Denis Knobel

Fat grafting accelerates revascularisation and decreases fibrosis following thermal injury

BACKGROUND Fat grafting has been shown clinically to improve the quality of burn scars. To date, no study has explored the mechanism of this effect. We aimed to do so by combining our murine model of fat grafting with a previously described murine model of thermal injury. METHODS Wild-type FVB mice (n=20) were anaesthetised, shaved and depilitated. Brass rods were heated to 100°C in a hot water bath before being applied to the dorsum of the mice for 10s, yielding a full-thickness injury. Following a 2-week recovery period, the mice underwent Doppler scanning before being fat/sham grafted with 1.5cc of human fat/saline. Half were sacrificed 4 weeks following grafting, and half were sacrificed 8 weeks following grafting. Both groups underwent repeat Doppler scanning immediately prior to sacrifice. Burn scar samples were taken following sacrifice at both time points for protein quantification, CD31 staining and Picrosirius red staining. RESULTS Doppler scanning demonstrated significantly greater flux in fat-grafted animals than saline-grafted animals at 4 weeks (fat=305±15.77mV, saline=242±15.83mV; p=0.026). Enzyme-linked immunosorbent assay (ELISA) analysis in fat-grafted animals demonstrated significant increase in vasculogenic proteins at 4 weeks (vascular endothelial growth factor (VEGF): fat=74.3±4.39ngml(-1), saline=34.3±5.23ngml(-1); p=0.004) (stromal cell-derived factor-1 (SDF-1): fat=51.8±1.23ngml(-1), saline grafted=10.2±3.22ngml(-1); p<0.001) and significant decreases in fibrotic markers at 8 weeks (transforming growth factor-ß1(TGF-ß): saline=9.30±0.93, fat=4.63±0.38ngml(-1); p=0.002) (matrix metallopeptidase 9 (MMP9): saline=13.05±1.21ngml(-1), fat=6.83±1.39ngml(-1); p=0.010). CD31 staining demonstrated significantly up-regulated vascularity at 4 weeks in fat-grafted animals (fat=30.8±3.39 vessels per high power field (hpf), saline=20.0±0.91 vessels per high power field (hpf); p=0.029). Sirius red staining demonstrated significantly reduced scar index in fat-grafted animals at 8 weeks (fat=0.69±0.10, saline=2.03±0.53; p=0.046). CONCLUSIONS Fat grafting resulted in more rapid revascularisation at the burn site as measured by laser Doppler flow, CD31 staining and chemical markers of angiogenesis. In turn, this resulted in decreased fibrosis as measured by Sirius red staining and chemical markers.

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.

Low-dose radiation augments vasculogenesis signaling through HIF-1-dependent and -independent SDF-1 induction

The inflammatory response to ionizing radiation (IR) includes a proangiogenic effect that could be counterproductive in cancer but can be exploited for treating impaired wound healing. We demonstrate for the first time that IR stimulates hypoxia-inducible factor-1α (HIF-1α) up-regulation in endothelial cells (ECs), a HIF-1α-independent up-regulation of stromal cell-derived factor-1 (SDF-1), as well as endothelial migration, all of which are essential for angiogenesis. 5 Gray IR-induced EC HIF-1α and SDF-1 expression was greater when combined with hypoxia suggesting an additive effect. While small interfering RNA silencing of HIF-1α mRNA and abolition of HIF-1α protein induction down-regulated SDF-1 induction by hypoxia alone, it had little effect on SDF-1 induction by IR, demonstrating an independent pathway. SDF-1-mediated EC migration in hypoxic and/or radiation-treated media showed IR induced strong SDF-1-dependent migration of ECs, augmented by hypoxia. IR activates a novel pathway stimulating EC migration directly through the expression of SDF-1 independent of HIF-1α induction. These observations might be exploited for stimulation of wound healing or controlling tumor angiogenesis.

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.

Melanoma Extirpation with Immediate Reconstruction: The Oncologic Safety and Cost Savings of Single-Stage Treatment.

BACKGROUND The timing of reconstruction following melanoma extirpation remains controversial, with some advocating definitive reconstruction only when the results of permanent pathologic evaluation are available. The authors evaluated oncologic safety and cost benefit of single-stage neoplasm extirpation with immediate reconstruction. METHODS The authors reviewed all patients treated with biopsy-proven melanoma followed by immediate reconstruction during a 3-year period (January of 2011 to December of 2013). Patient demographic data, preoperative biopsies, operative details, and postoperative pathology reports were evaluated. Cost analysis was performed using hospital charges for single-stage surgery versus theoretical two-stage surgery. RESULTS During the study period, 534 consecutive patients were treated with wide excision and immediate reconstruction, including primary closure in 285 patients (55 percent), local tissue rearrangement in 155 patients (30 percent), and skin grafting in 78 patients (15 percent). The mean patient age was 67 years (range, 19 to 98 years), and the median follow-up time was 1.2 years. Shave biopsy was the most common diagnostic modality, resulting in tumor depth underestimation in 30 patients (6.0 percent). Nine patients (2.7 percent) had positive margins on permanent pathologic evaluation. The only variables associated with positive margins were desmoplastic melanoma (p = 0.004) and tumor location on the cheek (p = 0.0001). The mean hospital charge for immediate reconstruction was

Augmenting neovascularization accelerates distraction osteogenesis.

22,528 compared with the theoretical mean charge of

Lacunocanalicular Fluid Flow Transduces Mechanical Tension Stress During Distraction Osteogenesis

35,641 for delayed reconstruction, leading to mean savings of 38.5 percent (SD, 7.9 percent). CONCLUSION This large series demonstrates that immediate reconstruction can be safely performed in melanoma patients with an acceptable rate of residual tumor requiring reoperation and significant health care cost savings. CLINICAL QUESTION/LEVEL OF EVIDENCE Therapeutic, IV.

1: LACUNOCANALICULAR FLOW AND DISTRACTION OSTEOGENESIS: THEORY AND THERAPEUTICS

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.

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

Abstract The mechanotransduction mechanisms linking distraction device activation to new bone formation remain unknown. We hypothesize that the tension stress of activation during distraction osteogenesis is transmitted through lacunocanalicular fluid flow to initiate the osteogenic signaling cascade. Adult Sprague-Dawley rats (N = 24) were subjected to mandibular osteotomy and application of an external distraction device. After a 3-day latency period, half the animals (n = 12) underwent device activation at 0.25 mm twice daily for 6 days (total activation, 3 mm), and the other half (n = 12) had no activation. On day 10, the animals were injected with fluorescent reactive red lacunocanalicular tracer before killing. Mandibles were harvested, embedded, and sectioned, and reactive red epifluorescence lacunocanalicular flow was measured. Protein was harvested for focal adhesion kinase 1 (FAK1), NESPRIN1, SUN1, LAMIN A/C, and SMAD1 Western blotting as well as for bone morphogenetic protein (BMP)-2 enzyme-linked immunosorbent assay and alkaline phosphatase assay. Lacunocanalicular fluid flow was significantly greater in the distracted samples (60.5 ± 14 vs 10.3 ± 4 molecules of equivalent soluble fluorochrome per megapixel, P = 0.01). Flow distribution demonstrated the highest lacunocanalicular flow near the center of the distraction gap. Increased lacunocanalicular flow resulted in increased FAK1 (P = 0.009), NESPRIN1 (P = 0.01), SUN1 (P = 0.01), and LAMIN A/C (P = 0.008) expression. Focal adhesion kinase 1 activation in the presence of BMP-2 protein expression (P = 0.001) resulted in increased intranuclear SMAD1 phosphorylation (P = 0.04) and alkaline phosphatase activity (P < 0.0001). These findings suggest that activation of the distraction osteogenesis device affects cellular response through changes in lacunocanalicular fluid flow.

81B: MITIGATING RADIATION-INDUCED SKIN INJURY: PREVENTING ANGIOSTASIS VIA TOPICAL CXCR3 SILENCING

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.