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

Exercise-induced acute compartment syndrome of the thigh is an uncommon entity. We present a rare case of bilateral exercise-induced three-compartment syndrome of the thighs that required fasciotomies. The objective of this study was to understand the history, physical examination, signs, symptoms, pathophysiology, diagnosis, and treatment of compartment syndrome and rhabdomyolysis. A 42-year-old man presented to the Emergency Department (ED) complaining of worsening pain and swelling in both thighs 45 h after performing a lower extremity exercise regimen. The patient's thighs were tender and swollen, but there was no ecchymosis or evidence of trauma. Admitting serum creatinine kinase (CK) was 106,289 U/L. Treatment for rhabdomyolysis was initiated. The next day, he complained of escalating bilateral thigh pain. Repeat serum CK was 346,580 U/L. The patient was diagnosed with bilateral thigh compartment syndrome and immediately taken to the operating room for fasciotomies. Postoperatively, the patient's symptoms improved rapidly and his serum CK quickly returned to normal. His incisions were closed and he returned to normal activities of daily living. Because exercise-induced compartment syndrome is an extremely rare diagnosis with a high risk of poor outcome, this article serves to emphasize the importance of considering this diagnosis during the work-up of patients presenting to the ED with rhabdomyolysis

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

Large temple and suprabrow lesions can pose a reconstructive challenge. When the lesion extends anterior to the hairline, esthetically acceptable local flaps may be difficult to design. We describe a modified scalp flap (ie, part Converse scalping flap and part scalp rotation flap) that can be tailored to reconstruct a variety of difficult temple and suprabrow lesions while simultaneously maintaining eyebrow position.The modified scalp flap is raised in a subgaleal plane until approximately 2.5 cm above the brow. At this level, dissection proceeds in the subcutaneous plane to protect the frontal branch of the facial nerve and to keep the flap thin. (The key to the modified scalp flap is the dissection plane change that protects the frontal branch of the facial nerve.) The extent of posterior subgaleal dissection is dictated by the amount of anterior rotation necessary. A temporal dog-ear is removed subfollicularly to permit modified flap rotation and preserve the superficial temporal artery.The modified scalp flap has been used to reconstruct temple and suprabrow lesions in 10 patients ranging in age from 4 months to 22 years. There were no complications. Four typical cases are presented.Temple and suprabrow lesions can be excised and successfully reconstructed in one stage using a modified scalp flap that is extended from the hair-bearing scalp onto the glabrous skin of the forehead. This novel modified scalp flap prevents eyebrow/hairline distortion and avoids facial nerve injury

Introduction: Bioengineering osseous tissue requires recapitulating the cellular, matrix, and lacunocanalicular components of bone. A construct must have a microvascular network which requires simultaneous co-culture of endothelial and osteogenic cells. Recreation of the matrix requires optimization of composition and microarchitecture. Engineering of constructs large enough to solve actual clinical problems requires novel strategies that address chemotransportative requirements by replicating lacunocanalicular flow. Methods: Cells: Adipose-derived mesenchymal stem cells (MSCs) were isolated and expanded from human lipoaspirate and differentiated into osteoprogenitor-rich (OPC) and endothelioprogenitor-rich (EPC), confirmed by RT-PCR. Normal human osteoblasts (NHOst) and human umbilical vein endothelial cells (HUVEC) served as terminally differentiated cell lines. The effects of coculture (e.g OPC + HUVEC, OPC + EPC etc) on capacity for bone formation was evaluated by von Kossa assay. Matrix: Murine alveolar defects were created. Scaffolds composed of either absorbable collagen sponge (ACS) or biphasic hydroxyapatite/tri-calcium phosphate (HA-TCP) in a 15/85 ratio were constructed and implanted. HA-TCP scaffolds were further investigated, comparing 15/85 and 60/40 HA/TCP in a rabbit calvarial model. Scaffold pore size (380/180 microns) and strut size (250/180 microns) were also investigated. New bone formation was analyzed histomorphometrically using micro-CT. Lacunocanalicular flow: We have developed a novel flow perfusion bioreactor designed to mimic lacunocanalicular flow. To validate, murine femurs were explanted to the bioreactor for 14 days. Viability and function were evaluated using thiazolyl blue tetrazolium bromide (MTT), DNA quantification, alkaline phosphatase (ALP) assay, and tetracycline labelling. Furthermore, optimal culture conditions were tested with MSC-seeded custom thick 3D HA-TCP scaffolds cultured in static conditions or in flow perfusion. Cellularity was assessed by SEM,!

We previously reported that topically applied calreticulin (CRT), a calcium-binding ER chaperone protein comprising N, P, and C domains, markedly enhances diabetic murine (db/db) and porcine cutaneous wound healing. Consistent with the potent wound healing effects, we further showed, in vitro, that exogenous CRT stimulated proliferation of keratinocytes and fibroblasts, induced concentration-dependent migration of these cells and monocytes and macrophages, and upregulated protein expression of collagen, fibronectin, and TGF-beta-3 in fibroblasts. Notably, all these broad-ranging effects purport novel non-ER functions for CRT that act from outside the cell inward. The current studies address: 1) whether the ER chaperone function of CRT is required for its extracellular functions, 2) the molecular structure(s) of CRT that function in its biological activities and 3) the in vitro effects of CRT on diabetic compared to normal mouse and human fibroblasts. Using CRT null mouse embryo fibroblasts (K42) compared to wild type (K41) in proliferation and migration assays (scratch plate and chamber), we show that exogenous CRT stimulates proliferation of null K42 cells to a similar extent as K41 cells (2-fold at 10 pg/ml). However, K42 cells require 100 times more CRT for a peak migratory response (1 vs 100 ng/ml), with a 20% decreased response. We also show that the C domain stimulates fibroblast proliferation to the same extent and peak response as the entire molecule. Finally, we show that fibroblasts isolated from db/db mouse skin and human fibroblasts cultured in high glucose, to simulate type II diabetes, respond to CRT by migration and proliferation albeit with 1/3 less robust response requiring 10-fold more CRT for peak responses compared to controls. The breath of novel non-ER functions of CRT, structure-function relationships, and effects on diabetic cells in vitro underscore this molecule as a potential potent agent for the topical treatment for healing diabetic wounds