the 2007 Abstract Presentations - Wound Healing Society

Abstracts
86
EXPRESSION OF THE A-SMA GENE AND CONTRACTILE
ABILITY OF SKIN FIBROBLASTS FROM DIABETIC MICE:
CORRELATION WITH WOUND CLOSURE RATES IN VIVO
AND IMPLICATIONS IN WOUND HEALING
Xiao Tian Wang, MD, Jin Bo Tang, Paul Y. Liu
Department of Surgery, Roger Williams Medical Center, Providence, RI
Purpose: Healing potential of the wounds in diabetic patients is very limited and
delay or non-healing of diabetic wounds are serious problems that lack efficient
treatments clinically. In this study we investigated how aSMA gene expression is
changed in diabetic skin fibroblasts and contractile ability of collagen gel matrix
decreased by the fibroblasts and correlations with in vivo wound closure rate,
and propose novel gene therapy approaches to reverse these detrimental efforts.
Methods: We used 10 db1/db- diabetic mice (BKS. Cg-m1/1Lepr db )and10
littermates. Two skin excision wounds, 0.8 0.8 cm each, were created on the
back of each mouse, and sizes of the wounds were recorded over a post-surgical
4-week period. Excised skin was cultured as explants to obtain skin fibroblasts.
The fibroblasts were seeded into three-dimension collagen gels and changes in
the gel dimensions were recorded for a 3-week period. Simultaneously RNA of
cultured skin fibroblasts was extracted to assess the levels of expression of the a-
SMA gene. a-SMA gene expression in the cells from their littermates (nondiabetic
mice) served as controls. Thereafter we transferred to diabetic skin
fibroblasts exogenous VEGF or PDGF genes and determined the changes of
the a-SMA gene expression after gene therapy.
Results: The closure rate of the skin excision wounds was significantly greater
in the db1/db1 mice (20.4 2.1 days) than in their littermates (15.5 1.9 days)
(p o 0.001). The fibroblasts from the db1/db1 mice showed very poor
contractile behavior in the three-dimensional collagen gels; in contract, the gel
seeded with the non-diabetic fibroblasts shrinked very remarkably. The
differences in dimensions of gels seeded with different cells are statistically
significant at 1, 2, and 3 weeks (p o 0.001). Levels of a-SMA gene expression
were significantly lower in the db1/db1 diabetic skin fibroblasts than in the
cells of their littermates (p o 0.05) and a-SMA gene expression was significantly
elevated by transfer of the VEGF and PDGF genes.
Conclusions: Down-regulation of a-SMA gene expression may be responsible
for the lower contractile ability of diabetic skin fibroblasts and delay in closure of
the skin wounds. This study demonstrated that transfer of growth factor genes
through appropriate gene therapy approaches increases a-SMA gene expression
and can be a potential method to enhance the healing rate of diabetic wound.
88
EFFICACY AND MECHANISMS OF NEGATIVE PRESSURE
(VAC) THERAPY IN PROMOTING WOUND HEALING: A
RODENT MODEL
S.M. Jacobs 1 , D. Matissen 1 ,F.Liu 1 , G. Niedt 2 , MD, J. K. Wu 1
1 Division of Plastic & Reconstructive Surgery,
2 Department of Dermatology, Columbia University College of Physicians &
Surgeons, New York, NY
Introduction: The Vacuum-Assisted Closure device (VAC) has revolutionized
wound care, although the exact mechanism is not well understood. We
hypothesize that mechanical stress imposed by the VAC device on wounds
induces production of pro-angiogenic factors, stimulates formation of granulation
tissue, and promotes secondary healing.
Methods: Full-thickness 2 2 cm excisional wounds were created on the dorsa
of rats and divided into three groups: 1) Control (Tegaderm dressing); 2) Special
Control (VAC foam and Tegaderm dressing); and 3) VAC (VAC dressing with
125 mm Hg continuous negative pressure). Wound tissues were harvested and
areas were measured on POD 0, 3, 5, 7. Wound closure rates (WCR) were
calculated. Tissues were stained for Factor VIII and Masson’s Trichrome for
vessel and collagen content, respectively. Protein was extracted from the
remaining tissue for western blot analysis of CD31, vascular endothelial growth
factor (VEGF), and basic fibroblast growth factor (bFGF) content.
Results: VAC-treated wounds had statistically significant WCR over all time
points compared to the control and special control wounds: POD 3: 31% versus
9% and 9%, respectively, (p o 0.0001); POD 5: 45% versus 24% and 23%,
respectively, (p = 0.0003); POD7: 54.4% versus 43.0%and 31.5%, respectively,
(p o 0.0001). VAC wounds had greater vascularity and collagen deposition
compared to the controls at all time points. Expression of CD31, VEGF, and
FGF-2 were higher in VAC wounds by POD 5; however, there was no
difference in POD 3 & 7.
Conclusion: A novel rodent animal model was used successfully to study VAC
wound healing. There is increased wound contraction and collagen deposition
by POD 3, as well as increased production of VEGF and bFGF by POD 5.
Further application of this rat model and analysis of wound tissue will further
elucidate the mechanism of the VAC device in promoting improved wound
healing.
87
IN VITRO EFFECTS OF CALRETICULIN CORROBORATES
ITS ROLE IN HEALING OF DIABETIC WOUNDS
M.R. Greives, C.L. Cadacio, K.M. Blechman, M. Rahman, J.P. Levine
L.I. Gold New York University School of Medicine, New York, NY USA
Defective wound healing with consequential morbidities has become an
increasingly serious clinical problem in urgent need of novel therapies. We have
discovered that the ER chaperone protein, calreticulin (CRT), enhances wound
healing. To determine whether CRT can specifically improve the healing of
diabetic wounds and the mechanisms involved, we analyzed the effect of CRT
in repair of excisional wounds in diabetic mice (lep-/lep-) and compared normal
and diabetic wound cells in in-vitro migration and proliferation assays. CRT
(50 mg/day for 4 days) was applied to dorsal wounds (6 mm) that were splinted
open to prevent wound contraction, the mice injected with BrDU, and the
wounds harvested 3,7,10,14, and 28 days post-wounding. CRT induced a
decrease in time to closure of the diabetic wounds (day 17 vs. 21; p o 0.05)
with a remarkable appearance of dermal appendages at day 28 that were
lacking in the untreated controls. Accordingly, epithelial gap was reduced at
days 7 and 10 (p 0.05) and granulation tissue was markedly increased at day
7 (p 0.0006). Histologically, the CRT-treated wounds appeared highly
cellular with increased BrDU positive proliferating basal keratinocytes and
fibroblasts (p 0.05). By picrosirius red staining, increased collagen organization
was observed. In-vitro, CRT induced chemotaxis of human fibroblasts,
keratinocytes and macrophages with maximal induction at 100 ng/ml, 10 pg/ml
and 1 ng/ml, respectively, and greater than positive controls (p o 0.05). Importantly,
whereas the diabetic cellular counterparts exhibited decreased
migration of positive controls, CRT partially restored their migratory capacity.
Furthermore, CRT (100 pg/ml) maximally induced proliferation of keratinocytes
and fibroblasts by 2.2-fold and 8.3-fold, respectively over the untreated
controls. The responses obtained in-vitro support the physiological mechanisms
involved in CRT-induced enhanced closure and cellularity of the diabetic
wounds. We conclude that CRT has the potential to be a powerful topical
therapeutic for the treatment of diabetic wounds through multiple biological
effects. This work was supported by Calretex, LLC.
Wound Rep Reg (2007) 15 A14–A54 c 2007 by the Wound Healing Society
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