John P. Tutela

Improved diabetic wound healing through topical silencing of p53 is associated with augmented vasculogenic mediators

Diabetes is characterized by several poorly understood phenomena including dysfunctional wound healing and impaired vasculogenesis. p53, a master cell cycle regulator, is upregulated in diabetic wounds and has recently been shown to play a regulatory roles in vasculogenic pathways. We have previously described a novel method to topically silence target genes in a wound bed with small interfering (si)RNA. We hypothesized that silencing p53 results in improved diabetic wound healing and augmentation of vasculogenic mediators. Paired 4‐mm stented wounds were created on diabetic db/db mice. Topically applied p53 siRNA, evenly distributed in an agarose matrix, was applied to wounds at postwound day 1 and 7 (matrix alone and nonsense siRNA served as controls). Animals were sacrificed at postwound days 10 and 24. Wound time to closure was photometrically assessed, and wounds were harvested for histology, immunohistochemistry, and immunofluorescence. Vasculogenic cytokine expression was evaluated via Western blot, reverse transcription‐polymerase chain reaction, and enzyme‐linked immunosorbent assay. The ANOVA/t‐test was used to determine significance (p≤ 0.05). Local p53 silencing resulted in faster wound healing with wound closure at 18±1.3 d in the treated group vs. 28±1.0 d in controls. The treated group demonstrated improved wound architecture at each time point while demonstrating near‐complete local p53 knockdown. Moreover, treated wounds showed a 1.92‐fold increase in CD31 endothelial cell staining over controls. Western blot analysis confirmed near‐complete p53 knockdown in treated wounds. At day 10, VEGF secretion (enzyme‐linked immunosorbent assay) was significantly increased in treated wounds (109.3±13.9 pg/mL) vs. controls (33.0±3.8 pg/mL) while reverse transcription‐polymerase chain reaction demonstrated a 1.86‐fold increase in SDF‐1 expression in treated wounds vs. controls. This profile was reversed after the treated wounds healed and before closure of controls (day 24). Augmented vasculogenic cytokine profile and endothelial cell markers are associated with improved diabetic wound healing in topical gene therapy with p53 siRNA.

Progenitor cell mobilization enhances bone healing by means of improved neovascularization and osteogenesis.

Background: Although bone repair is a relatively efficient process, a significant portion of patients fail to heal their fractures. Because adequate blood supply is essential to osteogenesis, the authors hypothesize that augmenting neovascularization by increasing the number of circulating progenitor cells will improve bony healing. Methods: Bilateral full-thickness defects were created in the parietal bones of C57 wild-type mice. Intraperitoneal AMD3100 (n = 33) or sterile saline (n = 33) was administered daily beginning on postoperative day 3 and continuing through day 18. Circulating progenitor cell number was quantified by fluorescence-activated cell sorting. Bone regeneration was assessed with micro–computed tomography. Immunofluorescent CD31 and osteocalcin staining was performed to assess for vascularity and osteoblast density. Results: AMD3100 treatment increased circulating progenitor cell levels and significantly improved bone regeneration. Calvarial defects of AMD3100-treated mice demonstrated increased vascularity and osteoblast density. Conclusions: Improved bone regeneration in this model was associated with elevated circulating progenitor cell number and subsequently improved neovascularization and osteogenesis. These findings highlight the importance of circulating progenitor cells in bone healing and may provide a novel therapy for bone regeneration.

Inhibition of Smad3 Expression in Radiation-Induced Fibrosis Using a Novel Method for Topical Transcutaneous Gene Therapy

OBJECTIVE To attempt to mitigate the effects of irradiation on murine skin after high-dose radiation using a novel transcutaneous topical delivery system to locally inhibit gene expression with small interfering RNA (siRNA) against Smad3. DESIGN Laboratory investigation. SETTING University laboratory. SUBJECTS Twenty-five wild-type C57 mice. INTERVENTION In an isolated skin irradiation model, the dorsal skin of C57 wild-type mice was irradiated (45 Gy). Just before irradiation, Smad3 and nonsense siRNA were applied to 2 separate dorsal skin areas and then reapplied weekly. Skin was harvested after 1 and 4 weeks. Smad3 expression were assessed by immunohistochemistry, and collagen deposition and architecture was examined using picrosirius red collagen staining. MAIN OUTCOME MEASURES Epidermal thickness was measured semiquantitatively at 4 weeks. Radiation-induced fibrosis was measured quantitatively via tensiometry. The Young modulus, a measure of cutaneous rigidity inversely related to elasticity, was determined, with normal irradiated skin serving as a control specimen. RESULTS Murine skin treated with topical Smad3 siRNA demonstrated effective Smad3 inhibition at 1 week and persistent suppression at 4 weeks. Collagen deposition and epidermal thickness were significantly decreased in skin treated with Smad3 siRNA compared with control irradiated skin. Tensiometry demonstrated decreased tension in Smad3 siRNA-treated skin, with a Young modulus of 9.29 MPa (nonirradiated normal skin, 7.78 MPa) compared with nonsense (control) siRNA-treated skin (14.68 MPa). CONCLUSIONS Smad3 expression can be effectively silenced in vivo using a novel topical delivery system. Moreover, cutaneous Smad3 inhibition mitigates radiation-induced changes in tissue elasticity, restoring a near-normal phenotype.

Topical prolyl hydroxylase domain-2 silencing improves diabetic murine wound closure

Prolyl hydroxylase domain 2 (PHD2) has been implicated in several pathways of cell signaling, most notably in its regulation of hypoxia‐inducible factor (HIF)‐1α stability. In normoxia, PHD2 hydroxylates proline residues on HIF‐1α, rendering it inactive. However, in hypoxia, PHD2 is inactive, HIF‐1α is stabilized and downstream effectors such as vascular endothelial growth factor and fibroblast growth factor‐2 are produced to promote angiogenesis. In the present study we utilize RNA interference to PHD2 to promote therapeutic angiogenesis in a diabetic wound model, presumably by the stabilization of HIF‐1α. Stented wounds were created on the dorsum of diabetic Lepr db/db mice. Mice were treated with PHD2 small interfering RNA (siRNA) or nonsense siRNA. Wounds were measured photometrically on days 0–28. Wounds were harvested for histology, protein, and RNA analysis. Diabetic wounds treated with siRNA closed within 21±1.2 days; sham‐treated closed in 28±1.5 days. By day 7, Western blot revealed near complete suppression of PHD protein and corresponding increased HIF‐1α. Angiogenic mediators vascular endothelial growth factor and fibroblast growth factor‐2 were elevated, corresponding to increased CD31 staining in the treated groups. siRNA‐mediated silencing of PHD2 increases HIF‐1α and several mediators of angiogenesis. This corresponded to improved time to closure in diabetic wounds compared with sham‐treated wounds. These findings suggest that impaired wound healing in diabetes can be ameliorated with therapeutic angiogenesis.

Regulators and mediators of radiation-induced fibrosis: Gene expression profiles and a rationale for Smad3 inhibition

OBJECTIVE: Radiotherapy, an essential modality in cancer treatment, frequently induces fibrotic processes in the skin, including accumulation of extracellular matrix. Transforming growth factor-β is essential in regulating extracellular matrix gene expression and is dependent on Smad3, an intracellular mediator/transcription factor. Our study characterized the genetic expression involved in extracellular matrix accumulation during radiationinduced fibrosis. We performed Smad3 gene silencing in an attempt to abrogate the effects of radiation. STUDY DESIGN: Laboratory research. SETTING: University laboratory. SUBJECTS AND METHODS: C57 murine dermal fibroblasts were irradiated with 20 Gy RNA isolated (0, 6, 12, 24, 48, 72 hours postirradiation) and mRNA analyzed (reverse transcriptase polymerase chain reaction) for known regulators (Smad3, interleu-kin-13 [IL-13]), tumor necrosis factor-α [TNF-α]) and mediators of fibrosis (collagen 1A1 [Col1A1]), TGF-β, matrix metalloprotease-1 and −2 (MMP-1, MMP-2), and tissue inhibitor of metallo-protease-1 (TIMP-1). Smad3 gene expression was silenced using siRNA in an effort to restore an unirradiated gene profile. RESULTS: Following irradiation, there was a steady increase in mRNA expression of Smad3, IL-13, TGF-β, Col1A1, MMP-2, TIMP-1, with peak at 12 to 24 hours and subsequent decline by 72 hours. TNF-α expression remained elevated throughout. MMP-1 showed minimal expression initially, which decreased to negligible by 72 hours. Inhibition of Smad3 significantly decreased expression of Col1A1, TGF-β, MMP-2, and TIMP-1. IL-13 and TNF-α expression was not affected by Smad3 silencing. CONCLUSION: We have characterized the early-phase mRNA expression profiles of the major mediators of radiation-induced fibrosis. Smad3 siRNA effectively abrogated the elevation of Col1A1, TGF-β, TIMP-1, and MMP-2. IL-13 and TNF-α were unaffected by Smad3 silencing and appear to be minor regulators in fibrosis. These findings suggest a therapeutic rationale for Smad3 silencing in vivo.

Augmenting neovascularization accelerates distraction osteogenesis.

Background: Distraction osteogenesis has revolutionized the treatment of craniofacial deformities, but it is limited by lengthy consolidation periods and tenuous healing in certain clinical settings, such as irradiated tissue. In this study, the authors aim to investigate whether increasing neovascularization by progenitor cell mobilization accelerates bone formation during distraction. Methods: Sprague-Dawley rats aged 8 weeks (n = 36) were subjected to unilateral mandibular distraction with 3-day latency, 7-day activation (0.25 mm twice daily), and 21-day consolidation periods. From the beginning of the consolidation period, animals received daily injections of either AMD3100 (bone marrow progenitor cell mobilizing agent) or sterile saline. Animals were euthanized on postoperative day 31; mandibles were harvested; and bone regeneration was assessed using micro–computed tomography, immunohistochemistry, bone morphogenetic protein-2 enzyme-linked immunosorbent assay, and mechanical testing. Results: Immunohistochemistry demonstrated that AMD3100 treatment increased vascular density and bone formation. Micro–computed tomography and dual-emission x-ray absorptiometry demonstrated that AMD3100-treated animals had improved bone generation compared with sham-treated controls. Greater force was required on three-point testing to break AMD3100-treated bone. Bone morphogenetic protein-2 expression was up-regulated with AMD3100. Interestingly, the nondistracted contralateral hemimandibles treated with AMD3100 were also stronger than sham-treated counterparts. Conclusions: Progenitor cell mobilization improves bone regeneration in a rat distraction model. Furthermore, because this effect is seen in healthy bone and in ischemic bone healing during distraction, the mechanism is not merely related to oxygenation, but could be a phenomenon of fluid flow.

Shaken Not Stirred? The Effect of Processing Techniques on Fat Graft Survival

36 CONCLUSION: Although HD fractions of centrifuged lipoaspirate contain fewer functional adipocytes than LD fractions, they survive to a greater extent following grafting. The mechanism responsible may be vasculogenic in nature, as baseline analysis found HD fractions to contain more progenitor cells/g as well as increased concentrations of several vasculogenic mediators (VEGF, SDF) compared to LD fractions. This study is the first of its kind, identifying graded densities of lipoaspirate with unique characteristics that directly influence fat graft survival. Shaken Not Stirred? The Effect of Processing Techniques on Fat Graft Survival

1: LACUNOCANALICULAR FLOW AND DISTRACTION OSTEOGENESIS: THEORY AND THERAPEUTICS

Introduction: Our hypothesis is that the tension stress of activation increases lacunocanalicular flow and upregulates the mechanotransductive osteogenic pathway. Furthermore, we hypothesize improving vascularization by endothelial progenitor cell (EPC) mobilization enhances lacunocanalicular flow in the consolidation period, maintaining upregulation of the mechanotransductive pathway to improve and accelerate osteogenesis.

112B: CHEWING THE FAT: THE IMPORTANCE OF PROCESSING TECHNIQUES ON FAT GRAFT SURVIVAL

Background: Lipoaspirate processing methods including centrifugation and Telfa®-rolling have been advocated without direct comparison of their efficacy. Adipocytes secrete proteins that are active participants in regulating angiogenesis and apoptosis. We hypothesize that processing adipose tissue on Telfa gauze creates a lipoaspirate with a more functional adipokine profile and comparable fat graft survival to centrifugation.

Lacunocanalicular Flow in Osseous Repair and Regeneration

INTRODUCTION: Our hypothesis is that the tension stress of activation increases lacunocanalicular flow and upregulates the mechanotransductive-osteogenic pathway. Furthermore, we hypothesize improving vascularization by endothelial progenitor cell (EPC) mobilization enhances lacunocanalicular flow in the consolidation period, maintaining upregulation of the mechanotransductive pathway to accelerate osteogenesis. We develop strategies to manipulate this mechanism to recapitulate lacunocanalicular flow and optimize the biomimicry of tissueengineered implants.