Surgical Trapping of a Large Complex Middle Cerebral Artery Aneurysm With Double-Barrel Bypass: 3-Dimensional Operative Video.

Large middle cerebral artery (MCA) bifurcation aneurysms are known vascular lesions that are usually symptomatic but often difficult to treat (whether with open or endovascular techniques), especially when the M2 branches originate from the aneurysm dome.1-7 The challenge lies in securing the aneurysm while fully maintaining the flow in the vessels arising from the dome. Standard microsurgical clipping or endovascular techniques are not feasible in perfectly treating these aneurysms. Revascularization of the MCA branches with bypass and trapping of the aneurysm is often necessary. Here, we present a case of a large complex partially thrombosed right MCA bifurcation aneurysm with both the superior and the inferior divisions arising from the dome. The patient initially presented with a right MCA stroke and left hemiparesis. Using radial artery as an interposition graft, 2 bypasses-internal maxillary artery to the inferior division and superficial temporal artery to the superior division-were performed. The aneurysm was trapped and decompressed by placing clips at the M1 terminus and the M2 origins. Intraoperative angiography and postoperative NOVA (VasSol Inc.) magnetic resonance angiography (MRA) confirmed patency and excellent flow in the bypass grafts. The patient's postoperative course was uncomplicated, and at 2-mo follow-up, had significant improvement of her hemiparesis. The patient provided informed consent for the procedure.

Minimally Invasive Approach for Resection of Masseteric Vascular Malformations.

OBJECTIVE: Vascular malformations (VMs) in the head and neck region often cause esthetic as well as functional problems for patients. Intramuscular VMs (IVM), such as those in the masseter, can cause severe facial asymmetry and typically are excised transcutaneously to facilitate wide exposure and safe dissection from the facial nerve. This requires extensive dissection, prolonged healing, and can lead to suboptimal facial scarring. METHODS: We describe the technique of resecting large IVMs of the masseter muscle in 3 patients using an entirely intraoral approach with continuous nerve monitoring and without visible facial scarring or secondary deformity. Preoperative injection of sclerotherapy was performed to reduce intra-operative bleeding and optimize resection. RESULTS: Successful excision was performed without complication in 3 patients to date. Total average operating room time was 120 minutes (range 95-145 minutes). Estimated blood loss was 213 mL (range 180-240 mL). The patients were discharged home either post-operative day (POD) 1 or 2, with 1 returning to work POD 4. Facial nerve function was normal postoperatively and no hematomas developed. Subjective masticatory function was equivalent to preoperative levels in all patients. CONCLUSIONS: Intraoral excision of VMs of the masseter muscle can be safely performed without added risk or complication. Continuous facial nerve monitoring allows minimally invasive approaches to be considered with less risk of iatrogenic facial nerve injury. We purport that this is a safe and effective method with substantially better esthetic outcomes compared with traditional transcutaneous approaches.

Improving Senescent Wound Healing With Local and Systemic Therapies.

The population is aging, and the prevalence of chronic wounds is increasing. Because neovascularization is essential for tissue repair and both local and systemic factors affect new blood vessel formation, we hypothesize that altering either pathway would reciprocally enhance wound healing in the aged. To test this hypothesis, p53 was locally suppressed and endothelial progenitor cells (EPCs) were systemically mobilized in a murine model of senescent wound healing.Bilateral 6-mm full-thickness stented wounds were made on the dorsum of Zmpste24 mice. Animals received weekly topical p53 small interfering RNA (siRNA) (n = 25), weekly topical nonsense siRNA (n = 25), daily subcutaneous AMD3100 injections (n = 25), or daily subcutaneous saline injections (n = 25). Wounds were photographically assessed and harvested for reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunostaining over 40 days. Circulating EPC levels were measured using fluorescence-activated cell sorting analysis.Local p53 siRNA significantly improved Zmpste24 wound healing (18 ± 2 vs 40 ± 3 days; P ≤ 0.0001). p53 siRNA significantly increased local provasculogenic factors (hypoxia-inducible factor 1 α, stromal cell-derived factor 1 α, and vascular endothelial growth factor; P ≤ 0.05) and decreased local proapoptotic factors (p53, PUMA, and Bax; P ≤ 0.05). Local p53 siRNA also significantly increased the number of circulating EPCs (8 ± 0.2% vs 2.6 ± 0.1%; P ≤ 0.0001). AMD3100 treatment also significantly improved wound healing (20 ± 2 vs 40 ± 3 days; P ≤ 0.0001) and increased EPCs mobilization (7.8 ± 0.4% vs 2.6 ± 0.1%; P ≤ 0.0001). In addition, systemic AMD3100 increased local provasculogenic factors (hypoxia-inducible factor 1 α, stromal cell-derived factor 1 α, and vascular endothelial growth factor; P ≤ 0.05) and decreased local proapoptotic factors (p53, PUMA, and Bax; P ≤ 0.05). Both treatments significantly increased the number of blood vessels in the wound bed (P ≤ 0.0001).The marked delay in Zmpste24 wound healing is significantly improved by local (p53 siRNA) and systemic (AMD3100) treatments. The resulting decrease in proapoptotic factors and increase in provasculogenic factors in the wound bed as well as the increased level of circulating EPCs appear to reverse age-related wound healing impairment by enhancing wound neovascularization.

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 $22,528 compared with the theoretical mean charge of $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.

Prospective Analysis of Payment per Hour in Head and Neck Reconstruction: Fiscally Feasible or Futile?

BACKGROUND: The authors assess the fiscal viability of complex head and neck reconstructive surgery by evaluating its financial reimbursement in the setting of resources used. METHODS: The authors prospectively assessed provider reimbursement for consecutive patients undergoing head and neck reconstruction. Total care time was determined by adding 15 minutes to the operative time for each postoperative hospital day and each postoperative follow-up appointment within the 90-day global period. Physician reimbursement was divided by total care time hours to determine an hourly rate of reimbursement. A control group of patients undergoing carpal tunnel release was evaluated using the same methods described. RESULTS: A total of 50 patients met the inclusion criteria for study. The payer was Medicaid for nine patients (18 percent), Medicare for 19 patients (38 percent), and commercial for 22 patients (44 percent). The average provider revenue per case was $3241.01 ± $2500.65. For all patients, the mean operative time was 10.6 ± 3.87 hours and the mean number of postoperative hospital days was 15.1 ± 8.06. The mean reimbursement per total care time hour was $254 ± $199.87. Statistical analysis demonstrated difference in reimbursement per total care time hour when grouped by insurance type (p = 0.002) or flap type (p = 0.033). Of the 50 most recent patients to undergo carpal tunnel release, the average revenue per case was $785.27. CONCLUSION: Total care time analysis demonstrates that physician reimbursement is not commensurate with resources used for complex head and neck reconstructive surgery.

Predictive Factors for Preoperative Percutaneous Endoscopic Gastrostomy Placement: Novel Screening Tools for Head and Neck Reconstruction.

OBJECTIVE: The treatment of head and neck cancer has varying impact on postoperative recovery and return of swallowing function. The authors aim to establish screening tools to assist in preoperatively determining the need for gastrostomy tube placement. METHODS: The authors prospectively assessed all patients undergoing complex head and neck reconstructive surgery during a 1-year study period. Only patients tolerating an oral diet, without preoperative gastrostomies, were enrolled for study. Eight parameters were assessed including: body mass index (BMI), prealbumin, albumin, smoking history, comorbidities [including coronary artery disease (CAD), chronic obstructive pulmonary disease (COPD), and diabetes mellitus (DM)], age, use of microvascular reconstruction, and type of defect. Two specific screening tools were assessed. In the first, a multivariate logistic regression model was employed to determine factor(s) that predict postoperative gastrostomy tube. In a second screening tool, the 8 parameters were scored between 0 to 1 points. The total score obtained for each patient was correlated with postoperative gastrostomy placement. RESULTS: Out of the 60 study patients enrolled in the study, 24 patients (40%) received a postoperative gastrostomy. In the logistic regression model, albumin level was the only factor that was significantly associated with need for postoperative gastrostomy (P < 0.0023). A score of 4 or greater was determined to have a sensitivity of 83% and specificity of 61% for postoperative gastrostomy. CONCLUSIONS: Patients with a score of 4 or more with this screening scoring system or those patients with an albumin level <3.5 g/dL were at high risk for postoperative feeding tube placement.

Acellular dermal matrix-based gene therapy augments graft incorporation.

BACKGROUND: Acellular dermal matrix (ADM) is widely used for structural or dermal replacement purposes. Given its innate biocompatibility and its potential to vascularize, we explored the possibility of ADM to function as a small interfering RNA (siRNA) delivery system. Specifically, we sought to improve ADM vascularization by siRNA-mediated inhibition of prolyl hydroxylase domain-2 (PHD2), a cytoplasmic protein that regulates hypoxia inducible factor-1α, and improve neovascularization. MATERIALS AND METHODS: Fluorescently labeled siRNA was used to rehydrate thin implantable ADM. Pharmacokinetic release of siRNA was determined. Twelve millimeter sections of ADM reconstituted with PHD2 siRNA (nonsense siRNA as control) and applied to dorsal wounds of 40 FVB mice. Grafts were sewn in, bolstered, and covered with occlusive dressings. Photographs were taken at 0, 7, and 14 d. Wounds were harvested at 7 and 14 d and analyzed (messenger RNA, protein, histology, and immunohistochemistry). RESULTS: Release kinetics was first-order with 80% release by 12 h. By day 14, PHD2-containing ADM appeared viable and adherent, whereas controls appeared nonviable and nonadherent. Real-time reverse transcription-polymerase chain reaction demonstrated near-complete knockdown of PHD2, whereas vascular endothelial growth factor and FGF-2 were increased 2.3- and 4.7-fold. On enzyme-linked immunosorbent assay, vascular endothelial growth factor was increased more than fourfold and stromal cell-derived factor doubled. Histology demonstrated improved graft incorporation in treated groups. Immunohistochemical demonstrated increased vascularity measured by CD31 staining and increased new cell proliferation by denser proliferating cell nuclear antigen staining in treated versus controls. CONCLUSIONS: We concluded that ADM is an effective matrix for local delivery of siRNA. Strategies to improve the matrix and/or genetically alter the local tissue environment can be envisioned.

Lacunocanalicular fluid flow transduces mechanical tension stress during distraction osteogenesis.

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.

Zmpste24-/- mouse model for senescent wound healing research.

BACKGROUND: The graying of our population has motivated the authors to better understand age-related impairments in wound healing. To increase research throughput, the authors hypothesized that the Hutchinson-Gilford progeria syndrome Zmpste24-deficient (Zmpste24(-/-)) mouse could serve as a model of senescent wound healing. METHODS: Using a stented excisional wound closure model, the authors tested this hypothesis on 8-week-old male Zmpste24(-/-) mice (n = 25) and age-matched male C57BL/6J wild-type mice (n = 25). Wounds were measured photogrammetrically and harvested for immunohistochemistry, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction, and circulating vasculogenic progenitor cells were measured by flow cytometry. RESULTS: Zmpste24(-/-) mice had a significant delay in wound closure compared with wild-type mice during the proliferative/vasculogenic phase. Zmpste24(-/-) wounds had decreased proliferation, increased 8-hydroxy-2'-deoxyguanosine levels, increased proapoptotic signaling (i.e., p53, PUMA, BAX), decreased antiapoptotic signaling (i.e., Bcl-2), and increased DNA fragmentation. These changes correlated with decreased local vasculogenic growth factor expression, decreased mobilization of bone marrow-derived vasculogenic progenitor cells, and decreased new blood vessel formation. Age-related impairments in wound closure are multifactorial. CONCLUSIONS: The authors' data suggest that the Hutchinson-Gilford progeria syndrome Zmpste24(-/-) progeroid syndrome shares mechanistic overlap with normal aging and therefore might provide a uniquely informative model with which to study age-associated impairments in wound closure.

Subcutaneous heparin does not increase postoperative complications in neurosurgical patients: An institutional experience.

INTRODUCTION: Prophylaxis for venous thromboembolic disease continues to pose a challenging management problem in postoperative neurosurgical patients, particularly those in the intensive care unit (ICU). This study evaluates neurosurgical patients admitted to the surgical ICU (SICU) at a tertiary hospital and compared those who had received subcutaneous unfractionated heparin (SQUFH) to those who did not. This study was conducted to better evaluate if the administration of SQUFH to neurosurgical patients is safe and whether the administration of SQUFH is an independent risk factor for bleeding in this patient population. METHODS: Retrospective analysis was performed on prospectively collected data on all postoperative neurosurgical patients admitted over the course of 11 years to the SICU at Long Island Jewish Medical Center. This study included neurosurgical patients who received SQUFH and those who did not. Data acquired included demographic information, hemodynamic monitoring, pharmacologic interventions, laboratory results, and survival outcomes as well as occurrences of heparin-induced thrombocytopenia and pulmonary embolism. Subcutaneous unfractionated heparin for venous thromboembolic prophylaxis were dosed according to previously established literature based hospital protocols. Data were analyzed by χ(2), Fisher exact test, Mann-Whitney U test, or the product limit method, where appropriate. RESULTS: Five hundred twenty-two neurosurgical patients were included in the study. Two hundred thirteen patients (40.8%) with mean age of 58 years received SQUFH (133 patients received SQUFH within 24 hours of surgery and 80 patients received SQUFH 24 hours postoperatively). Once SQUFH was initiated, it was continued until discharge from the hospital. Three hundred nine patients (59.2%) with mean age 57.8 years received no anticoagulation. In the SQUFH patient population, 72 patients (33.8%) had a diagnosis of subarachnoid hemorrhage compared with 125 patients (40.5%) from the group who had not received anticoagulation. There was no significant difference in ICU length of stay between the groups, 5.8 ± 5.4 (no deep vein thrombosis prophylaxis), and those receiving SQUFH, 6.7 ± 6.1 (over 24 hours) and 5.9 ± 4.8 (over 24 hours). No postoperative hemorrhages were observed (confirmed by computed tomography of the brain) in any of the neurosurgical patients with subarachnoid hemorrhage, intracerebral hemorrhage, or subdural or epidural hemorrhage. No instances of heparin-induced thrombocytopenia (HIT) or pulmonary embolism (PE) were observed. CONCLUSIONS: Administration of SQUFH dosed according to the risk for thromboembolism does not appear to contribute to postoperative hemorrhage in neurosurgical patients. This study supports the concept that the administration of SQUFH is safe in postoperative neurosurgical population.

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.

Bleeding risk secondary to deep vein thrombosis prophylaxis in patients with lower gastrointestinal bleeding.

INTRODUCTION: Deep vein thrombosis (DVT) and pulmonary embolism (PE) continue to pose a major burden on the health care system in the United States. The precise timing of anticoagulation initiation in critically ill patients with recent or active lower gastrointestinal bleeding (LGIB) is not well defined. We set out to study the safety and efficacy of early heparin administration for DVT prophylaxis in these patients. METHODS: A review of all patients admitted to the ICU with a diagnosis of LGIB over a 13-year period was performed. A total of 60 patients received subcutaneous heparin along with mechanical prophylaxis, whereas 59 patients had intermittent pneumatic compression devices alone. RESULTS: There was no difference in morbidity or mortality between the groups who received heparin and the nonheparin cohort. Neither of the groups developed a DVT or PE during the study period. Patients who received heparin had a median ICU LOS of 3 days, when compared with 2 days for patients who did not receive heparin (P < .0118). There was a significant association between units of blood received during the first 24 hours in the ICU and heparin usage (P < .0229). Those administered heparin received more units (median 3) than those who did not receive heparin (median 2). CONCLUSIONS: Administration of subcutaneous heparin increases the transfusion requirements and LOS in ICU patients with LGIB. After 24 hours, however, the blood transfusion requirements are equivalent. DVT prophylaxis in patients with a diagnosis of LGIB should be initiated after 24 hours of ICU admission.

The effect of low body mass index on outcome in critically ill surgical patients.

BACKGROUND: Body mass index (BMI) has been correlated with complications and outcome in surgical patients at the two extremes of the nutrition spectrum. OBJECTIVE: To study the relationship between BMI, outcome, hospital length of stay, and complications in patients admitted to the surgical intensive care unit (SICU). DESIGN: Review of prospectively acquired data in SICU patients. Data acquired included weight, height, age, gender, Acute Physiology and Chronic Health Evaluation (APACHE) II-III scores, Simplified Acute Physiology II (SAPS II) scores, and morbidity and mortality. Patients who stayed in the unit <24 hours were excluded. RESULTS: Of 793 patients, 706 had a normal BMI (NBMI; mean 22.12 kg/m²) and 87 were underweight (UBMI; mean 16.81 kg/m²). There was no statistically significant difference in APACHE II-III and SAPS scores. The NBMI group had more infections, and the UBMI group had more pulmonary complications (χ(2), P < .0087). There was no significant difference in acute respiratory distress syndrome, atrial fibrillation, myocardial infarction, septicemia, or ventilator- associated pneumonia (Fisher exact test, P = 0.38; χ(2), P = .41). The ICU length of stay between the 2 groups was not significantly different (6.7 vs 5.8 days; P = .64). Overall, there was 11.1% (88/793) SICU mortality; 74 of 706 (10.5%) patients expired in the NBMI group, and 14 of 87 (16.1%) patients expired in the UBMI group. CONCLUSIONS: Low BMI is associated with increased mortality in SICU patients. A BMI <18.5 kg/m² is an independent factor affecting outcome in surgical critical care patients.

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.

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.

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.