Faculty Bibliography

OBJECTIVE: Progenitor cells (PCs) contribute to postnatal neovascularization and tissue repair. Here, we explore the mechanism contributing to decreased diabetic circulating PC number and propose a novel treatment to restore circulating PC number, peripheral neovascularization, and tissue healing. RESEARCH DESIGN AND METHODS: Cutaneous wounds were created on wild-type (C57BL/J6) and diabetic (Lepr(db/db)) mice. Blood and bone marrow PCs were collected at multiple time points. RESULTS: Significantly delayed wound closure in diabetic animals was associated with diminished circulating PC number (1.9-fold increase vs. 7.6-fold increase in lin(-)/sca-1(+)/ckit(+) in wild-type mice; P < 0.01), despite adequate numbers of PCs in the bone marrow at baseline (14.4 +/- 3.2% lin(-)/ckit(+)/sca1(+) vs. 13.5 +/- 2.8% in wild-type). Normal bone marrow PC mobilization in response to peripheral wounding occurred after a necessary switch in bone marrow stromal cell-derived factor-1alpha (SDF-1alpha) expression (40% reduction, P < 0.01). In contrast, a failed switch mechanism in diabetic bone marrow SDF-1alpha expression (2.8% reduction) resulted in impaired PC mobilization. Restoring the bone marrow SDF-1alpha switch (54% reduction, P < 0.01) with plerixafor (Mozobil, formerly known as AMD3100) increased circulating diabetic PC numbers (6.8 +/- 2.0-fold increase in lin(-)/ckit(+), P < 0.05) and significantly improved diabetic wound closure compared with sham-treated controls (32.9 +/- 5.0% vs. 11.9 +/- 3% at day 7, P > 0.05; 73.0 +/- 6.4% vs. 36.5 +/- 7% at day 14, P < 0.05; and 88.0 +/- 5.7% vs. 66.7 +/- 5% at day 21, P > 0.05, respectively). CONCLUSIONS: Successful ischemia-induced bone marrow PC mobilization is mediated by a switch in bone marrow SDF-1alpha levels. In diabetes, this switch fails to occur. Plerixafor represents a potential therapeutic agent for improving ischemia-mediated pathology associated with diabetes by reducing bone marrow SDF-1alpha, restoring normal PC mobilization and tissue healing

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

BACKGROUND: Management of severe traumatic lower extremity injuries remains a considerable challenge. Free tissue transfer is now a standard part of reconstruction for Gustilo IIIB and IIIC injuries. There is limited information on arterial injury patterns in this population. We undertook a review of our experience to gain insight on vascular injury patterns and surgical outcomes. STUDY DESIGN: A 26-year retrospective analysis was performed of all lower extremity Gustilo IIIB and IIIC injuries requiring microvascular reconstruction at New York University Medical Center. Patient demographics, Gustilo classification, angiographic findings (conventional/computed tomographic angiography/magnetic resonance angiography), recipient vessels, elapsed time from injury, flap choices, and outcomes were examined. RESULTS: Two hundred twenty-two free flaps on 191 patients were performed from September 1982 until March 2008. There were 151 males and 40 females ranging in age from 4 to 83 years (median age 33 years). Patients sustained either Gustilo IIIB (170 patients) or IIIC (21 patients) open fractures. One hundred fifty-four patients had angiograms (78.2% IIIB, 100% IIIC). Sixty-six (42.9%) had normal 3-vessel runoff and 88 (57.1%) were abnormal. Sixty-one patients (31.9%) had anterior tibial injuries, 17 patients (8.9%) had posterior tibial injuries, and 30 (15.7%) had peroneal injuries. Sixty-three complications occurred (11 early thrombosis, 33 requiring secondary procedures, and 10 requiring amputation). CONCLUSIONS: Angiography of severe lower extremity injuries requiring free flap reconstruction usually revealed arterial injury and is generally indicated. In our experience, the anterior tibial artery is most commonly injured and the posterior tibial artery is most likely to be spared and used as a recipient

Purpose: Cutaneous radiation injury occurs during the treatment of cancer, or in rare environmental exposure. As the acute wound heals, fibrosis is induced and extracellular matrix (ECM) is deposited. The fibrotic pathway is mediated by the transforming growth factor-beta (TGF-beta) cascade, and is dependent on Smad3, a transcription factor for ECM. We characterized gene expression of this cascade after radiation injury and performed in vitro and in vivo gene silencing of Smad3 in an attempt to reverse the fibrotic pathway. Methods: Wild-type murine dermal fibroblasts were irradiated with 20Gy and harvested at serial time-points. RT-PCR was performed for known regulators and mediators of fibrosis. Smad3 was silenced by transfection with siRNA. For the in vivo experiment, dorsal skin of wild-type mice was irradiated with 45 Gy. Five weeks later, siRNA was applied to the fibrotic areas for one week. Skin was harvested and tissue analyzed by RT-PCR and Western blotting, as well as tissue tensiometry, which quantitatively measures rigidity. Results: Following irradiation, there was a steady increase in mRNA expression of Smad3, TGFbeta, and ECM genes collagen 1A1, metalloprotease2, and tissue inhibitor of metalloprotease-1, with peak expression at 12-24 hours. Inhibition of Smad3 with siRNA significantly decreased expression of Smad3, TGFbeta, and ECM genes. In the mouse model, topical treatment with siRNA again significantly decreased expression of these genes. Tensiometry demonstrated decreased stiffness in Smad3 siRNA treated skin, with a Young's modulus nearer to normalcompared to untreated and nonsense siRNA treated skin. Conclusion: Following initiation of the fibrotic pathway by radiation, Smad3 siRNA treatment both in vitro and in vivo effectively reversed gene expression. Furthermore, cutaneous Smad3 inhibition mitigated radiation-induced fibrotic stiffening. These findings suggest a therapeutic role for Smad3 silencing for cancer patients treated with radiation as well as those accidentally exposed to radiation

BACKGROUND/AIMS: Neovascularization involves angiogenesis and vasculogenesis mediated by cytokines and soluble chemokines. The predominant stimulus is ischemia, however, recent data suggest that ionizing radiation (IR) has angiogenic potential. In this study we evaluated whether IR increases vascularity and perfusion in vivo. METHODS: In wild-type mice, a full-thickness, pedicled skin flap was created and isolated for localized irradiation at a dose of 5 Gy. Serial Doppler analysis of the flap was performed. The skin flaps were then harvested at various time points for vascularity and histologic analysis. Blood was concurrently harvested for serum and hematopoietic progenitor cell population analysis. RESULTS: IR to an ischemic flap augmented the angiogenic cytokines SDF-1 and VEGF. Serum MMP-9 and s-kit levels, which are critical for progenitor cell mobilization, were also increased. When hematopoietic progenitor cells were evaluated by Sca1+/Flk1+ cells, a correlate 2-fold increase was seen compared to controls. When the flaps were examined, both vascularity and perfusion were increased. CONCLUSION: In this study we demonstrate that local, low-dose IR upregulates angiogenic chemokines and results in progenitor cell mobilization to the systemic circulation. There is a resultant increase in the vascularity of the irradiated flap, suggesting that the pro-angiogenic effects of IR can be harnessed locally

Blood vessel growth is regulated by angiogenic and angiostatic CXC chemokines, and radiation is a vasculogenic stimulus. We investigated the effect of radiation on endothelial cell chemokine signaling, receptor expression, and migration and apoptosis. Human umbilical vein endothelial cells were exposed to a single fraction of 0, 5, or 20Gy of ionizing radiation (IR). All vasculogenic chemokines (CXCL1-3/5-8) increased 3-13-fold after 5 or 20Gy IR. 20Gy induced a marked increase (1.6-4-fold) in angiostatic CXC chemokines. CXCR4 expression increased 3.5 and 7-fold at 48h after 5 and 20Gy, respectively. Bone marrow progenitor cell chemotaxis was augmented by conditioned media from cells treated with 5Gy IR. Whereas 5Gy markedly decreased intrinsic cell apoptosis (0Gy=16%+/-3.6 vs. 5Gy=4.5%+/-0.3), 20Gy increased it (21.4%+/-1.2); a reflection of pro-survival angiogenic chemokine expression. Radiation induces a dose-dependent increase in pro-angiogenic CXC chemokines and CXCR4. In contrast, angiostatic chemokines and apoptosis were induced at higher (20Gy) radiation doses. Cell migration improved significantly following 5Gy, but not 20Gy IR. Collectively, these data suggest that lower doses of IR induce an angiogenic cascade while higher doses produce an angiostatic profile

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 x 4 x 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.

OBJECTIVE: Determine long-term loss of mandible height with use of stress-shielding reconstruction plates for free fibula flap mandible reconstruction. DESIGN: Retrospective single-blinded medical record review. SUBJECTS: Seventy patients who had fibula free flap mandible reconstructions performed for 10 years. Patients who underwent radiotherapy were excluded. METHODS: Review of 70 fibula free flap mandible reconstructions performed for the last 10 years in a city hospital revealed 7 patients (10%) who had resections for benign odontogenic diseases. All had a three-dimensional cast model made, on which the reconstruction plate was bent to the desired shape preoperatively. Free fibula height on panoramic x-ray images taken preoperatively and at 2 and 12 months postoperatively. RESULTS: Seven (10%) patients met criteria for the study. Bone height was maintained at 2 months postoperatively, but at 12 months, there was a statistically significant loss of fibular bone height averaging 20% in the anterior, body, and ramus areas (P < 0.05). Despite this, all patients were considered eligible for dental rehabilitation, and 4 of 7 patients have had osseointegrated implants placed. CONCLUSIONS: As opposed to miniplates, increased resorption may have been due to the stress-shielding phenomenon unique to a reconstruction plates. However, this did not seem to affect the ability to place osseointegrated implants.