In vitro-analysis of radiation-induced dermal wounds

Program Oral Presentations

Effect of TGF-1 antisense therapy on aberrant wound healing Haneen Sadick, MD, PhD (presenter); Gregor Bran, MD; Ulrich Goessler, MD, PhD; Karl Hoermann, MD; Frank Riedel, MD OBJECTIVES: Wound healing is a physiological event in which tissue injury initiates a repair process leading to collagen formation and degradation. Keloids are a fibroproliferative disorder of excessive wound healing due to an imbalance between matrix metalloproteinases and their specific inhibitors, TIMPs, towards an excessive expression of MMPs with extensive deposition of scar tissue. TGF-1 has been identified as an important regulator of this process with a strong pro-fibrotic function which additionally suppresses the TIMP function. METHODS: Tissue staining for MMP was investigated by immunohistochemistry. The effect of TGF-1 antisense therapy on the expression of MMPs in keloid-derived fibroblasts was analysed by ELISA and multiplex RT-PCR. RESULTS: Immunohistochemistry revealed a stronger staining of MMP in keloid-tissue than in normal skin samples. The treatment of keloid-fibroblast with TGF-1 antisense oligonucleotides (ONDs) in vitro efficiently down-regulated the expression of MMP-1, MMP-3, and MMP-9 but not of MMP-2 and MMP-13. In the supernatans of keloid-derived fibroblasts, the TGF-1 antisense therapy showed decreased secretion levels of all MMP proteins apart from MMP-13. So the synthesis of MMP-2 and MMP-13 does not seem to be influenced by TGF-1 antisense ONDs in the supernatans of keloid-fibroblasts. CONCLUSIONS: TGF-1 targeting may be a potential therapeutic option for the inhibition of proteolytic tissue destruction in keloids. The results should be regarded as a further implication for the treatment of keloids. Identification of valid housekeeping genes in human myoblasts Jens Stern-Straeter, MD (presenter); Alexander Sauter, MD; Stefan Kassner, MD;

Karl Hormann, MD; Gabriel Alejandro Bonaterra, MD; Ulrich Goessler, MD, PhD OBJECTIVES: Analysis of gene expression using real-time PCR (qRT-PCR) requires normalization by genes that are continuously expressed [housekeeping genes (HKG)]. For precise qRT-PCR measurements it is most important that the reference gene expression is invariant under the investigated experimental conditions. It becomes more evident that HKG expression may vary considerably under different culture conditions, which leads to inaccurate measurements and misinterpretation of the obtained results. In this study, we investigated six HKG, which have been reported in the literature to be invariant, during the differentiation of human myoblast cultures in order to identify the most suitable HKG for valid qRT-PCR studies. METHODS: qRT-PCR was used to assess the levels of mRNA encoding ␤-actin (ACTB), ␤-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO). The geNorm and NormFinder softwares were used to calculate the most suitable reference genes. RESULTS: Using the geNorm program, RPLPO and TBP were found to be the most stable genes. In addition, the NormFinder software showed that RPLPO was the most stable, whereas TBP ranked second. CONCLUSIONS: In conclusion, differences in gene expression between human myoblasts under differentiation conditions can be identified when the RNA expression of the target gene is corrected with RPLPO as normalization factor. In vitro-analysis of radiation-induced dermal wounds Ulrich Goessler, MD, PhD (presenter); Stefan Kassner, MD; Gregor Bran, MD; Ulrich Sommer; Jens Stern-Straeter, MD; Haneen Sadick, MD, PhD; Karl Hormann, MD OBJECTIVES: Radiotherapy as a valuable tool in the treatment of head and neck cancer is increasingly implemented in treatment regimens. As a consequence, clinicians are confronted with a rising amount of radiogenic complications. The aim of our study was the analysis of the pathophysiology of radiation-induced wounds of the head and neck at a molecular level. METHODS: Keratinocytes and fibroblasts from chronic nonhealing ulcers in radiated areas as well as from healthy skin areas in the same patient were harvested from five patients during surgical procedures and isolated in cell culture. A proliferation assay was performed. Gene-expression was analysed by RT-PCR and Microarray, protein-expression as a control by immunohistochemistry.

ORALS

necrotic superficial layer over the replanted nose and this layer fell off and beneath the live tissue was apparently visible. At the third week, post-auricular skin was used as a full thickness skin graft to cover the bare tissue. RESULTS: The amputated segment survived except for a small area over the left ala. Normal appearance of the face was restored. CONCLUSIONS: Composite grafting of the disrupted segment has been used to treat these patients. It seems it has acceptable results especially when it saves the invaluable time. The darkened skin usually indicates only partial thickness loss and the majority of the graft would survive. Late skin grafting could compensate for tissue loss.

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Otolaryngology-Head and Neck Surgery, Vol 141, No 3S1, September 2009

RESULTS: Keratinocytes and fibroblasts from radiogenic wounds showed reduced proliferation. Keratinocytes from radiogenic wounds showed a shift from the high molecular keratins 1 and 10 to the low molecular keratins 5 and 14 compared with normal control skin. Keratinocytes and fibroblasts from nonhealing wounds showed a decreased expression of mediators of cellular proliferation such as TGFa, TGFb1, FGF1, FGF2, and KGF. Mediators of angiogenesis such as VEGF and HGF also showed decreased expression in radiated tissue. The matrix-metalloproteinases MMP 2, 12 and 13 showed increased expression in radiated keratinocytes and fibroblasts. CONCLUSIONS: Our data showed a change of keratinocytes to a less differentiated state due to radiation. In addition, it seems that radiation-induced dermal injuries often fail to heal because of decreased proliferation, impaired angiogenesis, and persistently high concentrations of MMPs. Mechanical venous anastomosis in head and neck reconstruction Yash J Patil, MD (presenter) OBJECTIVES: This retrospective review examines a single surgeon’s experience with 200 consecutive free flaps used for head and neck reconstruction. In each flap a mechanical venous anastomosis is employed for venous outflow. This study examines the safety of a coupled venous anastomosis as the sole method for establishing venous outflow. Specifically, data were analyzed and stratified by the type of donor flap used, recipient vasculature, associated complications, vessel diameter, patient age and gender. METHODS: A retrospective review of all the operative reports and clinical records for 200 consecutive patients who underwent microvascular free flap reconstruction from 2003 to 2008 was performed with IRB approval. All reconstructions were performed by a single surgeon. Data were analyzed and stratified by the type of donor flap used, recipient vasculature, associated complications, vessel diameter, patient age and gender. RESULTS: There was greater than 98% flap survival using mechanical venous anastomosis as the primary means for venous outflow in this series of 200 consecutive patients. CONCLUSIONS: Mechanical venous anastomosis can provide a highly effective means for venous outflow in head and neck microvascular reconstruction. Microvascular reconstruction for advanced mandibular osteoradionecrosis Jeffrey D Suh, MD (presenter); Vishad Nabili, MD; Keith Blackwell, MD; Joel Sercarz, MD OBJECTIVES: The objective of this study is to assess the outcomes and complications of free tissue transfers for treat-

ment of advanced mandibular osteoradionecrosis (ORN) in head and neck cancer patients. METHODS: Retrospective case series. 29 patients with ORN of the mandible were treated by segmental mandibulectomy and microsurgical free tissue transfer between 1995 and 2007. All patients received XRT for previous HN cancer, and six of 29 patients received concurrent chemotherapy. All patients had either failed to respond to conservative management, which included HBO, long-term antibiotics, debridement, or had evidence of a pathologic fracture. There were 16 males and 13 females, with a median age of 63 years. Flap donor sites included fibula (25), latissimus dorsi/serratus anterior/rib (3), and iliac crest (1). Median follow-up was 48 months. RESULTS: There were no free flap failures. The incidence of reconstructive complications in this series was 38%. Median time to complication was 14.1 months. 10 patients (34%) required removal of their reconstruction plate for hardwarerelated complications. Four patients (13%) required additional mandibular resections for residual or recurrent ORN at the margins of the initial MISSING CONCLUSIONS: This present study confirms that microvascular free flaps are reliable in the treatment of advanced mandibular ORN. Nevertheless, there remains a 38% incidence of wound-healing complications, usually occurring in a delayed fashion after the immediate perioperative period. The incidence of hardware-related complications is increased in ORN patients, compared to patients undergoing microvascular flap reconstruction of the mandible for other indications. The lack of objective clinical criteria to judge the appropriate amount of mandible resection in patients with ORN remains an unresolved issue that results in the development of recurrent ORN in 13% of patients who undergo an inadequate mandibular resection. Molecular signature of keloids Denise Rae Wong, MD (presenter); David Hom, MD; Holly C Boyer, MD; Jizhen Lin, MD OBJECTIVES: Little is known about the global gene expression and the molecular signature specific for keloids. In this study, we sought to use Affymetrix microarrays for analysis of the specific growth factors, signaling pathways, and effectors in keloid tissue. 1) Understand the role of TGF-Beta-1 in the upregulation of extracellular matrix proteins in keloid tissue. 2) Identify candidate genes involved in extracellular matrix remodeling in keloid tissue. METHODS: The molecular pathogenesis of keloid formation was studied in a university basic science laboratory. Three keloid specimens and two control tissues were used for Affymetrix microarray analysis. The gene list retrieved from microarrays was then verified with reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry, and luciferase assays using human fibroblast cell culture in vitro. RESULTS: The extracellular matrix (ECM) proteins were