Phuong D. Nguyen

Human fat grafting alleviates radiation skin damage in a murine model.

Background: Autogenous fat grafting has been observed to alleviate the sequelae of chronic radiodermatitis. To date, no study has replicated this finding in an animal model. Methods: The dorsa of adult wild-type FVB mice were shaved and depilated. The dorsal skin was then distracted away from the body and irradiated (45 Gy). Four weeks after irradiation, 1.5-cc fat or sham grafts were placed in the dorsal subcutaneous space. Gross results were analyzed photometrically. The animals were euthanized at 4 and 8 weeks after fat or sham grafting and their dorsal skin was processed for histologic analysis. Results: Hyperpigmentation and ulceration were grossly improved in fat-grafted mice compared with sham-grafted controls. This improvement manifested histologically in a number of ways. For example, epidermal thickness measurements demonstrated decreased thickness in fat-grafted animals at both time points (20.6 ± 1.5 &mgr;m versus 55.2 ± 5.6 &mgr;m, p = 0.004; 17.6 ± 1.1 &mgr;m versus 36.3 ± 6.1 &mgr;m, p = 0.039). Picrosirius red staining demonstrated a diminished scar index in fat-treated animals at both time points as well (0.54 ± 0.05 versus 0.74 ± 0.07, p = 0.034; and 0.55 ± 0.06 versus 0.93 ± 0.07, p = 0.001). Conclusion: Fat grafting attenuates inflammation in acute radiodermatitis and slows the progression of fibrosis in chronic radiodermatitis.

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.

A Novel Mouse Model of Cutaneous Radiation Injury

Background: Radiation therapy is a cornerstone of oncologic treatment. Skin tolerance is often the limiting factor in radiotherapy. To study these issues and create modalities for intervention, the authors developed a novel murine model of cutaneous radiation injury. Methods: The dorsal skin was isolated using a low-pressure clamp and irradiated. Mice were followed for 8 weeks with serial photography and laser Doppler analysis. Sequential skin biopsy specimens were taken and examined histologically. Tensiometry was performed and Youngs modulus calculated. Results: High-dose radiation isolated to dorsal skin causes progressive changes in skin perfusion, resulting in dermal thickening, fibrosis, persistent alopecia, and sometimes ulceration. There is increased dermal Smad3 expression, and decreased elasticity and bursting strength. Conclusions: This model of cutaneous radiation injury delivers reproducible localized effects, mimicking the injury pattern seen in human subjects. This technique can be used to study radiation-induced injury to evaluate preventative and therapeutic strategies for these clinical issues.

Establishment of a Critical-Sized Alveolar Defect in the Rat : A Model for Human Gingivoperiosteoplasty

Background: Despite technical advancement, treatment of congenital alveolar clefts has remained controversial. Currently, primary alveolar cleft repair (i.e., gingivoperiosteoplasty) has a 41 to 73 percent success rate. However, the remaining patients have persistent alveolar bone defects requiring secondary grafting procedures. Morbidity of secondary procedures includes pain, graft resorption, extrusion or infection, and graft or tooth loss. The authors present a novel rat alveolar defect model designed to facilitate investigation of therapeutics aimed at improving bone formation following primary alveolar cleft repair in humans. Methods: Sixteen 8-week-old Sprague-Dawley rats underwent creation of a 7 × 4 × 3-mm complete alveolar defect from the maxillary incisors to the zygomatic arch. Four animals were humanely killed at each of the following time points: 0, 4, 8, and 12 weeks. Morphometric analysis of the alveolar defect was determined by means of micro-computed tomography and histology. Results: Micro-computed tomography demonstrated that new bone filled 43 ± 5.6 percent of the alveolar defect at 4 weeks, 53 ± 8.3 percent at 8 weeks, and 48 ± 3.5 percent at 12 weeks. Histologically, at 4 weeks, proliferating fibroblasts and polymorphonuclear cells were scattered throughout the disorganized collagen in the intercalary gap. By 8 weeks, nascent woven bone spicules extended from the edges of the defect. At 12 weeks, the woven spicules had remodeled, with scant additional bone deposition. Conclusion: This model creates a critical-size alveolar defect that is similar in size and location to human alveolar defects and is suitable for studying proposed therapeutics.

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.

Grading lipoaspirate: is there an optimal density for fat grafting?

Background: Clinical results of fat grafting have been unpredictable. In this article, the authors hypothesize that centrifugation creates “graded densities” of fat with varying characteristics that influence lipoaspirate persistence and quality. Methods: Aliquots of human female lipoaspirate (10 cc) were centrifuged for 3 minutes at 1200 g. The bloody and oil fractions were discarded. Subsequently, 1.0 cc of the highest density and lowest density fat was separated for lipoinfiltration or analysis. Highest density or lowest density fat grafted into adult FVB mice was harvested at 2 and 10 weeks to quantify short- and long-term persistence, respectively. Progenitor cell number and expression of vascular endothelial growth factor, stromal cell–derived factor-1&agr;, platelet-derived growth factor, and adiponectin were analyzed by flow cytometry and enzyme-linked immunosorbent assay, respectively. Results: Greater percentages of highest density fat grafts remain at 2 and 10 weeks after injection compared with lowest density fat grafts (85.4 ± 1.9 percent versus 62.3 ± 0.1 percent, p = 0.05; and 60.8 ± 4.9 versus 42.2 ± 3.9, p < 0.05, respectively). Highest density fractions contain more progenitor cells per gram than lowest density fractions (2.0 ± 0.2-fold increase, p < 0.01). Furthermore, concentrations of vascular endothelial growth factor, stromal vascular fraction, platelet-derived growth factor, and adiponectin are all elevated in highest density compared with lowest density fractions (34.4 percent, p < 0.01; 34.6 percent, p < 0.05; 52.2 percent, p < 0.01; and 45.7 percent, p < 0.05, respectively). Conclusions: Greater percentages of highest density fractions of lipoaspirate persist over time compared with lowest density fractions. A vasculogenic mechanism appears to contribute significantly, as highest density fractions contain more progenitor cells and increased concentrations of several vasculogenic mediators than lowest density fractions.

Microvascular lifeboats: a stepwise approach to intraoperative venous congestion in DIEP flap breast reconstruction.

Summary: The deep inferior epigastric perforator (DIEP) flap is becoming a widely practiced method of autologous breast reconstruction. Although it has been shown to be a safe and reliable technique with acceptable morbidity, disadvantages include a comparatively higher incidence of venous congestion and total flap loss compared with autologous reconstruction with a pedicled or free transverse rectus abdominis myocutaneous flap. Venous congestion is reported in up to one-third of cases of breast reconstruction with a DIEP flap. If venous congestion is detected and addressed intraoperatively compared with postoperatively, outcomes are significantly improved. A wide variety of techniques have been introduced to augment venous drainage to treat congestion and prevent flap failure. Here, the authors offer a comprehensive review of techniques available to address intraoperative venous congestion in DIEP flaps for breast reconstruction. From this review, the authors propose a stepwise, algorithmic approach to diagnosing and treating this potentially devastating complication.

Scaffold-based rhBMP-2 therapy in a rat alveolar defect model: implications for human gingivoperiosteoplasty.

Background: Primary alveolar cleft repair has a 41 to 73 percent success rate. Patients with persistent alveolar defects require secondary bone grafting. The authors investigated scaffold-based therapies designed to augment the success of alveolar repair. Methods: Critical-size, 7 × 4 × 3-mm alveolar defects were created surgically in 60 Sprague-Dawley rats. Four scaffold treatment arms were tested: absorbable collagen sponge, absorbable collagen sponge plus recombinant human bone morphogenetic protein-2 (rhBMP-2), hydroxyapatite–tricalcium phosphate, hydroxyapatite–tricalcium phosphate plus rhBMP-2, and no scaffold. New bone formation was assessed radiomorphometrically and histomorphometrically at 4, 8, and 12 weeks. Results: Radiomorphometrically, untreated animals formed 43 ± 6 percent, 53 ± 8 percent, and 48 ± 3 percent new bone at 4, 8, and 12 weeks, respectively. Animals treated with absorbable collagen sponge formed 50 ± 6 percent, 79 ± 9 percent, and 69 ± 7 percent new bone, respectively. Absorbable collagen sponge plus rhBMP-2–treated animals formed 49 ± 2 percent, 71 ± 6 percent, and 66 ± 7 percent new bone, respectively. Hydroxyapatite–tricalcium phosphate treatment stimulated 69 ± 12 percent, 86 ± 3 percent (p < 0.05), and 87 ± 14 percent new bone, respectively. Histomorphometry demonstrated an increase in bone formation in animals treated with hydroxyapatite–tricalcium phosphate plus rhBMP-2 (p < 0.05; 4 weeks) compared with empty scaffold. Conclusions: Radiomorphometrically, absorbable collagen sponge and hydroxyapatite–tricalcium phosphate scaffolds induced more bone formation than untreated controls. The rhBMP-2 added a small but significant histomorphometric osteogenic advantage to the hydroxyapatite–tricalcium phosphate scaffold.

Combination Therapy Accelerates Diabetic Wound Closure

Background Non-healing foot ulcers are the most common cause of non-traumatic amputation and hospitalization amongst diabetics in the developed world. Impaired wound neovascularization perpetuates a cycle of dysfunctional tissue repair and regeneration. Evidence implicates defective mobilization of marrow-derived progenitor cells (PCs) as a fundamental cause of impaired diabetic neovascularization. Currently, there are no FDA-approved therapies to address this defect. Here we report an endogenous PC strategy to improve diabetic wound neovascularization and closure through a combination therapy of AMD3100, which mobilizes marrow-derived PCs by competitively binding to the cell surface CXCR4 receptor, and PDGF-BB, which is a protein known to enhance cell growth, progenitor cell migration and angiogenesis. Methods and Results Wounded mice were assigned to 1 of 5 experimental arms (n = 8/arm): saline treated wild-type, saline treated diabetic, AMD3100 treated diabetic, PDGF-BB treated diabetic, and AMD3100/PDGF-BB treated diabetic. Circulating PC number and wound vascularity were analyzed for each group (n = 8/group). Cellular function was assessed in the presence of AMD3100. Using a validated preclinical model of type II diabetic wound healing, we show that AMD3100 therapy (10 mg/kg; i.p. daily) alone can rescue diabetes-specific defects in PC mobilization, but cannot restore normal wound neovascularization. Through further investigation, we demonstrate an acquired trafficking-defect within AMD3100-treated diabetic PCs that can be rescued by PDGF-BB (2 μg; topical) supplementation within the wound environment. Finally, we determine that combination therapy restores diabetic wound neovascularization and accelerates time to wound closure by 40%. Conclusions Combination AMD3100 and PDGF-BB therapy synergistically improves BM PC mobilization and trafficking, resulting in significantly improved diabetic wound closure and neovascularization. The success of this endogenous, cell-based strategy to improve diabetic wound healing using FDA-approved therapies is inherently translatable.