skin tumors and induce remission in some inflammatory disorders, and some recently published data indicate that skin venous ulcers can also be targeted. Our department has a specific unit dedicated to healing skin ulcers, in which we have been using multisession ALA-PDT for 3 years to treat ulcers of all pathogenetic causes. In order to establish the role of ALA-PDT in wound healing, we experimentally evaluated the efficiency of red light versus red light 1 ALA, the antibacterial activity of ALA-PDT, and the activity of ALA-PDT versus a standard dressing in treating 13 cases of chronic venous ulcers, four of autoimmune ulcers, three of hypertensive arterial ulcers, and two cases of ulcerated radiodermatitis. All of the selected patients had multiple and/or large ulcers (area [20 cm2). After obtaining the patients’ formal informed consent, multiple ulcers were divided into groups and treated with: (1) our standard dressing alone; (2) our standard dressing and weekly ALA-PDT; and (3) our standard dressing and weekly red light exposure. The ALA-PDT treatment consisted of the application of 10% ALA in polyethylenglycol (PEG) ointment in occlusion for 24 hours, followed by exposure to diode red light at 630 nm, irradiance 160 mW at 50 mm, for 8 minutes, delivering 75 J/cm2. Microbiologic samples were collected from all of the ulcers for the primary isolation of Gram-negative bacilli, Gram-positive cocci, and mycetes before the application of ALA or dressing, after 24 hours’ ALA or dressing occlusion, and after exposure to red light. On the basis of our 3-year experience, we can say that multisession ALA-PDT heals all of the treated ulcers, and in half the time required by the standard dressing in the same patient; red light exposure alone does not promote wound healing; and ALA-PDT does not have any antibacterial activity. These in vivo results demonstrate the favorable activity of ALA-PDT in wound healing, although the underlying biologic mechanisms are still unclear.
Wounds are a major cause of morbidity and impaired quality of life. Nonhealing wounds can lead to prolonged periods of distress, permanent debilitation, and death. Every year, 6.5 million Americans are afflicted with chronic wounds caused by pressure, venous stasis, or diabetes. Electrical stimulation has been shown to be beneficial to nonhealing wounds. Using a well established porcine model for wound healing, we evaluated the effects of a bioelectric (CMB) wound dressing on deep partial thickness wounds using an epidermal migration assay and real-time reverse transcriptase-polymerase chain reaction studies with porcine specific oligonucleotides. Six female specific pathogenefree animals were used in our experiments. Wounds (10 3 10 3 0.5 mm) were created using a specialized electrokeratome. Wounds were treated with sterile dressings (CMB vs nonactive) and assessed using an epidermal migration assay beginning on day 4 (postwounding). Biopsies were taken for molecular analysis. Treatment with the CMB dressing resulted in 65% of wounds being completely epithelialized on day 5 as compared to 20% of wounds in the control group (P \ .001). Gene expression analysis of interleukin-1a (IL-1a) indicated that treatment with active dressing results in delayed and reduced peak expression. Although an initial inflammatory response is normal in adult fibronic healing, scarless fetal wound healing is characterized by the absence of the inflammatory phase of healing. Comparative analysis of matrix metalloproteinase9 (MMP-9) indicated that gene expression was reduced in the active dressing group. Over expression of MMP-9 has been associated with chronic wounds. Others have shown MMP-9 levels in acute wounds are up-regulated in scarless wound healing. These results suggest that the CMB bioelectric dressing may have important clinical implications. Additional studies, including clinical, are warranted.
Commercial support: None identified.
Commercial support: Sponsored by Silverleaf.
AB200
J AM ACAD DERMATOL
MARCH 2009
P3803
Development of a good manufacturing process facility for cell therapy of chronic nonhealing wounds Tatyana Yufit, MD, Roger Williams Medical Center, Providence, RI, United States; Polly Carson, Roger Williams Medical Center, Providence, RI, United States; Taehee Kwak, MD, Roger Williams Medical Center, Providence, RI, United States; Vincent Falanga, MD, Roger Williams Medical Center, Providence, RI, United States With increasing emphasis on translational bedside research, the need for appropriate regulatory oversight and approval has become essential. The requirements of the US Food and Drug Administration (FDA) for investigational new drug (IND) studies that are investigator-initiated have become increasingly stringent. In this report, we outline the steps that are required for developing an IND submission to the FDA and discuss the preparation and manufacturing issues facing investigators new to this type of research. At this time, the FDA is treating academic investigators (research) the same way they deal with the pharmaceutical industry. A substantial obstacle is the development of good manufacturing practice (GMP) facilities. This requires the establishment of standard operating procedures (SOPs) for each piece of equipment, procedures, quality assurance, personnel training, and documentation. The GMP facility needs to be approved and is subject to inspection and auditing at any time. There is a strongly intertwined relationship between the GMP, the clinical research team, the institutional review board (IRB), and FDA regulations. For the purpose of illustrating these challenges and how to overcome them, we will take the example of culturing autologous bone marrowederived mesenchymal stem cells for topical application to nonhealing human wounds. In this work, we have shown that cells delivered at a concentration of [1.5 million per cm2 of wound surface lead to accelerated healing of previously nonhealing wounds. The cells are delivered in a modified fibrin spray construct. All components of treatment, including cells, media, and fibrin are subject to SOP and regulatory issues. The development of the GMP facility for this work and the subsequent IND submission to the FDA is a formidable task with which academic investigators are not yet familiar. We will discuss how this can be accomplished.
Effects of aloe vera extract on fibroblasts and keratinocytes Jie Li, MD, University of Miami Miller School of Medicine, Miami, FL, United States; Eric Teplicki, University of Miami Miller School of Medicine, Miami, FL, United States; Qianli Ma, University of Miami Miller School of Medicine, Miami, FL, United States; Ran Huo, University of Miami Miller School of Medicine, Miami, FL, United States
Commercial support: None identified.
Background: Aloe vera (AV) extract has been incorporated in a vast number of topical preparations for wound healing and skin care; however, its effects on fibroblasts and keratinocytes, two integral cells in wound healing, remain unclear. Objective: To determine the in vitro effects of an AV extract on fibroblast and keratinocyte proliferation and migration. Methods: To assess proliferation, normal human fibroblasts and keratinocytes were treated with normal medium, or medium containing a preservative (normally mixed in with AV; contains sodium benzoate, potassium sorbate, and phosphoric acid) or AV/preservative. Media were changed every 24 hours. Cell numbers and viability were quantified with a cell counter. To assess cell migration, cross-shaped wounds were made on confluent cultures of treated and nontreated cells and photographed. Percentages of wound gap area covered by migrating cells were determined. Results: In fibroblast proliferation, fibroblast cultures with 1:50 dilutions of preservative, compared to normal media, showed 46% and 74% decreases in the number of viable fibroblast cells at days 3 and 5, respectively. When treated with AV/preservative at the same conditions, 31% and 32% increases in fibroblasts were observed. No significant effects of preservative or AV/preservative on fibroblast migration were found. For keratinocyte proliferation, 1:100 dilutions of preservative lead to 92% and 96% decreases in viable cells at days 3 and 5, respectively, while AV/preservative lead to 28 and 13% decreases (P \.01). In keratinocyte migration, no significant difference in migration was found by 1:100 preservative at 24 hours, while the corresponding AV/preservative group showed a 45% increase in migration. Conclusions: Surprisingly, the preservative normally mixed with AV led to significant inhibition of fibroblast and keratinocyte proliferation. However, AV was able to reverse these effects, and the AV/preservative group actually significantly increased fibroblast proliferation and keratinocyte migration over normal medium control. These in vitro data suggest that AV may have a beneficial role in wound healing, and that its effects may be even more pronounced in the presence of a less inhibitory preservative. Commercial support: None identified.
P3802
P3804
Photodynamic therapy in skin ulcer closure Marcello Monti, MD, Universita` degli Studi di Milano, Istituto Clinico Humanitas, Italy, Rozzano, Milano, Italy; Stefania Motta, Universita` degli Studi di Milano, Istituto Clinico Humanitas, Rozzano, Milano, Italy
Assessment of the effects on wound healing and gene expression of a bioelectric dressing using a porcine wound model and real time reverse transcriptase-polymerase chain reaction Stephen Davis, MD, University of Miami Miller School of Medicine, Miami, FL, United States; Joel Gil, University of Miami Miller School of Medicine, Miami, FL, United States; Jose Valdes, University of Miami Miller School Medicine, Miami, FL, United States; Robert Perez, University of Miami Miller School of Medicine, Miami, FL, United States; Yan Rivas, University of Miami Miller School of Medicine, Miami, FL, United States
Wound healing is the process of restoring integrity to traumatized tissue and involves the biologic principles of tissue response to injury with all of its associated inflammatory, immune, biochemical, cell biology, and signal transduction mechanisms. Photodynamic therapy with 5-aminolevulinic acid (ALA-PDT) is widely used in dermatology to destroy malignant skin tumors and induce remission in some inflammatory disorders, and some recently published data indicate that skin venous ulcers can also be targeted. Our department has a specific unit dedicated to healing skin ulcers, in which we have been using multisession ALA-PDT for 3 years to treat ulcers of all pathogenetic causes. In order to establish the role of ALA-PDT in wound healing, we experimentally evaluated the efficiency of red light versus red light 1 ALA, the antibacterial activity of ALA-PDT, and the activity of ALA-PDT versus a standard dressing in treating 13 cases of chronic venous ulcers, four of autoimmune ulcers, three of hypertensive arterial ulcers, and two cases of ulcerated radiodermatitis. All of the selected patients had multiple and/or large ulcers (area [20 cm2). After obtaining the patients’ formal informed consent, multiple ulcers were divided into groups and treated with: (1) our standard dressing alone; (2) our standard dressing and weekly ALA-PDT; and (3) our standard dressing and weekly red light exposure. The ALA-PDT treatment consisted of the application of 10% ALA in polyethylenglycol (PEG) ointment in occlusion for 24 hours, followed by exposure to diode red light at 630 nm, irradiance 160 mW at 50 mm, for 8 minutes, delivering 75 J/cm2. Microbiologic samples were collected from all of the ulcers for the primary isolation of Gram-negative bacilli, Gram-positive cocci, and mycetes before the application of ALA or dressing, after 24 hours’ ALA or dressing occlusion, and after exposure to red light. On the basis of our 3-year experience, we can say that multisession ALA-PDT heals all of the treated ulcers, and in half the time required by the standard dressing in the same patient; red light exposure alone does not promote wound healing; and ALA-PDT does not have any antibacterial activity. These in vivo results demonstrate the favorable activity of ALA-PDT in wound healing, although the underlying biologic mechanisms are still unclear.
Wounds are a major cause of morbidity and impaired quality of life. Nonhealing wounds can lead to prolonged periods of distress, permanent debilitation, and death. Every year, 6.5 million Americans are afflicted with chronic wounds caused by pressure, venous stasis, or diabetes. Electrical stimulation has been shown to be beneficial to nonhealing wounds. Using a well established porcine model for wound healing, we evaluated the effects of a bioelectric (CMB) wound dressing on deep partial thickness wounds using an epidermal migration assay and real-time reverse transcriptase-polymerase chain reaction studies with porcine specific oligonucleotides. Six female specific pathogenefree animals were used in our experiments. Wounds (10 3 10 3 0.5 mm) were created using a specialized electrokeratome. Wounds were treated with sterile dressings (CMB vs nonactive) and assessed using an epidermal migration assay beginning on day 4 (postwounding). Biopsies were taken for molecular analysis. Treatment with the CMB dressing resulted in 65% of wounds being completely epithelialized on day 5 as compared to 20% of wounds in the control group (P \ .001). Gene expression analysis of interleukin-1a (IL-1a) indicated that treatment with active dressing results in delayed and reduced peak expression. Although an initial inflammatory response is normal in adult fibronic healing, scarless fetal wound healing is characterized by the absence of the inflammatory phase of healing. Comparative analysis of matrix metalloproteinase9 (MMP-9) indicated that gene expression was reduced in the active dressing group. Over expression of MMP-9 has been associated with chronic wounds. Others have shown MMP-9 levels in acute wounds are up-regulated in scarless wound healing. These results suggest that the CMB bioelectric dressing may have important clinical implications. Additional studies, including clinical, are warranted.
Commercial support: None identified.
Commercial support: Sponsored by Silverleaf.
AB200
J AM ACAD DERMATOL
MARCH 2009