A new technique of ex vivo gene delivery of VEGF to wounds using genetically modified skin particles promotes wound angiogenesis.
BACKGROUND
Transplantation of genetically modified keratinocytes has been shown to accelerate wound healing. However, this method is labor-intensive and time-consuming. We have developed a new technique of intraoperative gene delivery to wounds that involves transplantation of transfected minced skin particles (MSPs) derived from harvested partial-thickness skin.


METHODS
MSPs measuring 0.8 × 0.8 × 0.35 mm were created from a split-thickness skin graft of a pig. In vitro transfection was carried out with adenoviral LacZ (Ad-LacZ) for qualitative and adenoviral vascular endothelial growth factor (Ad-VEGF) for quantitative analysis. Transfected MSPs were transplanted to each of 2.5 × 2.5 cm full-thickness wounds on the dorsum of the pig. Nontransfected MSPs served as controls. Wound chambers were applied and injected with saline to create a wet environment.


RESULTS
LacZ expression was detected in migrating cells originating from MSPs both in vitro and in vivo. VEGF expression in the wound fluid of Ad-VEGF-MSP-transplanted wounds on each of days 2 to 4 (mean ± SEM 6.74 ± 1.89 ng/mL, day 2; 9.88 ± 2.27 ng/mL, day 3; 9.87 ± 1.28 ng/mL, day 4) was significantly higher (p < 0.0001) compared with wounds transplanted with either untransfected MSPs, Ad-LacZ-MSPs, or untransplanted controls. In vitro VEGF expression was significantly higher (p < 0.0001) in Ad-VEGF 1 × 10(10) transfected MSPs compared with either Ad-VEGF 1 × 10(9) transfected MSPs or untransfected MSPs. Wounds transplanted with Ad-VEGF-MSPs showed significantly higher (p < 0.0001) numbers of newly formed blood vessels (12.6 ± 0.9 vessels/high power field [HPF]) compared with wounds transplanted with either Ad-LacZ-MSPs (4.4 ± 0.5 vessels/HPF) or untransfected MSPs (5.2 ± 0.7 vessels/HPF). All MSP-transplanted wounds (Ad-VEGF-MSPs, untransfected MSPs, Ad-LacZ-MSPs) showed significantly higher re-epithelialization compared with untransplanted wounds on days 10 and 14 (p < 0.0001).


CONCLUSIONS
We demonstrated successful transfection of MSPs that can be transplanted to wounds as a source of gene-expressing cells. This technique can be used to deliver growth-modulating genes in wound healing.
Division of Plastic Surgery, Brigham and Women's Hospital, Boston, MA, USA.
Taro
T
Koyama
Florian
F
Hackl
Pejman
P
Aflaki
Juri
J
Bergmann
Baraa
B
Zuhaili
Emily
E
Waisbren
Usha
U
Govindarajulu
Feng
F
Yao
Elof
E
Eriksson
D000818:Animals; D004195:Disease Models, Animal; D005260:Female; D018014:Gene Transfer Techniques; D018919:Neovascularization, Physiologic / Q000235:genetics*; D016038:Skin Transplantation; D013552:Swine; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*; D014945:Wound Healing / Q000235:genetics*
D042461:Vascular Endothelial Growth Factor A
Am J Pathol. 1998 Jun;152(6):1445-52
9626049
Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12188-92
7991604
Gene Ther. 2009 Jan;16(1):34-42
18701914
Plast Reconstr Surg Transplant Bull. 1959 Mar;23(3):226-39
13645241
J Invest Dermatol. 2007 May;127(5):998-1008
17435785
Hum Gene Ther. 1998 Sep 1;9(13):1939-50
9741432
Transplantation. 2002 Apr 15;73(7):1033-41
11965028
J Invest Dermatol. 1965 Jun;44:413-9
14319922
J Invest Dermatol. 1995 Dec;105(6):756-63
7490468
J Invest Dermatol. 2008 Jun;128(6):1360-2
18478013
Mol Ther. 2009 Jan;17(1):1-2
19116631
Tissue Eng Part A. 2008 Jan;14(1):19-27
18333801
Burns. 1994 Dec;20(6):526-8
7880418
Wound Repair Regen. 2007 Sep-Oct;15(5):657-64
17971011
J Surg Res. 2002 Mar;103(1):19-29
11855913
Br J Cancer. 1998 Mar;77(6):956-64
9528841
Wound Repair Regen. 2001 Mar-Apr;9(2):66-76
11350644
AMA Arch Surg. 1958 Dec;77(6):870-4
13593982
Surgery. 1957 Jul;42(1):218-27
13442893
Science. 1989 Dec 8;246(4935):1306-9
2479986
J Invest Dermatol. 2004 Dec;123(6):1182-91
15610532
Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9307-11
7937761
J Gene Med. 2008 Nov;10(11):1247-52
18767030
Free Radic Biol Med. 2006 Apr 1;40(7):1250-63
16545694
Wound Repair Regen. 2005 Jan-Feb;13(1):51-60
15659036
Blood. 1997 Nov 15;90(10):4153-61
9354686
J Surg Res. 1998 Aug;78(2):85-91
9733623
FASEB J. 1990 Nov;4(14):3245-50
2227214
Wound Repair Regen. 2008 Mar-Apr;16(2):288-93
18318812
Hum Gene Ther. 1998 Mar 1;9(4):529-39
9525314
Mol Biol Cell. 1992 Feb;3(2):211-20
1550962
Science. 1987 Sep 18;237(4821):1476-9
3629250