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Electroporation improves transfection efficiency in rat wound healing model
S58
Plastic Surgery I
J Am Coll Surg
regulation of transgene expression in a dose-dependent manner by a HSV-1 recombinant system. METHODS: QR9TOhEGF is a novel replication-defective virus that encodes hEGF under control of the tetO-containing CMV promoter (T-REx, Invitrogen) and the tetracycline repressor protein tetR. Without tetracycline, tetR binds to tetO, leading to effective repression of hEGF expression, while in the presence of tetracycline this repression is released. Vero cells were infected with QR9TOhEGF in the presence and absence of tetracycline. Porcine full-thickness wounds sealed with a polyvinyl chamber were infected with QR9TOhEGF in the presence and absence of tetracycline. Wound fluid was assayed at 24 and 48h using hEGF-ELISA. RESULTS: hEGF concentration (pg/ml) at 24 and 48hrs following infection shows up to at least 400-fold regulation. Sensitivity limit: 3pg/ml Vero cells
24 h 48 h
Tⴚ
Tⴙ
6 3.5
1360 1400
226 400
0.0096 0.0003
2.06 ⴛ 107 PFU/wound
In vivo
24 h 48 h
Fold regulation
p Value (unpaired t-test)
8 6
41 36
12 5.8
0.0281 0.0241
EGF-ELISA of conditioned medium (Vero cells) and wound fluid (In vivo) at 24 and 48 hours, in the presence and absence of tetracycline. Lower limit of sensitivity: 3pg/ml.
CONCLUSIONS: We have constructed an HSV-1 vector that allows the highest degree of regulation of gene expression in a single vector system. The feasibility of regulatable gene transfer to wounds has been demonstrated. Our method of in vivo delivery is currently being optimized to achieve higher expression.
Autologous cultured keratinocytes suspensions accelerate re-epithelialization in the diabetic pig Patrik E Velander MD, AFRCSI, Christoph Theopold MD, Raphael Gheerardyn MD, Oliver Bleiziffer MD, Feng Yao PhD, Elof Eriksson MD, FACS Harvard Medical School, Brigham and Women’s Hospital Boston, MA INTRODUCTION: Diabetes is one of the leading causes of chronic wounds in the U.S.A. Its incidence is increasing dramatically due to obesity and old age. Healing of these impaired wounds often represent a difficult challenge with the currently available treament. We recently established a delayed wound-healing model in the Yorkshire pig rendered diabetic by administration of Streptozotocin. This study evaluated epidermal regeneration in fluid treated skin wounds treated with suspensions of cultured autologous keratinocytes.
METHODS: Diabetes was induced by injecting Streptozotocin into a three months old female Yorkshire pig. Full thickness wounds were created on the dorsum and dressed with polyvinyl chambers to keep the wounds wet and to allow for wound fluid monitoring. Suspensions of keratinocytes were seeded into half of the wounds and Normal Saline into the remaining wounds. Serum glucose and wound fluid glucose concentrations were monitored daily. Wound contraction was monitored and biopsies taken on day 12. RESULTS: The serum glucose was significantly increased for the duration of the experiment (⬎350 mg/dl). Wound fluid glucose closely followed serum glucose concentration. Transplantation of keratinocytes suspensions significantly accelerated reepithelialization in the diabetic pig. There was no statistical difference between the contraction rates of wounds treated with a keratinocyte suspension or with normal saline.
Day 12
Normal Saline
Keratinocytes suspension
Re-epithelialization Wound contraction
47.8% 33.2%
82.6% 32.9%
CONCLUSIONS: This study shows that wound healing is delayed in the streptozotocin-induced diabetic pig model. A single-cell suspension of keratinocytes not only survives in the hyperglycemic wound environment but also contributes to the formation of a neo-epithelium, accelerating the rate of healing over saline-treated controls.
Electroporation improves transfection efficiency in rat wound healing model Michael P Lin MS3, Guy P Marti MD AIHT, Rami Dieb MD, Jiaai Wang BS, Rabia Qaiser MD, Pramod Bonde MD MS FRCS, Mark D Duncan MD FACS, John W Harmon MD FACS Johns Hopkins University School of Medicine Baltimore, MD INTRODUCTION: We are investigating the use of electroporation in vivo to enhance transfection efficiency and improve wound healing with DNA plasmid expression vectors for growth factors. METHODS: For electroporation (EP) transfection efficiency and wound healing we used luciferase and keratinocyte growth factor (KGF) DNA plasmid expression vectors respectively (gWIZ-Lux vector controlled by CMV promoter, Invitrogen, Carlsbad, CA). Animals were electroporated at the site of injection within two minutes of plasmid administration, using a square wave electroporator (ECM 830, BTX Genetronics, San Diego, CA). For luciferase, photon emission was quantified using the IVIS Imaging System (Xenogen Corporation, Alameda, CA). For the KGF experiment, healing 4 cutaneous 8mm punch biopsies was assessed using image analysis software based on NIH image (Scion Image, Frederick, MD). A Sprague Dawley rat sepsis model of wound healing with partial cecal ligation was utilized. We found in previous experiments that wound healing was delayed in septic animals. RESULTS: Single EP enhanced average luciferase expression threefold compared to plasmid without EP (p⬍0.05) and double EP (two
Vol. 199, No. 3S, September 2004
Plastic Surgery I
plasmid transfections with EP) had similar results; Table 1. Single EP transfection of a KGF plasmid vector improved wound healing as evidenced by an average of 60.0% smaller wound areas on Day 12 in the KGF⫹EP vs KGF animals (p⬍0.009). Luciferase expression
Day 8
Day 17
Day 24
Day 30
Control
4.2 ⫾ 0.5
5.8 ⫾ 0.7
3.4 ⫾ 0.3
3.6 ⫾ 0.2
4.3 ⫾ 0.3
Plasmid
18.8 ⫾ 8.5
7.0 ⫾ 0.8
13.1 ⫾ 8.0
4.8 ⫾ 0.5
11.1 ⫾ 3.0
Plasmid ⫹ EP
47.1 ⫾ 13.9
28.2 ⫾ 9.8
51.5 ⫾ 21.0
16.3 ⫾ 4.3
35.7 ⫾ 6.9
2 (plasmid ⫹ EP)
38.1 ⫾ 12.7
20.2 ⫾ 7.0
75.5 ⫾ 32.6
24.7 ⫾ 12.1
39.6 ⫾ 9.6
Kruskal-Wallis One-Way ANOVA on ranks
Differences in values among treatment groups are greater than would be expected by chance; there is a statistically significant difference (p ⬍ 0.001).
Pairwise multiple comparison (Dunn’s method)
Control vs plasmid ⫹ EP (p ⬍ 0.05); control vs 2 (plasmid ⫹ EP) (p ⬍ 0.05); plasmid vs plasmid ⫹ EP (p ⬍ 0.05); plasmid vs 2 (plasmid ⫹ EP) (p ⬍ 0.05).
Average
Wound day 12
Average wound size
p-value
KGF⫹EP vs KGF
460 ⫾ 78 vs. 1149 ⫾ 260
0.009 (student’s t-test)
EP ⫽ electroporation, KGF ⫽ keratinocyte growth factor. Luciferase expression values represent average ⫻ 105 ⫾ SEM (photons/sec). Values were recorded as total photon flux from each wound, with 4 wounds per rat. Wound size values represent average ⫾ SEM (pixels).
CONCLUSIONS: These results encourage us to explore further the possible benefit of electroporation-facilitated transfection of growth factors to improve wound healing.
KGF-1 plasmid delivered with electroporation accelerates wound closure in diabetic mice Guy P Marti MD, AIHT, Michael P Lin MS3, Rabia Qaiser MD, Rami Dieb MD, Jiaai Wang BS, Shah Parth MD, Pramod Bonde MD MS FRCS, Mark D Duncan MD FACS, John W Harmon MD FACS Johns Hopkins University School of Medicine Baltimore, MD INTRODUCTION: Correcting the wound healing deficit of diabetes is an unmet medical challenge. Diminished levels of important growth factors are found in wounds of diabetic individuals. METHODS: We optimized in vivo electroporation (EP) parameters to six 100 microsecond pulses of 1750 volts/cm (ECM830, BTX Genetronics, San Diego CA). EP increased expression of Luciferase Plasmid DNA by 10 fold. We utilized electroporation to increase transfection efficiency of a DNA expression vector for Keratinocyte Growth Factor (KGF-1, Invitrogen Carlsbad CA) using the gWIZ vector controlled by the CMV promoter. We assessed closure of 5mm full thickness wounds on the backs of 60 BKS.Cg-m Leprdb/db diabetic mice by calculating wound areas (pixels) using image analysis software based on NIH image (Scion Image, Frederick, MD). RESULTS: The wounds treated with 40ug KGF-1 and electroporated (KGF-1/EP) closed faster than the untreated wounds (open area 220⫹-74 versus 576⫹-90, p⬍0.05 at day 12). In a second experiment wounds with KGF-1/EP healed faster than with KGF-1 alone (open area 637⫹-154 versus 971⫹-215, p⬍0.05 at day 12). Histological assessment was done with a grading scale for quality of epithelialization (Grade 1 : incomplete epithelium with micro ulcers, Grade 2 : thin epithelium,
S59
unresolved inflammation, Grade 3 : intact, not reticulated epithelium, Grade 4 : reticulated mature epithelium). The KGF-1/EP group healed better than control (3.3⫹-0.4 versus 1.5⫹-0.1 p⬍0.05). CONCLUSIONS: We conclude that electroporation assisted delivery of KGF-1 expression vector can improve closure of wounds in a diabetic mouse model.
Biolistic augmentation of impaired wound healing In steroid treated rats Yongbo Liu MD, Scott Dulchavsky MD, FACS, Scott Henry MD, PhD, Deborah Dulchavsky PA-C, Subhash Gautam PhD Henry Ford Health System Detroit, MI INTRODUCTION: Systemic corticosteroid treatment has effects on wound healing. Biolistic delivery of epidermal growth factor (EGF) and eukaryotic initiation factor 4E (eIF4E) mRNA’s to acute midline abdominal wounds in normal and steroid treated rats has been shown to augment wound healing. This study investigates the efficacy of the combined delivery of these agents. METHODS: Male Sprague-Dawley rats (300-350 grams)were used, intraperitoneal injections of dexamethasone sodium phosphate (16mg / kg / day) beginning two days prior to acute wounding and continuing daily until sacrifice. Midline abdominal wounds were created through the abdominal skin, fascia and muscle, then fascia was sutured. The Gene Gun was used to biolistically deliver mRNA treatment of EGF and eIF4E along the full length of the wounds. At seven and fourteen days animals were euthanized and wound strip of abdominal wall harvested, sectioned into 1 x 4cm strips for wound bursting strength (WBS) analysis via a tensiometer. RESULTS: Steroid treatment produced a significant decrease in WBS when compared to control animals at 7 or 14 days. Treatment of steroid exposed rats with biolistically delivered EGF or eIF4E alone produced a modest increase in WBS which was not statistically significant. Combination therapy of EGF and eIF4E significantly increased WBS at 14 days. CONCLUSIONS: At fourteen days, treatment with EGF ⫹ 4E produced synergic effect in WBS. Western blot studies of wounds treated with EGF⫹4E demonstrated dramatically increased type I collagen expression compared to control.
KSR modulates survival in the fibroblast-populated collagen matrix Mark A Carlson MD, FACS, Michael T Longaker MD, MBA, FACS University of Nebraska Medical Center Omaha, NE INTRODUCTION: Kinase suppressor of Ras (KSR) is a scaffold which coordinates growth factor-stimulated Erk signaling, which upregulates survival, proliferation, and other outputs. We hypothesized that KSR also would play a role in mechano-regulated survival in the fibroblast-populated collagen matrix (FPCM).
Plastic Surgery I
J Am Coll Surg
regulation of transgene expression in a dose-dependent manner by a HSV-1 recombinant system. METHODS: QR9TOhEGF is a novel replication-defective virus that encodes hEGF under control of the tetO-containing CMV promoter (T-REx, Invitrogen) and the tetracycline repressor protein tetR. Without tetracycline, tetR binds to tetO, leading to effective repression of hEGF expression, while in the presence of tetracycline this repression is released. Vero cells were infected with QR9TOhEGF in the presence and absence of tetracycline. Porcine full-thickness wounds sealed with a polyvinyl chamber were infected with QR9TOhEGF in the presence and absence of tetracycline. Wound fluid was assayed at 24 and 48h using hEGF-ELISA. RESULTS: hEGF concentration (pg/ml) at 24 and 48hrs following infection shows up to at least 400-fold regulation. Sensitivity limit: 3pg/ml Vero cells
24 h 48 h
Tⴚ
Tⴙ
6 3.5
1360 1400
226 400
0.0096 0.0003
2.06 ⴛ 107 PFU/wound
In vivo
24 h 48 h
Fold regulation
p Value (unpaired t-test)
8 6
41 36
12 5.8
0.0281 0.0241
EGF-ELISA of conditioned medium (Vero cells) and wound fluid (In vivo) at 24 and 48 hours, in the presence and absence of tetracycline. Lower limit of sensitivity: 3pg/ml.
CONCLUSIONS: We have constructed an HSV-1 vector that allows the highest degree of regulation of gene expression in a single vector system. The feasibility of regulatable gene transfer to wounds has been demonstrated. Our method of in vivo delivery is currently being optimized to achieve higher expression.
Autologous cultured keratinocytes suspensions accelerate re-epithelialization in the diabetic pig Patrik E Velander MD, AFRCSI, Christoph Theopold MD, Raphael Gheerardyn MD, Oliver Bleiziffer MD, Feng Yao PhD, Elof Eriksson MD, FACS Harvard Medical School, Brigham and Women’s Hospital Boston, MA INTRODUCTION: Diabetes is one of the leading causes of chronic wounds in the U.S.A. Its incidence is increasing dramatically due to obesity and old age. Healing of these impaired wounds often represent a difficult challenge with the currently available treament. We recently established a delayed wound-healing model in the Yorkshire pig rendered diabetic by administration of Streptozotocin. This study evaluated epidermal regeneration in fluid treated skin wounds treated with suspensions of cultured autologous keratinocytes.
METHODS: Diabetes was induced by injecting Streptozotocin into a three months old female Yorkshire pig. Full thickness wounds were created on the dorsum and dressed with polyvinyl chambers to keep the wounds wet and to allow for wound fluid monitoring. Suspensions of keratinocytes were seeded into half of the wounds and Normal Saline into the remaining wounds. Serum glucose and wound fluid glucose concentrations were monitored daily. Wound contraction was monitored and biopsies taken on day 12. RESULTS: The serum glucose was significantly increased for the duration of the experiment (⬎350 mg/dl). Wound fluid glucose closely followed serum glucose concentration. Transplantation of keratinocytes suspensions significantly accelerated reepithelialization in the diabetic pig. There was no statistical difference between the contraction rates of wounds treated with a keratinocyte suspension or with normal saline.
Day 12
Normal Saline
Keratinocytes suspension
Re-epithelialization Wound contraction
47.8% 33.2%
82.6% 32.9%
CONCLUSIONS: This study shows that wound healing is delayed in the streptozotocin-induced diabetic pig model. A single-cell suspension of keratinocytes not only survives in the hyperglycemic wound environment but also contributes to the formation of a neo-epithelium, accelerating the rate of healing over saline-treated controls.
Electroporation improves transfection efficiency in rat wound healing model Michael P Lin MS3, Guy P Marti MD AIHT, Rami Dieb MD, Jiaai Wang BS, Rabia Qaiser MD, Pramod Bonde MD MS FRCS, Mark D Duncan MD FACS, John W Harmon MD FACS Johns Hopkins University School of Medicine Baltimore, MD INTRODUCTION: We are investigating the use of electroporation in vivo to enhance transfection efficiency and improve wound healing with DNA plasmid expression vectors for growth factors. METHODS: For electroporation (EP) transfection efficiency and wound healing we used luciferase and keratinocyte growth factor (KGF) DNA plasmid expression vectors respectively (gWIZ-Lux vector controlled by CMV promoter, Invitrogen, Carlsbad, CA). Animals were electroporated at the site of injection within two minutes of plasmid administration, using a square wave electroporator (ECM 830, BTX Genetronics, San Diego, CA). For luciferase, photon emission was quantified using the IVIS Imaging System (Xenogen Corporation, Alameda, CA). For the KGF experiment, healing 4 cutaneous 8mm punch biopsies was assessed using image analysis software based on NIH image (Scion Image, Frederick, MD). A Sprague Dawley rat sepsis model of wound healing with partial cecal ligation was utilized. We found in previous experiments that wound healing was delayed in septic animals. RESULTS: Single EP enhanced average luciferase expression threefold compared to plasmid without EP (p⬍0.05) and double EP (two
Vol. 199, No. 3S, September 2004
Plastic Surgery I
plasmid transfections with EP) had similar results; Table 1. Single EP transfection of a KGF plasmid vector improved wound healing as evidenced by an average of 60.0% smaller wound areas on Day 12 in the KGF⫹EP vs KGF animals (p⬍0.009). Luciferase expression
Day 8
Day 17
Day 24
Day 30
Control
4.2 ⫾ 0.5
5.8 ⫾ 0.7
3.4 ⫾ 0.3
3.6 ⫾ 0.2
4.3 ⫾ 0.3
Plasmid
18.8 ⫾ 8.5
7.0 ⫾ 0.8
13.1 ⫾ 8.0
4.8 ⫾ 0.5
11.1 ⫾ 3.0
Plasmid ⫹ EP
47.1 ⫾ 13.9
28.2 ⫾ 9.8
51.5 ⫾ 21.0
16.3 ⫾ 4.3
35.7 ⫾ 6.9
2 (plasmid ⫹ EP)
38.1 ⫾ 12.7
20.2 ⫾ 7.0
75.5 ⫾ 32.6
24.7 ⫾ 12.1
39.6 ⫾ 9.6
Kruskal-Wallis One-Way ANOVA on ranks
Differences in values among treatment groups are greater than would be expected by chance; there is a statistically significant difference (p ⬍ 0.001).
Pairwise multiple comparison (Dunn’s method)
Control vs plasmid ⫹ EP (p ⬍ 0.05); control vs 2 (plasmid ⫹ EP) (p ⬍ 0.05); plasmid vs plasmid ⫹ EP (p ⬍ 0.05); plasmid vs 2 (plasmid ⫹ EP) (p ⬍ 0.05).
Average
Wound day 12
Average wound size
p-value
KGF⫹EP vs KGF
460 ⫾ 78 vs. 1149 ⫾ 260
0.009 (student’s t-test)
EP ⫽ electroporation, KGF ⫽ keratinocyte growth factor. Luciferase expression values represent average ⫻ 105 ⫾ SEM (photons/sec). Values were recorded as total photon flux from each wound, with 4 wounds per rat. Wound size values represent average ⫾ SEM (pixels).
CONCLUSIONS: These results encourage us to explore further the possible benefit of electroporation-facilitated transfection of growth factors to improve wound healing.
KGF-1 plasmid delivered with electroporation accelerates wound closure in diabetic mice Guy P Marti MD, AIHT, Michael P Lin MS3, Rabia Qaiser MD, Rami Dieb MD, Jiaai Wang BS, Shah Parth MD, Pramod Bonde MD MS FRCS, Mark D Duncan MD FACS, John W Harmon MD FACS Johns Hopkins University School of Medicine Baltimore, MD INTRODUCTION: Correcting the wound healing deficit of diabetes is an unmet medical challenge. Diminished levels of important growth factors are found in wounds of diabetic individuals. METHODS: We optimized in vivo electroporation (EP) parameters to six 100 microsecond pulses of 1750 volts/cm (ECM830, BTX Genetronics, San Diego CA). EP increased expression of Luciferase Plasmid DNA by 10 fold. We utilized electroporation to increase transfection efficiency of a DNA expression vector for Keratinocyte Growth Factor (KGF-1, Invitrogen Carlsbad CA) using the gWIZ vector controlled by the CMV promoter. We assessed closure of 5mm full thickness wounds on the backs of 60 BKS.Cg-m Leprdb/db diabetic mice by calculating wound areas (pixels) using image analysis software based on NIH image (Scion Image, Frederick, MD). RESULTS: The wounds treated with 40ug KGF-1 and electroporated (KGF-1/EP) closed faster than the untreated wounds (open area 220⫹-74 versus 576⫹-90, p⬍0.05 at day 12). In a second experiment wounds with KGF-1/EP healed faster than with KGF-1 alone (open area 637⫹-154 versus 971⫹-215, p⬍0.05 at day 12). Histological assessment was done with a grading scale for quality of epithelialization (Grade 1 : incomplete epithelium with micro ulcers, Grade 2 : thin epithelium,
S59
unresolved inflammation, Grade 3 : intact, not reticulated epithelium, Grade 4 : reticulated mature epithelium). The KGF-1/EP group healed better than control (3.3⫹-0.4 versus 1.5⫹-0.1 p⬍0.05). CONCLUSIONS: We conclude that electroporation assisted delivery of KGF-1 expression vector can improve closure of wounds in a diabetic mouse model.
Biolistic augmentation of impaired wound healing In steroid treated rats Yongbo Liu MD, Scott Dulchavsky MD, FACS, Scott Henry MD, PhD, Deborah Dulchavsky PA-C, Subhash Gautam PhD Henry Ford Health System Detroit, MI INTRODUCTION: Systemic corticosteroid treatment has effects on wound healing. Biolistic delivery of epidermal growth factor (EGF) and eukaryotic initiation factor 4E (eIF4E) mRNA’s to acute midline abdominal wounds in normal and steroid treated rats has been shown to augment wound healing. This study investigates the efficacy of the combined delivery of these agents. METHODS: Male Sprague-Dawley rats (300-350 grams)were used, intraperitoneal injections of dexamethasone sodium phosphate (16mg / kg / day) beginning two days prior to acute wounding and continuing daily until sacrifice. Midline abdominal wounds were created through the abdominal skin, fascia and muscle, then fascia was sutured. The Gene Gun was used to biolistically deliver mRNA treatment of EGF and eIF4E along the full length of the wounds. At seven and fourteen days animals were euthanized and wound strip of abdominal wall harvested, sectioned into 1 x 4cm strips for wound bursting strength (WBS) analysis via a tensiometer. RESULTS: Steroid treatment produced a significant decrease in WBS when compared to control animals at 7 or 14 days. Treatment of steroid exposed rats with biolistically delivered EGF or eIF4E alone produced a modest increase in WBS which was not statistically significant. Combination therapy of EGF and eIF4E significantly increased WBS at 14 days. CONCLUSIONS: At fourteen days, treatment with EGF ⫹ 4E produced synergic effect in WBS. Western blot studies of wounds treated with EGF⫹4E demonstrated dramatically increased type I collagen expression compared to control.
KSR modulates survival in the fibroblast-populated collagen matrix Mark A Carlson MD, FACS, Michael T Longaker MD, MBA, FACS University of Nebraska Medical Center Omaha, NE INTRODUCTION: Kinase suppressor of Ras (KSR) is a scaffold which coordinates growth factor-stimulated Erk signaling, which upregulates survival, proliferation, and other outputs. We hypothesized that KSR also would play a role in mechano-regulated survival in the fibroblast-populated collagen matrix (FPCM).