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Surgical Forum Abstracts
J Am Coll Surg
Methods: An E1E3 deleted serotype 5 replication incompetent adenovirus with the cDNA for the E.coli -galactosidase reporter gene (AdLac-Z) was used. Six millimeter excisional wounds were created on the dorsal ear of New Zealand white rabbits, exposing the cartilage. The wounds were treated with 108 plaque forming units (pfu) of AdLac-Z as either a SC injection around the perimeter of the wound, a collagen-alginate gel or a collagen gel with an avidin-antibody binding system loaded with AdLac-Z applied to the wound. The wounds were evaluated 3,7 and 14 days later for gene expression and the epithelial gap. Results: Expression of -galactosidase was similar at 3 days among all treatment groups. By 7 days the collagen-avidin-antibody group (A) had lost most transgene expression, but the injected (I) and collagenalginate (C) groups still demonstrated trace gene expression. At 14 days none of the groups showed transgene expression. Table 1 shows the mean epithelial gap ⫾SD for each group and time point: Table 1 Group Days Gap in mm Group Days Gap in mm Group Days Gap in mm C A I
3 3 3
4.4 ⫾ 0.68 4.98 ⫾ 0.66 5.32 ⫾ 0.88
C A I
7 7 7
2.35 ⫾ 1.0 3.86 ⫾ 1.25 4.07 ⫾ 0.39
C A I
14 14 14
0.56 ⫾ 0.78 0.60 ⫾ 0.77 0.48 ⫾ 0.69
The C group had a significantly smaller epithelial gap at 3 days compared to the I group, p⬍0.01. The C group had a significantly smaller epithelial gap at 7 days than both the I and the A groups, p⬍0.01. At 14 days there was no significant difference between any of the groups. Conclusions: The collagen-alginate gel adenoviral delivery system had the best transgene expression and least adverse effects on wound healing. Collagen based gel system for adenoviral delivery may be beneficial for clinical applications of gene therapy in wound healing.
Reversible regulation of HEGF expression in porcine full-thickness wounds Michael Chen, Feng Yao, PhD, Starck Johnson, MD, Scott Visovatti, Andreas Wenger, Elof Eriksson, MD, PhD, FACS. Harvard Medical School, Division of Plastic Surgery, 75 Francis Street, Boston, MA 02115, USA (617) 732-5093 Introduction: Epidermal growth factor plays a key role in wound healing. We have cloned hEGF under the control of the tet-On switch. Previous work in this lab has shown the efficacy of this system in vitro. This study determines whether EGF expression can be controlled in the wound microenvironment using this novel switch via ex vivo gene transfer. Methods: 32 full-thickness skin wounds were created on a Yorkshire pig. Wounds were transplanted with a double stable cell line, R11/ OEGF, which expresses hEGF under the control of the tet-On switch. Appropriate wounds received tetracycline/saline. hEGF-specific ELISA’s were performed to determine levels of hEGF present in wound fluid collected daily. Student’s T-test was used to determine signficance. Results: 3 days post-transplant, hEGF expression in wounds receiving tetracycline increased 500-fold over that in wounds not receiving tetracycline(Fig 1). In addition, to determine the efficacy of rerepression of EGF expression after removal of tetracycline, we gave tetracycline from days 3 to 6 only. Upon removal of tetracycline on day 6, hEGF expression appropriately decreased, and 2 days after removal of tetracycline, the levels of EGF expression were only 5-fold over that of cells never given tetracycline (Fig 2).
Conclusions: Using a novel genetic switch, we were able to precisely control hEGF expression in the wound microenvironment through ex vivo gene transfer. Our results suggest the potential use of this switch to precisely control specific growth factor expression in the wound microenvironment.
Adenoviral-mediated PDGF-B over-expression enhanced re-epithelialization is mediated by TGF-␣ Jagajan Karmacharya, Bernard Martin, Yukia Kitano, Antoneta Radu, Meenhard Herlyn, Richard Kirschner, Timothy M Crombleholme. The Children’s Institute for Surgical Science, Divisions of General, Thoracic, and Fetal Surgery and Plastic and Reconstructive Surgery. The Children’s Hospital of and The Wistar Institute, Philadelphia, PA USA Introduction: We have previously demonstrated that adenoviralmediated overexpression of PDGF-B in excisional wound in diabetic (db/db) mice enhances healing and AD-PDGF-B appears to stimulate keratinocyte proliferation and re-epithelialization. We hypothesized that the mechanism by which PDGF induces these proliferative and migratory changes in keratinocytes is by the induction of keratinocyte mitogen gene expression in adjacent fibroblasts. Methods: In order to test this hypothesis we studied the effect of Ad-PDGF-B on the expression of keratinocyte growth factors in db/db mice. 10 mm excisional wounds were created in db/db mice (n ⫽ 9) which were harvested at 1, 3, and 7 days for extraction of mRNA for RT-PCR and immunohistochemisty. The effect of Ad-PDGFB on keratinocyte proliferation were examined in primary cultures of human fibroblasts transfected with Ad-PDGF-B. The keratinocyte proliferation index (KPI) was determined by Ki67 immunostaining (a proliferation marker). Results: RT-PCR of demonstrated marked induction of TGF-a but not KGF gene expression at 1 day post-wounding. Immunohistochemisty demonstrated increased expression of TGF-a in the fibroblasts adjacent to the advancing edge of keratinocytes at 1 day post wounding. The KPI of keratinocytes exposed Ad-PDGF-B 43.9%. Neutralizing antibodies to TGF-a added to the media decreased the KPI to 9.7% but neutralizing antibodies to KGF, EGF, and IGF-1 had no effect on KPI suggesting the increased proliferation observed in response to PDGF-B is mediated by TGF-␣. Conclusions: We conclude that Ad-PDGF-B induced over-expression in fibroblasts induces TGF-a gene expression and protein production which has both proliferative and migrational effects on keratinocytes. In db/db mice Ad-PDGF-B stimulates re-epithelialization by induction of TGF-a gene expression and growth factor production in fibroblasts at the leading edge of re-epithelializing keratinocytes.
Biolistic ‘‘gene gun’’ delivery of eukaryotic initiation factor 4E (eIF 4E) mRNA enhances wound healing Karl W Schwarz, MD⌽, Mary, T Murray, PhD⌽, Paul K Hwang, BS⌽, Lawrence N Diebel, MD⌽, Scott A Dulchavsky, MD, PhD⌽. Departments of Surgery⌽ and The Center for Molecular Medicine & Genetics Wayne State University, Detroit, MI; Designated contact person: Scott A. Dulchavsky, M.D., Ph.D., Detroit Receiving Hospital Dept. of Surgery, 4201 St. Antoine, Detroit, MI 48201, USA. (313) 745-1350 Introduction: Initiation of translation is the rate limiting step in protein synthesis; eIF 4E increases translational efficiency by facilitating ribosome scanning. eIF4E is present in cells in rate limiting amounts; chronic over-expression of eIF4E causes cell transformation by upregulating growth-related proteins. Biolistic delivery of Epidermal Growth Factor (EGF) increases wound healing; transiently increasing wound eIF4E levels with biolistic mRNA transmission may further augment wound healing without oncogenesis. Methods: Midline fascial wounds were created in rats and biolistically treated with gold particles carrying mRNA encoding for hEGF with or without eIF4E prior to suture closure; control animals received blank bullets. The animals were sacrificed at 7 or 14 days for determination of peak wound bursting strength on a tensiometer. Results are expressed as mean ⫾ standard deviation; statistics are via analysis of variance.
Vol. 191, No. 4S, October 2000
Results: Peak Tensile Wound Strength (Newtons) Treatment Group (n ⴝ 6) Control Normal ⫹ EGF Normal ⫹ EGF & eIF 4E
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Surgical Forum Abstracts
7 days
14 days
4.5 ⫾ 1.3 6.5 ⫾ 2.9 8.4 ⫾ 3.6
9.8 ⫾ 3.9 11.1 ⫾ 3.4 18.8 ⫾ 3.5*
* p ⬍ 0.01 compared to control or EGF by ANOVA
Conclusions: Simultaneous biolistic delivery of EGF mRNA with eIF4E mRNA significantly increases wound breaking strength compared to control animals or treatment with EGF mRNA alone without risk of cellular transformation. Further studies of translational activation to augment wound healing are warranted.
Composite tissue allograft (CTA): tolerance induction without graft vs host disease (GvHD) Vijay Gorantla MD, Gustavo Perez-Abadia MD, Kaustubha Prabhune MD, Haldun Orhun MD, Thanos Kakoulidis MD, Ramsey Majzoub MD, Claudio Maldonado PhD, Gary Anderson PhD, Lynn Ogden MD, Warren Breidenbach MD, Suzanne Ildstad MD, John Barker MD, PhD. Division of Plastic and Reconstructive Surgery, Institute for Cellular Therapeutics, Department of Surgery, University of Louisville & Christine M. Kleinert Institute. University of Louisville, 511 South Floyd St, 320 MDR Bldg, Louisville, KY, 40292, USA. Tel (502) 852-0166. Introduction: Successful human hand transplants have recently been performed using immunosuppression. However, the risks of the nonspecific immunosuppressants required for graft survival are subject to controversy. Mixed allogeneic chimerism (MAC) induces donorspecific tolerance to solid organ allografts. CTAs add another variable in that the mature donor immune system (bone marrow (BM)) accompanies the graft, potentially resulting in GvHD. The aim of this study was to achieve a GvHD free tolerant state using MAC in a rat hind-limb model. Methods: Group I: naïve WF rats received hind limbs from unmanipulated ACI rats (n ⫽ 6). Group II: host WF rats were irradiated with 950cGy and reconstituted with MHC disparate ACI BM cells (␣-␥␦ T-cell depleted) (n ⫽ 8). Flow cytometry after 28 days confirmed chimerism. These rats then received non-irradiated ACI limbs. Group III: (ACI ⫹ WF) rats, received similar treatment as in Group II but were transplanted with ACI Limbs pretreated with 1050cGy TBI (n ⫽ 8). Results: Group I rejected their limbs in 5.7 ⫾ 1.5 days. Group II: 7/8 (ACI ⫹ WF) rats (⬎85% chimerism) exhibited rejection-free survival of non-irradiated CTAs for 22.4 ⫾ 2.8 days, but developed severe GvHD only after the CTA and died. Group III: 7/8 (ACI ⫹ WF) rats showed no sign of rejection or GvHD at 77 ⫾ 1.8 days. One rat died of infection at 34 days post-op with no sign of GvHD. Notably, this is significantly prolonged beyond the time of onset of GvHD in Group II. Conclusions: Partial conditioning of the host alone led to high levels of MAC without GvHD before CTA. The bone marrow in unmanipulated limbs resulted in GvHD. After transplantation of irradiated limbs, MAC prevented acute rejection and resulted in a GvHD free state. Taken together, these data show that immunocompetent donor cells transferred with the CTA can mediate GvHD and that inactivation of these cells by irradiation could prevent GvHD.
Tolerance to limb allografts in a miniature swine model
12 days of cyclosporine. The aims of this study were 1) to determine if tolerance to allografts could be achieved with the addition of a vascularized skin component, and 2) to evaluate the role of the donor bone marrow following long-term tolerance. Methods: Limb grafts from 4 swine with a 100 cm2 cutaneous paddle were heterotopically transplanted into MHC matched, minor antigen mismatched miniature swine and treated with 12 days of cyclosporine. Biweekly biopsies were performed and evaluated histologically. Pig allelic antigen (PAA), a non-MHC-linked marker, was used to study bone marrow migration in PAA⫹ recipients with PAA⫺ allografts. Swine demonstrating long-term tolerance to their allografts were sacrificed, and the graft marrow was analyzed for the migration of recipient cells into graft marrow space using the PAA marker. Results: All animals accepted the musculoskeletal portion of their grafts by gross inspection and histologic analysis. One animal demonstrated tolerance to the skin (⬎120 days). Three animals sloughed the graft epidermis (days 39, 41, and 50) leaving an intact dermis. The host skin began to re-epithelialize the graft dermis. At sacrifice, the allograft bone marrow harvest yielded viable marrow. All hematopoietic cells identified in the graft marrow were PAA positive and therefore of recipient origin. Conclusions: Tolerance to limb allografts with a skin component can be achieved with a 12-day course of cyclosporine and MHC matching. Immune modulation of the skin component may be necessary for clinical success. Furthermore, recipient hematopoietic cells repopulate the graft marrow space, and tolerance to limb allografts does not appear to depend on the presence of donor bone marrow cells.
Chondrogenesis of human adipo-derived mesodermal stem cells Jerry I Huang, BS, Marc H Hedrick, MD, H. Peter Lorenz, MD, Min Zhu, MD, Prosper Benhaim, MD. Division of Plastic and Reconstructive Surgery; University of California, Los Angeles 200 UCLA Medical Plaza, Suite #140; Los Angeles, CA 90095-6902, USA; Phone (310) 206-4468 Introduction: The repair of cartilaginous defects remains a significant clinical problem. Human bone marrow contains pluripotent mesenchymal stem cells capable of differentiation down multiple mesodermal lineages, including bone and cartilage. However, bone marrow harvest is painful and yields few cells. The purpose of this study is to determine if human adipose tissue contains a similar population of stem cells with osteochondrogenic potential. Methods: Human liposuction aspirate from five patients were washed and digested, yielding fibroblast-like cells. Cells were psssaged three times in basal media, then induced toward chondrogenesis by placement in high-density micromass cultures supplemented with TGF-1, insulin, transferrin, and ascorbic acid. Chondrogenesis was determined by histologic evaluation at 2, 7, and 14 days with H&E, Alcian blue, and Goldner’s trichrome staining. In addition, immunohistochemistry was performed using antibodies against type II collagen, chondroitin-4-sulfate, and keratan sulfate. Intensity of histologic and immunohistochemistry staining was subjectively graded as: none (⫺); background (⫹/⫺); light (⫹); moderate (⫹⫹); heavy (⫹⫹⫹). Results: Cartilaginous nodules with a perichondral cellular border formed within 48 hours in all treated cultures. Untreated control cells showed no evidence of chondrogenic differentiation. Time Stages of chondrogenic Alcian Trichrome Chondroitin- Keratan Type II point differentiation blue (collagen) 4-sulfate sulfate collagen
David W Mathes, MD, Judy L Bourget, MD, Mark A Randolph, MA, Mario G Solari, BA, Anette, Wu, MD, David H Sachs, MD, PhD, WP Andrew, Lee, MD. Massachusetts General Hospital, Harvard Medical School, Boston, MA, WACC 453, Boston, MA 02114, USA Phone: 617-726-6943
2d Cellular condensation 7d Spheroid formation 14 d Increased extracellular matrix secretion
Introduction: The recent human hand transplants depend on longterm immunosuppression that entails significant risks to the recipients. We have demonstrated long-term tolerance to vascularized musculoskeletal allografts in MHC matched miniature swine treated with only
Conclusions: Human adipose tissue contains mesodermal stem cells capable of in vitro chondrogenesis. This abundant and easily obtained source of cells has considerable potential for future tissue engineering strategies in cartilage repair.
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