Pierre B Saadeh, M.D.

TOPICAL SIRNA AND STEM CELL THERAPY REDUCE REACTIVE OXYGEN SPECIES AND ACCELERATE HEALING IN A SENESCENT WOUND
Butala, P.; Knobel, D.; Crawford, J. L.; Szpalski, C.; Marchac, A.; Sultan, S. M.; Wetterau, M. T.; Davidson, E. H.; Saadeh, P. B.; Warren, S. M.
2011 MAR-APR ;19(2):A16-A16, Wound repair & regeneration
— id: 129008, year: 2011, vol: 19, page: A16, stat: Journal Article,

Augmenting neovascularization accelerates distraction osteogenesis
Davidson, Edward H; Sultan, Steven M; Butala, Parag; Tutela, John Paul; Canizares, Orlando; Wagner, I Janelle; Knobel, Denis; Saadeh, Pierre B; Warren, Stephen M
2011 Aug;128(2):406-414, Plastic & reconstructive surgery
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
— id: 135583, year: 2011, vol: 128, page: 406, stat: Journal Article,

RADIATION-INDUCED SKIN FIBROSIS IS MEDIATED BY IL-13 AND MITIGATED BY LOCAL IL-13 GENE SUPPRESSION
Layliev, J.; Knobel, D.; Patel, M.; Sagebin, F.; Weinstein, A.; Cohen, O.; Wetterau, M.; Barr, J.; Henderson, R.; Warren, S.; Saadeh, P.
2011 MAR-APR ;19(2):A32-A32, Wound repair & regeneration
— id: 129009, year: 2011, vol: 19, page: A32, stat: Journal Article,

3D Volume Assessment Techniques and Computer-Aided Design and Manufacturing for Preoperative Fabrication of Implants in Head and Neck Reconstruction
Patel, Ashish; Otterburn, David; Saadeh, Pierre; Levine, Jamie; Hirsch, David L
2011 Nov;19(4):683-709, Facial plastic surgery clinics of North America
Cases in subdisciplines of craniomaxillofacial surgery-corrective jaw surgery, maxillofacial trauma, temporomandibular joint/skull base, jaw reconstruction, and postablative reconstruction-illustrate the ease of use, cost effectiveness, and superior results that can be achieved when using computer-assisted design and 3D volumetric analysis in preoperative surgical planning. This article discusses the materials and methods needed to plan cases, illustrates implementation of guides and implants, and describes postoperative analysis in relation to the virtually planned surgery
— id: 139041, year: 2011, vol: 19, page: 683, stat: Journal Article,

Fat grafting accelerates revascularisation and decreases fibrosis following thermal injury
Sultan SM; Barr JS; Butala P; Davidson EH; Weinstein AL; Knobel D; Saadeh PB; Warren SM; Coleman SR; Hazen A
2011 Feb;65(2):219-227, Journal of plastic, reconstructive & aesthetic surgery : JPRAS
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 degrees 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
— id: 138703, year: 2011, vol: 65, page: 219, stat: Journal Article,

Human Fat Grafting Alleviates Radiation Skin Damage in a Murine Model
Sultan SM; Stern CS; Allen RJ; Thanik VD; Chang CC; Nguyen PD; Canizares O; Szpalski C; Saadeh PB; Warren SM; Coleman SR; Hazen A
2011 Aug;128(2):363-372, Plastic & reconstructive surgery
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 depilitated. The dorsal skin was then distracted away from the body and radiated (45Gy) using a Varian 2300 Linear Accelerator. Four weeks following radiation, 1.5cc fat or sham grafts were placed in the dorsal subcutaneous space. Gross results were analyzed photometrically. The animals were sacrificed at 4 and 8 weeks following fat or sham grafting and their dorsal skin was processed for histologic analysis. Inflammation was assessed by epidermal thickness measurements on H&E stained sections. Vascular density was assessed using CD31 staining. Fibrosis was assessed using Smad-3 and Picrosirius Red staining. RESULTS: Hyperpigmentation and ulceration were grossly improved in fat-grafted mice compared to sham-grafted controls. Epidermal thickness measurements demonstrated decreased thickness in fat-grafted animals at both timepoints (20.6+/-1.5mum vs 55.2+/-5.6mum, p=0.004; 17.6+/-1.1mum vs 36.3+/-6.1mum, p=0.039). Vascular density was decreased in fat-grafted mice compared to sham-grafted at both timepoints (17.5+/-1.3 vessels/hpf vs 29+/-3.5, p=0.055; 13.25+/-1.4 vs 17.0+/-1.6, p=0.003). Intensity of Smad3 staining was significantly decreased in fat-grafted animals at both timepoints (2.77+/-0.3% vs 4.98+/-0.9%, p=0.004; 3.05+/-0.2% vs 5.81+/-0.3%, p=0.011). Picrosirius red staining demonstrated a diminished scar-index in fat-treated animals at both timepoints (.54+/-0.05 vs .74+/-0.07, p=0.034; .55+/-0.06 vs .93+/-.07, p=0.001). CONCLUSIONS: Fat grafting attenuates inflammation in acute radiodermatitis and slows the progression of fibrosis in chronic radiodermatitis
— id: 134340, year: 2011, vol: 128, page: 363, stat: Journal Article,

Interval cranioplasty: comparison of current standards
Sultan, Steven M; Davidson, Edward H; Butala, Parag; Schachar, Jeffrey S; Witek, Lukasz; Szpalski, Caroline; Ricci, Jack L; Saadeh, Pierre B; Warren, Stephen M
2011 May;127(5):1855-1864, Plastic & reconstructive surgery
BACKGROUND: : Although different cranioplasty storage methods are currently in use, no study has prospectively compared these methods. The authors compare freezing and subcutaneous storage methods in a rat model. METHODS: : Trephine defects (10 mm) were created in 45 Sprague-Dawley rats. The cranial bone grafts were stored in an autologous subcutaneous pocket (n = 15), frozen at -80 degrees C (n = 15), immediately analyzed (n = 12), or immediately replanted into the defect (n = 3). After 10 days of storage, the subcutaneous or frozen grafts were either replanted (subcutaneous, n = 3; frozen, n = 3) or analyzed (subcutaneous, n = 12; frozen, n = 12). Grafts underwent histologic analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, alkaline phosphatase assay, mechanical testing, and micro-computed tomographic imaging. RESULTS: : After 10 days of storage, physiologic assays demonstrated a significant decrease in cellular functionality (e.g., alkaline phosphatase assay concentration: fresh, 18.8 +/- 0.77 mM/mg; subcutaneous, 12.2 +/- 0.63 mM/mg; frozen, 8.07 +/- 1.1 mM/mg; p < 0.012 for all comparisons). Mechanical integrity (maximal load) of fresh grafts was greatest (fresh, 9.26 +/- 0.29 N; subcutaneous, 6.27 +/- 0.64 N; frozen, 4.65 +/- 0.29 N; fresh compared with frozen, p < 0.001; fresh compared with subcutaneous, p = 0.006). Replantation of subcutaneously stored and frozen grafts resulted in limited bony union and considerable resorption after 12 weeks; in contrast, replanted fresh grafts demonstrated bony union and little resorption. CONCLUSIONS: : Current preservation methods for interval cranioplasty do not maintain bone graft viability. Subcutaneous storage appears to provide a small advantage compared with freezing
— id: 131968, year: 2011, vol: 127, page: 1855, stat: Journal Article,

OBESITY IMPAIRS BLOOD VESSEL FORMATION
Szpalski, C.; Wetterau, M.; Cohen, O.; Patel, M.; Layliev, J.; Saadeh, P. B.; Warren, S. M.
2011 MAR-APR ;19(2):A56-A56, Wound repair & regeneration
— id: 129011, year: 2011, vol: 19, page: A56, stat: Journal Article,

Use of Virtual 3-Dimensional Surgery in Post-Traumatic Craniomaxillofacial Reconstruction
Tepper OM; Sorice S; Hershman GN; Saadeh P; Levine JP; Hirsch D
2011 Mar;69(3):733-741, Journal of oral & maxillofacial surgery
Traumatic craniofacial injuries often present as difficult reconstructive challenges for maxillofacial surgeons. Reconstruction is often complicated by significant soft tissue loss, comminuted bony fragments, a tenuous blood supply, and wound contamination. For panfacial injuries, restoration of normal facial width, facial height, and sagittal projection may be difficult to achieve. Marked swelling may limit the surgeons' ability to palpate and recognize subtle bony defects and malunion. Furthermore, a true 3-dimensional assessment of bony alignment may not be possible with traditional surgical exposures to the craniofacial skeleton. This article builds on previous work that introduced the use of 3-dimensionally guided surgery for microvascular free-flap reconstruction of the craniofacial skeleton. Use of this technology improves the planning, timing, and overall precision of microvascular reconstructive surgery. Based on this experience, a similar approach to reconstructing patients with significant craniofacial trauma has been adopted
— id: 121304, year: 2011, vol: 69, page: 733, stat: Journal Article,

Progenitor cell mobilization enhances bone healing by means of improved neovascularization and osteogenesis
Wang, Xiao Xia; Allen, Robert J Jr; Tutela, John Paul; Sailon, Alexander; Allori, Alexander C; Davidson, Edward H; Paek, Gina K; Saadeh, Pierre B; McCarthy, Joseph G; Warren, Stephen M
2011 Aug;128(2):395-405, Plastic & reconstructive surgery
BACKGROUND: : Although bone repair is a relatively efficient process, a significant portion of patients fail to heal their fractures. Because adequate blood supply is essential to osteogenesis, the authors hypothesize that augmenting neovascularization by increasing the number of circulating progenitor cells will improve bony healing. METHODS: : Bilateral full-thickness defects were created in the parietal bones of C57 wild-type mice. Intraperitoneal AMD3100 (n = 33) or sterile saline (n = 33) was administered daily beginning on postoperative day 3 and continuing through day 18. Circulating progenitor cell number was quantified by fluorescence-activated cell sorting. Bone regeneration was assessed with micro-computed tomography. Immunofluorescent CD31 and osteocalcin staining was performed to assess for vascularity and osteoblast density. RESULTS: : AMD3100 treatment increased circulating progenitor cell levels and significantly improved bone regeneration. Calvarial defects of AMD3100-treated mice demonstrated increased vascularity and osteoblast density. CONCLUSIONS: : Improved bone regeneration in this model was associated with elevated circulating progenitor cell number and subsequently improved neovascularization and osteogenesis. These findings highlight the importance of circulating progenitor cells in bone healing and may provide a novel therapy for bone regeneration
— id: 135582, year: 2011, vol: 128, page: 395, stat: Journal Article,

Social networking services: Implications for the next generation of physicians
Weinstein, Andrew L.; Saadeh, Pierre B.; Warren, Stephen M.
2011 JUL ;150(1):15-16, Surgery
— id: 135204, year: 2011, vol: 150, page: 15, stat: Journal Article,

Topical prolyl hydroxylase domain-2 silencing improves diabetic murine wound closure
Wetterau M; George F; Weinstein A; Nguyen PD; Tutela JP; Knobel D; Cohen Ba O; Warren SM; Saadeh PB
2011 Jul;19(4):481-486 L, Wound repair & regeneration
Prolyl hydroxylase domain 2 (PHD2) has been implicated in several pathways of cell signaling, most notably in its regulation of hypoxia-inducible factor (HIF)-1alpha stability. In normoxia, PHD2 hydroxylates proline residues on HIF-1alpha, rendering it inactive. However, in hypoxia, PHD2 is inactive, HIF-1alpha 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-1alpha. 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-1alpha. 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-1alpha 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
— id: 134339, year: 2011, vol: 19, page: 481, stat: Journal Article,

TOPICAL SMAD3 SILENCING IMPROVES HEALING IN A NOVEL IRRADIATED WOUND MODEL
Wetterau, M. T.; Szpalski, C.; Knobel, D.; Albano, N.; Cohen, O.; Patel, M.; Layliev, J.; Warren, S. M.; Levine, J. P.; Saadeh, P. B.
2011 MAR-APR ;19(2):A59-A59, Wound repair & regeneration
— id: 129012, year: 2011, vol: 19, page: A59, stat: Journal Article,

Improved fat graft survival with mobilization of progenitor cells
Butala, Parag; Sultan, Steven M.; Davidson, Edward H.; Crawford, James L.; Szpaiski, Caroline; Knobel, Denis; Saadeh, Pierre B.; Warren, Stephen M.; Coleman, Sydney; Hazen, Alexes
2010 SEP ;211(3):S94-S95, Journal of the American College of Surgeons
— id: 113916, year: 2010, vol: 211, page: S94, stat: Journal Article,

Modeling senescent wound healing with the Zmpste24 transgenic mouse
Butala, Parag; Szpalski, Caroline; Knobel, Denis; Crawford, James L.; Marchac, Alexandre; Davidson, Edward H.; Sultan, Steven M.; Wetterau, Meredith; Saadeh, Pierre B.; Warren, Stephen M.
2010 SEP ;211(3):S78-S79, Journal of the American College of Surgeons
— id: 113915, year: 2010, vol: 211, page: S78, stat: Journal Article,

Treatment of metachronous lower extremity defects with delay and splitting of a previously advanced reverse sural artery flap
Capla, Jennifer M; Michaels, Joseph 4th; Ceradini, Daniel J; Levine, Jamie P; Saadeh, Pierre B
2010 Dec;126(6):322e-323e, Plastic & reconstructive surgery
— id: 114865, year: 2010, vol: 126, page: 322e, stat: Journal Article,

Lower extremity arterial injury patterns and reconstructive outcomes in patients with severe lower extremity trauma: a 26-year review
Haddock, Nicholas T; Weichman, Katie E; Reformat, Derek D; Kligman, Brad E; Levine, Jamie P; Saadeh, Pierre B
2010 Jan;210(1):66-72, Journal of the American College of Surgeons
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
— id: 107272, year: 2010, vol: 210, page: 66, stat: Journal Article,

Perforator vessel recipient options in the lower extremity: an anatomically based approach to safer limb salvage
Haddock, Nicholas; Garfein, Evan S; Reformat, Derek; Hecht, Elizabeth; Levine, Jamie; Saadeh, Pierre
2010 Sep;26(7):461-469, Journal of reconstructive microsurgery
When free tissue transfer is employed for defects of the lower third of the leg, recipient anastomoses are typically performed to major vessels. The aim of this study was to assess soleal perforators located in the distal half of the leg as potential vessels for free flap recipient vessels. Six fresh cadavers (12 limbs) were dissected. Perforators of adequate size (>or=1 mm) were documented as was the location and ease of dissection. Lower extremity magnetic resonance angiograms (MRAs) of 18 extremities were retrospectively reviewed. Two free tissue transfers to lower extremity perforators were presented. Soleal perforators most reliably matched our recipient vessel requirements. Perforators were of adequate size to support free tissue transfer, easy to dissect, and were located at mid/distal fibula level. MRA evaluation confirmed these results. One free tissue reconstruction was performed for trauma (posterior tibial perforator) and one was performed for a chronic radiation wound (peroneal perforator). The soleus muscle is easily exposed and is supplied distally by perforators from both the posterior tibial and the peroneal artery systems. These perforating branches are more accessible than the major lower extremity arteries, making the exposure and anastomosis technically easier and sparing potential iatrogenic injury to critical vessels
— id: 111961, year: 2010, vol: 26, page: 461, stat: Journal Article,

Inhibition of Smad3 expression in radiation-induced fibrosis using a novel method for topical transcutaneous gene therapy
Lee, Judy W; Tutela, John P; Zoumalan, Richard A; Thanik, Vishal D; Nguyen, Phuong D; Varjabedian, Leon; Warren, Stephen M; Saadeh, Pierre B
2010 Jul;136(7):714-719, Archives of otolaryngology, head & neck surgery
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
— id: 111363, year: 2010, vol: 136, page: 714, stat: Journal Article,

Regulators and mediators of radiation-induced fibrosis: Gene expression profiles and a rationale for Smad3 inhibition
Lee, Judy W; Zoumalan, Richard A; Valenzuela, Cristian D; Nguyen, Phuong D; Tutela, John P; Roman, Benjamin R; Warren, Stephen M; Saadeh, Pierre B
2010 Oct;143(4):525-530, Otolaryngology, head & neck surgery
OBJECTIVE: Radiotherapy, an essential modality in cancer treatment, frequently induces fibrotic processes in the skin, including accumulation of extracellular matrix. Transforming growth factor-beta is essential in regulating extracellular matrix gene expression and is dependent on Smad3, an intracellular mediator/transcription factor. Our study characterized the genetic expression involved in extracellular matrix accumulation during radiation-induced fibrosis. We performed Smad3 gene silencing in an attempt to abrogate the effects of radiation. STUDY DESIGN: Laboratory research. SETTING: University laboratory. SUBJECTS AND METHODS: C57 murine dermal fibroblasts were irradiated with 20 Gy RNA isolated (0, 6, 12, 24, 48, 72 hours postirradiation) and mRNA analyzed (reverse transcriptase polymerase chain reaction) for known regulators (Smad3, interleukin-13 [IL-13]), tumor necrosis factor-alpha [TNF-alpha]) and mediators of fibrosis (collagen 1A1 [Col1A1]), TGF-beta, matrix metalloprotease-1 and -2 (MMP-1, MMP-2), and tissue inhibitor of metalloprotease-1 (TIMP-1). Smad3 gene expression was silenced using siRNA in an effort to restore an unirradiated gene profile. RESULTS: Following irradiation, there was a steady increase in mRNA expression of Smad3, IL-13, TGF-beta, Col1A1, MMP-2, TIMP-1, with peak at 12 to 24 hours and subsequent decline by 72 hours. TNF-alpha expression remained elevated throughout. MMP-1 showed minimal expression initially, which decreased to negligible by 72 hours. Inhibition of Smad3 significantly decreased expression of Col1A1, TGF-beta, MMP-2, and TIMP-1. IL-13 and TNF-alpha expression was not affected by Smad3 silencing. CONCLUSION: We have characterized the early-phase mRNA expression profiles of the major mediators of radiation-induced fibrosis. Smad3 siRNA effectively abrogated the elevation of Col1A1, TGF-beta, TIMP-1, and MMP-2. IL-13 and TNF-alpha were unaffected by Smad3 silencing and appear to be minor regulators in fibrosis. These findings suggest a therapeutic rationale for Smad3 silencing in vivo
— id: 113665, year: 2010, vol: 143, page: 525, stat: Journal Article,

Low-dose radiation augments vasculogenesis signaling through HIF-1-dependent and -independent SDF-1 induction
Lerman, Oren Z; Greives, Matthew R; Singh, Sunil P; Thanik, Vishal D; Chang, Christopher C; Seiser, Natalie; Brown, Daniel J; Knobel, Denis; Schneider, Robert J; Formenti, Silvia C; Saadeh, Pierre B; Levine, Jamie P
2010 Nov 4;116(18):3669-3676, Blood
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-1alpha (HIF-1alpha) up-regulation in endothelial cells (ECs), a HIF-1alpha-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-1alpha and SDF-1 expression was greater when combined with hypoxia suggesting an additive effect. While small interfering RNA silencing of HIF-1alpha mRNA and abolition of HIF-1alpha 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-1alpha induction. These observations might be exploited for stimulation of wound healing or controlling tumor angiogenesis
— id: 138185, year: 2010, vol: 116, page: 3669, stat: Journal Article,

Improved diabetic wound healing through topical silencing of p53 is associated with augmented vasculogenic mediators
Nguyen, Phuong D; Tutela, John Paul; Thanik, Vishal D; Knobel, Denis; Allen, Robert J Jr; Chang, Christopher C; Levine, Jamie P; Warren, Stephen M; Saadeh, Pierre B
2010 Nov-Dec;18(6):553-559, Wound repair & regeneration
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
— id: 138168, year: 2010, vol: 18, page: 553, stat: Journal Article,

Radiation-induced fibrosis isrescued by sirna blockade of SMAD3
Roman B.R.; Lee J.W.; Zoumalan R.A.; Tutella J.P.; Paek G.K.; Immerman S.; Knobel D.; Wetterau M.; Crawford J.; Warren S.M.; Saadeh P.B.
2010 ;18(2):A20-A20, Wound repair & regeneration
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
— id: 135598, year: 2010, vol: 18, page: A20, stat: Journal Article,

Cranial bone defects: current and future strategies
Szpalski, Caroline; Barr, Jason; Wetterau, Meredith; Saadeh, Pierre B; Warren, Stephen M
2010 Dec;29(6):E8-E8, Neurosurgical focus
Bony defects in the craniomaxillofacial skeleton remain a major and challenging health concern. Surgeons have been trying for centuries to restore functionality and aesthetic appearance using autografts, allografts, and even xenografts without entirely satisfactory results. As a result, physicians, scientists, and engineers have been trying for the past few decades to develop new techniques to improve bone growth and bone healing. In this review, the authors summarize the advantages and limitations of current animal models; describe current materials used as scaffolds, cell-based, and protein-based therapies; and lastly highlight areas for future investigation. The purpose of this review is to highlight the major scaffold-, cell-, and protein-based preclinical tools that are currently being developed to repair cranial defects
— id: 114859, year: 2010, vol: 29, page: E8, stat: Journal Article,

Decreased circulating progenitor cell number and failed mechanisms of stromal cell-derived factor-1alpha mediated bone marrow mobilization impair diabetic tissue repair
Tepper, Oren M; Carr, Jacquelyn; Allen, Robert J Jr; Chang, Christopher C; Lin, Clarence D; Tanaka, Rica; Gupta, Sanjeev M; Levine, Jamie P; Saadeh, Pierre B; Warren, Stephen M
2010 Aug;59(8):1974-1983, Diabetes
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
— id: 111581, year: 2010, vol: 59, page: 1974, stat: Journal Article,

Cutaneous low-dose radiation increases tissue vascularity through upregulation of angiogenic and vasculogenic pathways
Thanik, Vishal D; Chang, Christopher C; Lerman, Oren Z; Greives, Matthew R; Le, Huong; Warren, Stephen M; Schneider, Robert J; Formenti, Sylvia C; Saadeh, Pierre B; Levine, Jamie P
2010 ;47(6):472-480, Journal of vascular research
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
— id: 113939, year: 2010, vol: 47, page: 472, stat: Journal Article,

DIABETIC WOUND HEALING RESULTS FROM IMPAIRED NEOVASCULARIZATION
Allen, RJ; Nguyen, PD; Canizares, O; Wagner, J; Levine, JP; Saadeh, PB; Warren, SM
2009 MAR-APR ;17(2):A23-A23, Wound repair & regeneration
— id: 97663, year: 2009, vol: 17, page: A23, stat: Journal Article,

FAT GRAFTING FOR THE TREATMENT OF MURINE RADIATION SKIN DAMAGE
Allen, RJ; Nguyen, PD; Varjabedian, L; Schachar, JS; Thanik, VD; Saadeh, PB; Coleman, SR; Hazen, A
2009 MAR-APR ;17(2):A18-A18, Wound repair & regeneration
— id: 97661, year: 2009, vol: 17, page: A18, stat: Journal Article,

Functional analysis of simultaneous dual-differentiation vs multilineage cell coculture for vascularized bone engineering
Allori, AC; Reformat, DD; Davidson, EH; Allen, RJ; Sailon, AM; Valenzuela, CD; Saadeh, PB; Levine, JP; Ricci, JL; Warren, SM
2009 SEP ;209(3):S90-S91, Journal of the American College of Surgeons
— id: 102459, year: 2009, vol: 209, page: S90, stat: Journal Article,

Dose-dependent effect of radiation on angiogenic and angiostatic CXC chemokine expression in human endothelial cells
Chang, Christopher C; Lerman, Oren Z; Thanik, Vishal D; Scharf, Carrie L; Greives, Matthew R; Schneider, Robert J; Formenti, Sylvia C; Saadeh, Pierre B; Warren, Stephen M; Levine, Jamie P
2009 Dec;48(3):295-302, Cytokine
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
— id: 104228, year: 2009, vol: 48, page: 295, stat: Journal Article,

The lower-extremity allen test
Haddock, Nicholas T; Garfein, Evan S; Saadeh, Pierre B; Levine, Jamie P
2009 Sep;25(7):399-403, Journal of reconstructive microsurgery
The Allen test is used to diagnose the relative contribution of the ulnar and radial arteries to each hand. We modified this test to investigate the relative vascular contributions to distal perfusion of the lower extremity. With the patient supine, a handheld Doppler is used to locate the first dorsal metatarsal artery. The posterior tibial artery (PT) and dorsalis pedis artery (DP) pulses are compressed. A persistent signal indicates collateral flow through the peroneal artery (PA). Sequential decompression is then used to evaluate the relative contribution of the PT and DP to distal circulation. We report a case in which angiography failed to predict reliance on the PT. In this case, performance of the lower-extremity Allen test (LEAT) led to an alternative recipient vessel choice. The LEAT is simple to perform and provides a valuable adjunct to angiographic data
— id: 103148, year: 2009, vol: 25, page: 399, stat: Journal Article,

The tear trough and lid/cheek junction: anatomy and implications for surgical correction
Haddock, Nicholas T; Saadeh, Pierre B; Boutros, Sean; Thorne, Charles H
2009 Apr;123(4):1332-1340, Plastic & reconstructive surgery
BACKGROUND: The tear trough and the lid/cheek junction become more visible with age. These landmarks are adjacent, forming in some patients a continuous indentation or groove below the infraorbital rim. Numerous, often conflicting procedures have been described to improve the appearance of the region. The purpose of this study was to evaluate the anatomy underlying the tear trough and the lid/cheek junction and to evaluate the procedures designed to correct them. METHODS: Twelve fresh cadaver lower lid and midface dissections were performed (six heads). The orbital regions were dissected in layers, and medical photography was performed. RESULTS: In the subcutaneous plane, the tear trough and lid/cheek junction overlie the junction of the palpebral and orbital portions of the orbicularis oculi muscle and the cephalic border of the malar fat pad. In the submuscular plane, these landmarks differ. Along the tear trough, the orbicularis muscle is attached directly to the bone. Along the lid/cheek junction, the attachment is ligamentous by means of the orbicularis retaining ligament. CONCLUSIONS: The tear trough and lid/cheek junction are primarily explained by superficial (subcutaneous) anatomical features. Atrophy of skin and fat is the most likely explanation for age-related visibility of these landmarks. 'Descent' of this region with age is unlikely (the structures are fixed to bone). Bulging orbital fat accentuates these landmarks. Interventions must extend significantly below the infraorbital rim. Fat or synthetic filler may be best placed in the intraorbicularis plane (tear trough) and in the suborbicularis plane (lid/cheek junction)
— id: 98782, year: 2009, vol: 123, page: 1332, stat: Journal Article,

Intracranial Microvascular Free Flaps
Levine, Steven; Garfein, Evan S; Weiner, Howard; Yaremchuk, Michael J; Saadeh, Pierre B; Gurtner, Geoffrey; Levine, Jamie P; Warren, Stephen M
2009 Feb;25(2):89-95, Journal of reconstructive microsurgery
Large acquired intracranial defects can result from trauma or surgery. When reoperation is required because of infection or tumor recurrence, management of the intracranial dead space can be challenging. By providing well-vascularized bulky tissue, intracranial microvascular free flaps offer potential solutions to these life-threatening complications. A multi-institutional retrospective chart and radiographic review was performed of all patients who underwent microvascular free-flap surgery for salvage treatment of postoperative intracranial infections between 1998 and 2006. A total of six patients were identified with large intracranial defects and postoperative intracranial infections. Four patients had parenchymal resections for tumor or seizure and two patients had posttraumatic encephalomalacia. All patients underwent operative debridement and intracranial free-flap reconstruction using the latissimus dorsi muscle ( N = 2), rectus abdominis muscle ( N = 2), or omentum ( N = 2). All patients had titanium ( N = 4) or Medpor ( N = 2) cranioplasties. We concluded that surgery or trauma can result in significant intracranial dead space. Treatment of postoperative intracranial infection can be challenging. Vascularized free tissue transfer not only fills the void, but also provides a delivery system for immune cells, antibodies, and systemically administered antibiotics. The early use of this technique when intracranial dead space and infection coexist is beneficial
— id: 90063, year: 2009, vol: 25, page: 89, stat: Journal Article,

MEDIATORS OF INCREASED APOPTOSIS IN STRESSED DIABETIC FIBROBLAS
Nguyen, PD; Allen, RJ; Tutela, JP; Thanik, VD; Haberman, ID; Valenzuela, C; Lee, JW; Levine, JP; Warren, SM; Saadeh, PB
2009 MAR-APR ;17(2):A10-A10, Wound repair & regeneration
— id: 97659, year: 2009, vol: 17, page: A10, stat: Journal Article,

Mechanisms of improved diabetic wound healing achieved with topical silencing of p53
Nguyen, PD; Tutela, JP; Thanik, VD; Allen, RJ; Cohen, OD; Wagner, IJ; Levine, JP; Warren, SM; Saadeh, PB
2009 SEP ;209(3):S73-S73, Journal of the American College of Surgeons
— id: 102458, year: 2009, vol: 209, page: S73, stat: Journal Article,

Establishment of a critical-sized alveolar defect in the rat: a model for human gingivoperiosteoplasty
Nguyen, Phuong D; Lin, Clarence D; Allori, Alexander C; Ricci, John L; Saadeh, Pierre B; Warren, Stephen M
2009 Mar;123(3):817-825, Plastic & reconstructive surgery
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 x 4 x 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
— id: 98780, year: 2009, vol: 123, page: 817, stat: Journal Article,

Scaffold-Based rhBMP-2 Therapy in a Rat Alveolar Defect Model: Implications forHuman Gingivoperiosteoplasty
Nguyen, Phuong D; Lin, Clarence D; Allori, Alexander C; Schachar, Jeffrey S; Ricci, John L; Saadeh, Pierre B; Warren, Stephen M
2009 Dec;124(6):1829-1839, Plastic & reconstructive surgery
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
— id: 105524, year: 2009, vol: 124, page: 1829, stat: Journal Article,

Improved bony healing with AMD3100 via neovascularization and osteogenesis
Paek, GK; Wang, XX; Allen, RJ; Nguyen, PD; Davidson, EH; Tutela, JP; Sailon, A; Saadeh, PB; Warren, SM
2009 SEP ;209(3):S62-S63, Journal of the American College of Surgeons
— id: 102455, year: 2009, vol: 209, page: S62, stat: Journal Article,

The Proximally Based Peroneal Vascular Bundle An Insulated Extension Cord for Free Flop Reconstruction
Sailon, AM; Reformat, DD; Hecht, EM; Garfein, ES; Spector, JA; Levine, JP; Saadeh, PB
2009 MAY ;62(5):556-559, Annals of plastic surgery
Large traumatic wounds around the proximal third of the lower extremity may have disrupted local vasculature, potentially obviating local pedicled options,. However, free-tissue transfer to this area is technically challenging given the resulting paucity of recipient options and the depth of principal blood vessels. We present an anatomic and radiographic study of the proximally based peroneal vascular bundle as a recipient option In the proximal leg. Optimal approach was prone, through an incision over the fibula with dissection between lateral and posterior compartments. Magnetic resonance angiography demonstrated consistent vascular anatomy between patients. A proximally based peroneal vascular bundle protected by a cuff of flexor hallucis longus was used as a recipient vessel in free flat) reconstruction of an open knee wound. The bundle itself does not require coverage by virtue of its own local muscle Cliff. Caveats for its Use include file need for adequate leg inflow and foot outflow
— id: 98848, year: 2009, vol: 62, page: 556, stat: Journal Article,

A murine model for studying diffusely injected human fat
Thanik, Vishal D; Chang, Christopher C; Lerman, Oren Z; Allen, Robert J Jr; Nguyen, Phuong D; Saadeh, Pierre B; Warren, Stephen M; Levine, Jamie P; Coleman, Sydney R; Hazen, Alexes
2009 Jul;124(1):74-81, Plastic & reconstructive surgery
BACKGROUND: The study of human autologous fat grafting has been primarily anecdotal. In this study, the authors aim to develop a murine model that recapitulates human fat grafting to study the fate of injected fat and the cell populations contained within. METHODS: The authors' method of fat harvesting and refinement has been described previously. The authors injected nude and tie2/lacZ mice with 2 ml of human lipoaspirate placed on the dorsal surface in a multipass, fan-like pattern. Fatty tissue was injected in small volumes of approximately 1/30 ml per withdrawal. The dorsal skin and associated fat was excised at various time points. Sections were stained with hematoxylin and eosin and cytochrome c oxidase IV. Transgenic tie2/lacZ samples were stained with X-galactosidase. At the 8-week time point, volumetric analysis was performed. RESULTS: Volumetric analysis at the 8-week time point showed 82 percent persistence of the original volume. Gross analysis showed it to be healthy, nonfibrotic, and vascularized. Hematoxylin and eosin analysis showed minimal inflammatory or capsular reaction, with viable adipocytes. Fat grafted areas were vascularized with multiple blood vessels. Cytochrome c oxidase IV human-specific stain and beta-galactosidase expression revealed these vessels to be of human origin. CONCLUSIONS: The authors have developed a murine model with which to study the fate of injected lipoaspirate. There is a high level of persistence of the grafted human fat, with minimal inflammatory reaction. The fat is viable and vascularized, demonstrating human-derived vessels in a mouse model. This model provides a platform for studying the populations of progenitor cells known to reside in lipoaspirate
— id: 100530, year: 2009, vol: 124, page: 74, stat: Journal Article,

IMPROVED DIABETIC WOUND HEALING VIA TOPICAL GENE THERA
Tutela, JP; Nguyen, PD; Thanik, VD; Canizares, O; Varjabedian, L; Wagner, J; Lee, JW; Davidson, EH; Haberman, ID; Cohen, OD; Warren, SM; Levine, JP; Saadeh, PB
2009 MAR-APR ;17(2):A11-A11, Wound repair & regeneration
— id: 97660, year: 2009, vol: 17, page: A11, stat: Journal Article,

Plating in microvascular reconstruction of the mandible: can fixation be too rigid?
Zoumalan, Richard A; Hirsch, David L; Levine, Jamie P; Saadeh, Pierre B
2009 Sep;20(5):1451-1454, Journal of craniofacial surgery
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
— id: 104227, year: 2009, vol: 20, page: 1451, stat: Journal Article,

RNA interference of p53 improves diabetic wound healing
Chang, CC; Thanik, VD; Branson, BR; Gupta, SM; Levine, JP; Warren, SM; Saadeh, PB
2008 MAR-APR ;16(2):A48-A48, Wound repair & regeneration
— id: 76412, year: 2008, vol: 16, page: A48, stat: Journal Article,

Functional reconstruction of glossectomy defects: the vertical rectus abdominus myocutaneous neotongue
Haddock, Nicholas T; DeLacure, Mark D; Saadeh, Pierre B
2008 Jul;24(5):343-350, Journal of reconstructive microsurgery
The vertical rectus abdominus myocutaneous (VRAM) flap is a valuable option for tongue reconstruction. However, the traditional inset (skin to remaining oral mucosa) obviates a more anatomic reconstruction. Eight patients underwent total or subtotal glossectomy with VRAM reconstruction. The muscle inset was supported at the inferior mandibular border attached to the remaining lingual mucosa or gingiva. The neotongue, consisting of skin and subcutaneous fat, was sutured posteriorly to the remaining tongue base, and the other surfaces were trimmed and left unsutured. Reconstruction was successful in all patients. The neotongue assumed palatal configuration, and within 2 weeks uniform granulation tissue followed by mucosalization occurred. One year postoperatively, all patients tolerated ad lib diets, spoke intelligibly, were gastrostomy tube and tracheotomy free and had no evidence of aspiration. This neotongue sits on the mandible under voluntary control, permitting effective obturation against the hard palate and providing successful speech and swallowing
— id: 91434, year: 2008, vol: 24, page: 343, stat: Journal Article,

Less Is More: VRAM Inset Modification in Glossectomy Reconstruction
Haddock, Nicholas T; Delacure, Mark D; Saadeh, Pierre B
2008 Aug;122(2):70e-72e, Plastic & reconstructive surgery
— id: 94600, year: 2008, vol: 122, page: 70e, stat: Journal Article,

Hedgehog signaling is essential for normal wound healing
Le, Huong; Kleinerman, Rebecca; Lerman, Oren Z; Brown, Daniel; Galiano, Robert; Gurtner, Geoffrey C; Warren, Stephen M; Levine, Jamie P; Saadeh, Pierre B
2008 Nov-Dec;16(6):768-773, Wound repair & regeneration
The hedgehog family of morphogens (sonic [Shh], Indian, and desert hedgehog) are central regulators of embryologic growth and tissue patterning. Although recent work implicates Shh in postnatal tissue repair and development, conclusive evidence is lacking. Here, we demonstrated the importance of Shh in wound repair, by examining the effects of cyclopamine, a specific inhibitor of the Shh signaling cascade, on tissue repair. Using a murine-splinted excisional wound model, which attenuates wound contraction in this loose-skinned rodent, we established that, by all measures (wound closure, epithelialization, granulation formation, vascularity, and proliferation), wound healing was profoundly impaired when Shh signaling was disrupted. Because embryonic disruption of Shh is associated with distinct phenotypic defects, our findings invite investigation of the potential role of Shh signaling under postnatal conditions associated with disregulated wound healing
— id: 91870, year: 2008, vol: 16, page: 768, stat: Journal Article,

Topical therapy for diabetic wounds using lineage-negative progenitor cells
Lin, CD; Macklin, JE; Lerman, OZ; Tepper, OT; Saadeh, PB; Levine, JP; Warren, SM
2008 MAR-APR ;16(2):A23-A23, Wound repair & regeneration
— id: 76410, year: 2008, vol: 16, page: A23, stat: Journal Article,

Topical lineage-negative progenitor-cell therapy for diabetic wounds
Lin, Clarence D; Allori, Alexander C; Macklin, Jared E; Sailon, Alexander M; Tanaka, Rica; Levine, Jamie P; Saadeh, Pierre B; Warren, Stephen M
2008 Nov;122(5):1341-1351, Plastic & reconstructive surgery
BACKGROUND: Impaired diabetic wound healing is due, in part, to defects in mesenchymal progenitor cell tracking. Theoretically, these defects may be overcome by administering purified progenitor cells directly to the diabetic wound. The authors hypothesize that these progenitor cells will differentiate into endothelial cells, increase wound vascularity, and improve wound healing. METHODS: Lineage-negative progenitor cells were isolated from wild-type murine bone marrow by magnetic cell sorting, suspended in a collagen matrix, and applied topically to full-thickness excisional dorsal cutaneous wounds in diabetic mice. Application of lineage-positive hematopoietic cells or acellular collagen matrix served as comparative controls (n = 16 for each group; n = 48 total). Time to closure and percentage closure were calculated by morphometry. Wounds were harvested at 7, 14, 21, and 28 days and then processed, sectioned, stained (lectin/DiI and CD31), and vascularity was quantified. RESULTS:: Wounds treated with lineage-negative cells demonstrated a significantly decreased time to closure (14 days) compared with lineage-positive (21 days, p = 0.013) and collagen controls (28 days, p = 0.004), and a significant improvement in percentage closure at 14 days compared with the lineage-positive group (p < 0.01) and the collagen control (p < 0.01). Fluorescently tagged lineage-negative cells remained viable in the wound for 28 days, whereas lineage-positive cells were not present after 7 days. Lineage-negative, but not lineage-positive, cells differentiated into endothelial cells. Vascular density and vessel cross-sectional area were significantly higher in lineage-negative wounds. CONCLUSION: Topical progenitor-cell therapy successfully accelerates diabetic wound closure and improves wound vascularity
— id: 90061, year: 2008, vol: 122, page: 1341, stat: Journal Article,

High dose radiation impairs hypoxia responsiveness and vascular recovery in vitro and in vivo
Nguyen, PD; Lerman, OZ; Chang, CC; Thanik, VD; Warren, SM; Saadeh, PB; Levine, JP
2008 MAR-APR ;16(2):A19-A19, Wound repair & regeneration
— id: 76409, year: 2008, vol: 16, page: A19, stat: Journal Article,

Alveolar bone regeneration in a gingivoperiosteoplasty model
Nguyen, PD; Lin, CD; Allori, AC; Reisler, T; Levine, JP; Saadeh, PB; Warren, SM
2008 MAY ;14(5):806-807, Tissue engineering. Part A
— id: 86863, year: 2008, vol: 14, page: 806, stat: Journal Article,

Interventions for alveolar bone regeneration in a gingivoperiosteoplasty model
Nguyen, PD; Lin, CD; Allori, AC; Reisler, T; Saadeh, PB; Levine, JP; Warren, SM
2008 MAR ;42(5):S81-S82, Bone
— id: 76444, year: 2008, vol: 42, page: S81, stat: Journal Article,

A novel murine model of isolated skin radiation injury
Nguyen, PD; Zoumalan, RA; Chang, CC; Allen, RJ; Sailon, AM; Warren, SM; Levine, JP; Saadeh, PB
2008 SEP ;207(3):S61-S62, Journal of the American College of Surgeons
— id: 88543, year: 2008, vol: 207, page: S61, stat: Journal Article,

Microsurgical correction of facial contour deformities in patients with craniofacial malformations: a 15-year experience
Saadeh, Pierre B; Chang, Christopher C; Warren, Stephen M; Reavey, Patrick; McCarthy, Joseph G; Siebert, John W
2008 Jun;121(6):368e-378e, Plastic & reconstructive surgery
BACKGROUND: Since their first review of microsurgical correction of facial contour deformities in 19 patients with craniofacial malformations, the authors have treated an additional 74 patients (n = 93). The authors review indications, choices, safety, efficacy, complications, and technical refinements. A treatment algorithm is presented. METHODS: A retrospective chart review of all patients who underwent microvascular reconstruction of the face and all patients with craniofacial dysmorphology was performed. Between 1989 and 2004, a total of 93 patients with the following diagnoses were identified: craniofacial microsomia (n = 73), Treacher Collins syndrome (n = 8), and severe orbitofacial cleft (n = 12). All patients underwent microsurgical facial reconstruction with a superficial inferior epigastric, groin, or circumflex scapular flap. Flap revisions, complications, and non-free flap related surgery were reviewed. RESULTS: The mean age at microvascular reconstruction was 11 years (range, 4 to 27 years). Flap choices included the following: superficial inferior epigastric (n = 4), groin (n = 3), and circumflex scapular (n = 105). Seventy-six patients underwent unilateral and 17 patients underwent bilateral (one of 17 simultaneous) reconstructions. Postoperative complications included partial flap loss (n = 1), reexploration (n = 1), hematoma (n = 5), and cellulitis (n = 5). All patients had subjective improvement in facial contour, symmetry, skin tone, and color. Most patients underwent additional non-free flap procedures including mandibular distraction and ear reconstruction. CONCLUSIONS: Microsurgical flaps have markedly improved the authors' ability to restore craniofacial contour in patients with craniofacial malformations. In selected patients, the authors choose primary midface augmentation with free vascularized tissue to restore form and function. Microsurgical flaps in patients with craniofacial malformations are safe, effective, and reliable
— id: 79461, year: 2008, vol: 121, page: 368e, stat: Journal Article,

Topically delivered siRNA for cutaneous gene suppression
Sailon, AM; Thanik, VD; Zoumalan, RA; Chang, CC; Levine, JP; Warren, SM; Saadeh, PB
2008 SEP ;207(3):S103-S103, Journal of the American College of Surgeons
— id: 88544, year: 2008, vol: 207, page: S103, stat: Journal Article,

Treatment of radiation skin damage with Coleman fat grafting
Chang, CC; Thanik, VD; Lerman, OZ; Saadeh, PB; Warren, SM; Coleman, SR; Hazen, A
2007 NOV ;25(12):3280-3281, Stem cells
— id: 75629, year: 2007, vol: 25, page: 3280, stat: Journal Article,

Cyclic mechanical strain increases production of regulators of bone healing in cultured murine osteoblasts
Singh, Sunil P; Chang, Edward I; Gossain, Arun K; Mehara, Babak J; Galiano, Robert D; Jensen, John; Longaker, Michael T; Gurtner, Geoffrey C; Saadeh, Pierre B
2007 Mar;204(3):426-434, Journal of the American College of Surgeons
BACKGROUND: The adaptive response of bone to mechanical strain, for which angiogenesis is required, is underscored during fracture healing. Vascular endothelial growth factor (VEGF) and transforming growth factor beta-1 (TGF-beta1) are critical regulators of angiogenesis. The purpose of this study was to examine the effect of strain on the production of VEGF and TGF-beta1. STUDY DESIGN: MC3T3-E1 mouse osteoblasts underwent cyclic strain (low, 0.1 Hz, or high, 0.2 Hz) for 24 or 48 hours. VEGF and TGF-beta1 protein levels were determined by ELISA, and Northern blot analysis was performed for VEGF mRNA. Alkaline phosphatase (an osteoblast differentiation marker) activity was determined by functional enzymatic assay. All measurements were standardized for cell number by crystal violet colorimetric assay. Statistical significance was determined by t-test, ANOVA, and the Tukey-Kramer test. RESULTS: Protein production of VEGF and TGF-beta1 was dose-dependently elevated by strain (p < 0.05); alkaline phosphatase did not rise significantly. Northern blot analysis of strained osteoblast cells demonstrated increased VEGF mRNA. Cyclic strain was found to be progressively destructive in a dose-dependent manner, causing 51% and 70% decreases in cell number under low and high strain, respectively (p < 0.01). CONCLUSIONS: We demonstrated simultaneous, dose-dependent increases in VEGF and TGF-beta1 protein production by osteoblastic cells in response to increasing strain. VEGF mRNA also increased in response to strain. This strain-induced increase in angiogenic cytokines suggests a potential mechanism by which injured bone may recruit a new blood supply. But we also found increasing strain to increase cellular toxicity, suggesting that cyclic mechanical strain may select for a subpopulation of osteoblasts
— id: 71863, year: 2007, vol: 204, page: 426, stat: Journal Article,

Topical matrix-based siRNA silences local gene expression in a murine wound model
Thanik, V D; Greives, M R; Lerman, O Z; Seiser, N; Dec, W; Chang, C C; Warren, S M; Levine, J P; Saadeh, P B
2007 Sep;14(17):1305-1308, Gene therapy
The ability to affect gene expression via topical therapy has profound therapeutic implications for conditions characterized by open wounds including cutaneous neoplasms, thermal injury, skin disorders and dysfunctional wound healing. Specifically targeting local gene expression avoids systemic toxicity and simplifies treatment. We have developed a new method of topical matrix-based short interfering RNA application to precisely and effectively silence local gene expression in nondelimited wounds
— id: 74663, year: 2007, vol: 14, page: 1305, stat: Journal Article,

A soft-tissue approach to midfacial hypoplasia associated with Treacher Collins syndrome
Saadeh, Pierre; Reavey, Patrick L; Siebert, John W
2006 May;56(5):522-525, Annals of plastic surgery
INTRODUCTION: Treacher Collins syndrome is an autosomal dominant mandibulofacial dysostosis with characteristic hard- and soft-tissue facial abnormalities. These include ocular malformations, ear malformations, and hypoplasia of the facial skeleton, especially of the malar bones and mandible. Traditionally, surgical correction of the facial abnormalities has focused on skeletal reconstruction to restore facial form and symmetry. In this report, we describe the use of customized parascapular free flaps, after standard reconstructive surgeries, for the correction of defects of facial contour in Treacher Collins patients. In most cases, bony reconstruction of the zygoma or periorbita is not required. METHODS: From June 1995 to December 2003, 8 patients with Treacher Collins syndrome underwent microsurgical correction of facial contour using 16 free flaps. In all patients, staged parascapular free flaps were used for reconstruction. The microvascular technique involved a 2-team approach with simultaneous ipsilateral parascapular flap harvest and facial pocket dissection. The flaps were contoured, revascularized (14 superficial temporal vessels, 2 facial vessels), and inset. No vein grafts were used. The patients were followed for a minimum of 1 year, and postoperative evaluation included medical photography, visual assessment, and evaluation by the patient and family. RESULTS: Seven patients had previous facial skeleton correction using craniofacial techniques. The age at operation ranged from 4-19 years. Sixteen parascapular free flaps were used in the 8 patients. Postoperative complications were limited to 1 hematoma. There were no partial or total flap losses. All of the patients had improved facial contour and symmetry. Overlying skin tone and color similarly improved. CONCLUSION: After traditional skeletal reconstruction for the complex craniofacial defects of Treacher Collins syndrome, deficiencies in facial contour and symmetry usually persist. Customized soft-tissue free flaps can be employed to differentially resurface these defects and achieve optimal esthetic results in these challenging patients
— id: 64748, year: 2006, vol: 56, page: 522, stat: Journal Article,

In-vivo gene silencing using topical delivery of siRNA
Thanik, V; Greives, M; Seiser, N; Lerman, O; Hazen, A; Levine, J; Saadeh, P
2006 SEP ;203(3):S55-S56, Journal of the American College of Surgeons
— id: 69819, year: 2006, vol: 203, page: S55, stat: Journal Article,

Rebalancing of forces as an adjunct to resection suspension arthroplasty for trapezial osteoarthritis
Saadeh, Pierre B; Kazanowski, Melissa A; Sharma, Sheel; Beasley, Robert W
2004 Jul;52(6):567-570, Annals of plastic surgery
The carpometacarpal (CM) joint of the thumb is commonly affected by osteoarthritis. The strength required for a first CM ligament reconstruction depends on the forces across the joint. If these forces are rebalanced to reduce the requirements necessary to prevent subluxation, reconstructive requirements are lowered and surgical dissections reduced. A method to achieve this goal based on Landsmeer's zig-zag compression concept is presented. Fifteen consecutive patients (11 women; mean age, 63 years) with pantrapezial osteoarthritis were selected over a 2-year period to undergo this novel procedure. After standard trapezial resection, trapezoidal hemiresection was performed, allowing for medial movement of the first metacarpal base. Following the zig-zag concept, the first metacarpophalangeal joint reciprocally fell into flexion, decreasing forces causing subluxation of the first metacarpal base. A saddle-like suspension under the metacarpal base was created using the trapezial capsule. All 15 patients had excellent outcomes with elimination of pain, early recovery of mobility and power, and no recurrent subluxations. The durability of the procedure was confirmed clinically and radiologically. The medial relocation of the first metacarpal base rebalances and attenuates the normal deforming forces thereby eliminating the need for a strong CM ligament reconstruction
— id: 46143, year: 2004, vol: 52, page: 567, stat: Journal Article,

Osteoblast expression of vascular endothelial growth factor is modulated by the extracellular microenvironment
Spector JA; Mehrara BJ; Greenwald JA; Saadeh PB; Steinbrech DS; Bouletreau PJ; Smith LP; Longaker MT
2001 Jan;280(1):C72-C80, American journal of physiology. Cell physiology
Angiogenesis, the formation of new blood vessels, is crucial to the process of fracture healing. Vascular disruption after osseous injury results in an acidic, hypoxic wound environment. We have previously shown that osteoblasts can produce vascular endothelial growth factor (VEGF) in response to a variety of stimuli. In this study we examined pH and lactate concentration, two components of the putative fracture extracellular microenvironment, and determined their relative contribution to regulation of rat calvarial osteoblast VEGF production under both normoxic and hypoxic conditions. Our results demonstrate that pH and lactate concentration do independently affect osteoblast VEGF mRNA and protein production. Acidic pH (7.0) significantly decreased VEGF production, under normoxic and hypoxic conditions (P < 0.05), compared with neutral pH (7.4). This decrease was primarily transcriptionally regulated, because the rate of VEGF mRNA degradation was unchanged at pH 7.0 vs. 7.4. Similarly, an elevated lactate concentration (22 mM) also depressed osteoblast elaboration of VEGF at both neutral and acidic pH (P < 0.001). Furthermore, the effects of increasing acidity and elevated lactate appeared to be additive
— id: 26828, year: 2001, vol: 280, page: C72, stat: Journal Article,

Hypoxia regulates osteoblast gene expression
Warren SM; Steinbrech DS; Mehrara BJ; Saadeh PB; Greenwald JA; Spector JA; Bouletreau PJ; Longaker MT
2001 Jul;99(1):147-155, Journal of surgical research
Vascular disruption secondary to fracture creates a hypoxic gradient of injury wherein the oxygen tension at the center of the wound is very low. In vivo this hypoxic microenvironment stimulates the expression of a variety of cytokines from inflammatory cells, fibroblasts, endothelial cells, and osteoblasts. In order to begin to dissect this complex system, we have examined the effects of hypoxia on isolated osteoblast gene expression in vitro. Understanding gene expression in this system may facilitate the development of targeted therapeutic modalities designed to accelerate fracture repair and reduce complications. Using an established model of in vitro hypoxia, we have analyzed the expression of genes involved in bone matrix production and turnover. Subconfluent neonatal rat calvarial osteoblasts were exposed to hypoxia (pO(2) = 35-40 mm Hg) and total cellular RNA was collected at 0, 3, 6, 24, and 48 h. Northern analysis was used to analyze the expression patterns of (1) transforming growth factors (TGFs)-beta1, -beta2, and -beta3 and their type I receptor; (2) collagens I and III; and (3) tissue inhibitor of metalloproteinase-1. We have demonstrated a marked elevation of TGF-beta1 gene expression within 3 h of hypoxia. Although neither TGF-beta2 nor TGF-beta3 expression was affected by hypoxia, the TGF-beta type I receptor was substantially upregulated within 6 h. In addition, extracellular matrix scaffolding molecules (collagens I and III) were markedly, but differentially, upregulated. Finally, we have demonstrated that the expression of an inhibitor of extracellular matrix turnover, the tissue inhibitor of metalloproteinase-1, was strikingly decreased in response to hypoxia. These results imply that hypoxia can affect osseous healing by altering the expression of cytokines, bone-specific extracellular matrix molecules, and their regulators
— id: 26733, year: 2001, vol: 99, page: 147, stat: Journal Article,

Differential expression of receptor tyrosine kinases and Shc in fetal and adult rat fibroblasts: toward defining scarless versus scarring fibroblast phenotypes
Chin GS; Kim WJ; Lee TY; Liu W; Saadeh PB; Lee S; Levinson H; Gittes GK; Longaker MT
2000 Mar;105(3):972-979, Plastic & reconstructive surgery
The remarkable ability of the fetus to heal early gestation skin wounds without scarring remains poorly understood. Taking advantage of recent advances in signal transduction, the tyrosine phosphorylation patterns of fetal rat fibroblasts, representing the scarless cutaneous repair phenotype, and adult rat fibroblasts, representing scarforming phenotype, were examined whether there were inherent differences in cellular signaling. Specifically, correlation of the phosphorylation patterns with the expression levels of the signaling molecules that transmit information from the plasma membrane receptor to the nucleus was sought. By using three different cell lines of explanted fibroblasts from gestational day 13 fetal rat skin (n = 24) and 1-month-old postnatal adult rat skin (n = 3), immunoblotting was performed to compare tyrosine phosphorylation patterns. The results revealed five major protein bands of interest in fetal rat fibroblasts, but not in the adult rat fibroblasts. These phosphorylated protein bands are of interest because of their possible role in wound repair and may have the potential to regulate cellular responses to the extracellular matrix and their secondary signaling molecules. It was hypothesized that these bands represented receptor tyrosine kinases, epidermal growth factor receptor, and discoidin domain receptor 1, and their downstream adaptor protein Shc that binds receptor tyrosine kinases to transduce signals intracellularly. Furthermore, elevated expression of platelet-derived growth factor receptor-beta in adult compared with fetal fibroblasts was demonstrated, suggesting that decreased expression of certain growth factors may also be important for the scarless phenomenon to occur
— id: 8523, year: 2000, vol: 105, page: 972, stat: Journal Article,

The effects of ionizing radiation on osteoblast-like cells in vitro
Dudziak ME; Saadeh PB; Mehrara BJ; Steinbrech DS; Greenwald JA; Gittes GK; Longaker MT
2000 Oct;106(5):1049-1061, Plastic & reconstructive surgery
The well-described detrimental effects of ionizing radiation on the regeneration of bone within a fracture site include decreased osteocyte number, suppressed osteoblast activity, and diminished vascularity. However, the biologic mechanisms underlying osteoradionecrosis and the impaired fracture healing of irradiated bone remain undefined. Ionizing radiation may decrease successful osseous repair by altering cytokine expression profiles resulting from or leading to a change in the osteoblastic differentiation state. These changes may, in turn, cause alterations in osteoblast proliferation and extracellular matrix formation. The purpose of this study was to investigate the effects of ionizing radiation on the proliferation, maturation, and cytokine production of MC3T3-E1 osteoblast-like cells in vitro. Specifically, the authors examined the effects of varying doses of ionizing radiation (0, 40, 400, and 800 cGy) on the expression of transforming growth factor-beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), and alkaline phosphatase. In addition, the authors studied the effects of ionizing radiation on MC3T3-E1 cellular proliferation and the ability of conditioned media obtained from control and irradiated cells to regulate the proliferation of bovine aortic endothelial cells. Finally, the authors evaluated the effects of adenovirus-mediated TGF-beta1 gene therapy in an effort to 'rescue' irradiated osteoblasts. The exposure of osteoblast-like cells to ionizing radiation resulted in dose-dependent decreases in cellular proliferation and promoted cellular differentiation (i.e., increased alkaline phosphatase production). Additionally, ionizing radiation caused dose-dependent decreases in total TGF-beta1 and VEGF protein production. Decreases in total TGF-beta1 production were due to a decrease in TGF-beta1 production per cell. In contrast, decreased total VEGF production was secondary to decreases in cellular proliferation, because the cellular production of VEGF by irradiated osteoblasts was moderately increased when VEGF production was corrected for cell number. Additionally, in contrast to control cells (i.e., nonirradiated), conditioned media obtained from irradiated osteoblasts failed to stimulate the proliferation of bovine aortic endothelial cells. Finally, transfection of control and irradiated cells with a replication-deficient TGF-beta1 adenovirus before irradiation resulted in an increase in cellular production of TGF-beta1 protein and VEGF. Interestingly, this intervention did not alter the effects of irradiation on cellular proliferation, which implies that alterations in TGF-beta1 expression do not underlie the deficiencies noted in cellular proliferation. The authors hypothesize that ionizing radiation-induced alterations in the cytokine profiles and differentiation states of osteoblasts may provide insights into the cellular mechanisms underlying osteoradionecrosis and impaired fracture healing
— id: 39534, year: 2000, vol: 106, page: 1049, stat: Journal Article,

Transforming growth factor beta superfamily members: role in cartilage modeling
Frenkel SR; Saadeh PB; Mehrara BJ; Chin GS; Steinbrech DS; Brent B; Gittes GK; Longaker MT
2000 Mar;105(3):980-990, Plastic & reconstructive surgery
Normal and abnormal extracellular matrix turnover is thought to result, in part, from the balance in the expression of metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). The clinical manifestations of an imbalance in these relationships are evident in a variety of pathologic states, including osteoarthritis, deficient long-bone growth, rheumatoid arthritis, tumor invasion, and inadequate cartilage repair. Articular cartilage defects commonly heal as fibrocartilage, which is structurally inferior to the normal hyaline architecture of articular cartilage. Transforming growth factor-beta 1 (TGF-beta1), a cytokine central to growth, repair, and inflammation, has been shown to upregulate TIMP-1 expression in human and bovine articular cartilage. Additionally, members of the TGF-beta superfamily are thought to play key roles in chondrocyte growth and differentiation. Bone morphogenetic protein-2 (BMP-2), a member of this superfamily, has been shown to regulate chondrocyte differentiation states and extracellular matrix composition. It was proposed that, by optimizing extracellular matrix composition, BMP-2 would enhance articular cartilage healing. After determining the release kinetics of BMP-2 from a collagen type I implant (Long-Evans male rats; two implants/rat, n = 14), it was found that, in a tissue engineering application, BMP-2 induced a hyaline-like repair of New Zealand White rabbit knee articular cartilage defects (3-mm full-thickness defects in the femoral trochlea; 2 defects/rabbit, n = 36). The quality of cartilage repair with BMP-2 (with or without chondrocytes) was significantly better than defects treated with BMP-2, as assessed by a quantitative scoring scale. Immunohistochemical staining revealed TIMP-1 production in the cartilage defects treated with BMP-2. When studied in vitro, it was found that BMP-2 markedly increased TIMP-1 mRNA by both bovine articular and human rib chondrocytes. Additionally, increased TIMP-1 mRNA was translated into increased TIMP-1 protein production by bovine chondrocytes. Taken together, these data suggest that BMP-2 may be a useful cytokine to improve healing of cartilaginous defects. Furthermore, these data suggest that the beneficial effects of BMP-2 may be, in part, related to alterations in extracellular matrix turnover
— id: 27858, year: 2000, vol: 105, page: 980, stat: Journal Article,

Biomolecular mechanisms of calvarial bone induction: immature versus mature dura mater
Greenwald JA; Mehrara BJ; Spector JA; Chin GS; Steinbrech DS; Saadeh PB; Luchs JS; Paccione MF; Gittes GK; Longaker MT
2000 Apr;105(4):1382-1392, Plastic & reconstructive surgery
The ability of newborns and immature animals to reossify calvarial defects has been well described. This capacity is generally lost in children greater than 2 years of age and in mature animals. The dura mater has been implicated as a regulator of calvarial reossification. To date, however, few studies have attempted to identify biomolecular differences in the dura mater that enable immature, but not mature, dura to induce osteogenesis. The purpose of these studies was to analyze metabolic characteristics, protein/gene expression, and capacity to form mineralized bone nodules of cells derived from immature and mature dura mater. Transforming growth factor beta-1, basic fibroblast growth factor, collagen type IalphaI, osteocalcin, and alkaline phosphatase are critical growth factors and extracellular matrix proteins essential for successful osteogenesis. In this study, we have characterized the proliferation rates of immature (6-day-old rats, n = 40) and mature (adult rats, n = 10) dura cell cultures. In addition, we analyzed the expression of transforming growth factor beta-1, basic fibroblast growth factor-2, proliferating cell nuclear antigen, and alkaline phosphatase. Our in vitro findings were corroborated with Northern blot analysis of mRNA expression in total cellular RNA isolated from snap-frozen age-matched dural tissues (6-day-old rats, n = 60; adult rats, n = 10). Finally, the capacity of cultured dural cells to form mineralized bone nodules was assessed. We demonstrated that immature dural cells proliferate significantly faster and produce significantly more proliferating cell nuclear antigen than mature dural cells (p < 0.01). Additionally, immature dural cells produce significantly greater amounts of transforming growth factor beta-1, basic fibroblast growth factor-2, and alkaline phosphatase (p < 0.01). Furthermore, Northern blot analysis of RNA isolated from immature and mature dural tissues demonstrated a greater than 9-fold, 8-fold, and 21-fold increase in transforming growth factor beta-1, osteocalcin, and collagen IalphaI gene expression, respectively, in immature as compared with mature dura mater. Finally, in keeping with their in vivo phenotype, immature dural cells formed large calcified bone nodules in vitro, whereas mature dural cells failed to form bone nodules even with extended culture. These studies suggest that differential expression of growth factors and extracellular matrix molecules may be a critical difference between the osteoinductive capacity of immature and mature dura mater. Finally, we believe that the biomolecular bone- and matrix-inducing phenotype of immature dura mater regulates the ability of young children and immature animals to heal calvarial defects
— id: 11780, year: 2000, vol: 105, page: 1382, stat: Journal Article,

Immature versus mature dura mater: II. Differential expression of genes important to calvarial reossification [In Process Citation]
Greenwald JA; Mehrara BJ; Spector JA; Fagenholz PJ; Saadeh PB; Steinbrech DS; Gittes GK; Longaker MT
2000 Sep;106(3):630-638, Plastic & reconstructive surgery
The ability of immature animals and newborns to orchestrate successful calvarial reossification is well described. This capacity is markedly attenuated in mature animals and in humans greater than 2 years of age. Previous studies have implicated the dura mater as critical to successful calvarial reossification. The authors have previously reported that immature, but not mature, dural tissues are capable of elaborating a high expression of osteogenic growth factors and extracellular matrix molecules. These findings led to the hypothesis that a differential expression of osteogenic growth factors and extracellular matrix molecules by immature and mature dural tissues may be responsible for the clinically observed phenotypes (i.e., immature animals reossify calvarial defects; mature animals do not). This study continues to explore the hypothesis through an analysis of transforming growth factor (TGF)-beta3, collagen type III, and alkaline phosphatase mRNA expression. Northern blot analysis of total RNA isolated from freshly harvested immature (n = 60) and mature (n = 10) dural tissues demonstrated a greater than three-fold, 18-fold, and nine-fold increase in TGF-beta3, collagen type III, and alkaline phosphatase mRNA expression, respectively, in immature dural tissues as compared with mature dural tissues. Additionally, dural cell cultures derived from immature (n = 60) and mature dura mater (n = 10) were stained for alkaline phosphatase activity to identify the presence of osteoblast-like cells. Alkaline phosphatase staining of immature dural cells revealed a significant increase in the number of alkaline phosphatase-positive cells as compared with mature dural tissues (p < 0.001). In addition to providing osteogenic humoral factors (i.e., growth factors and extracellular matrix molecules), this finding suggests that immature, but not mature, dura mater may provide cellular elements (i.e., osteoblasts) that augment successful calvarial reossification. These studies support the hypothesis that elaboration of osteogenic growth factors (i.e., TGF-beta33) and extracellular matrix molecules (i.e., collagen type III and alkaline phosphatase) by immature, but not mature, dural tissues may be critical for successful calvarial reossification. In addition, these studies suggest for the first time that immature dural tissues may provide cellular elements (i.e., osteoblasts) to augment this process
— id: 11499, year: 2000, vol: 106, page: 630, stat: Journal Article,

A rat model of gingivoperiosteoplasty
Mehrara BJ; Saadeh PB; Steinbrech DS; Dudziak M; Grayson BH; Cutting CB; McCarthy JG; Gittes GK; Longaker MT
2000 Jan;11(1):54-58, Journal of craniofacial surgery
The ability to avoid a subsequent bone graft makes the use of gingivoperiosteoplasty (GPP) at the time of cleft lip repair an attractive technique. The use of GPP, in combination with presurgical orthodontics, has been shown to result in successful bony union in the majority of patients. However, secondary bone grafting is still necessary in 30% to 40% of patients due to persistent alveolar bony defects. The elucidation of methods to improve the success rates of these procedures has been hampered by the lack of reproducible animal models. The purpose of this study was, therefore, to develop a rodent model of GPP that would facilitate the investigation of methods to improve osteogenesis in alveolar defects. We report a surgically produced rat model (9 x 5 x 3-mm alveolar defect) that is reproducible, inexpensive (relative to large-animal models), and simple technically. In addition, healing in this model occurs in a predictable manner during a 12-week period, thus enabling analysis of methods designed to accelerate or facilitate osseous regeneration
— id: 20721, year: 2000, vol: 11, page: 54, stat: Journal Article,

Mechanisms of fibroblast growth factor-2 modulation of vascular endothelial growth factor expression by osteoblastic cells
Saadeh, PB; Mehrara, BJ; Steinbrech, DS; Spector, JA; Greenwald, JA; Chim, GS; Ueno, H; Gittes, GK; Longaker, MT
2000 JUN ;141(6):2075-2083, Endocrinology
Normal bone growth and repair is dependent on angiogenesis. Fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGF beta) have all been implicated in the related processes of angiogenesis, growth, development, and repair. The purpose of this study was to investigate the relationships between FGF-2 and both VEGF and TGF beta in nonimmortalized and clonal osteoblastic cells. Northern blot analysis revealed 6-fold peak increases in VEGF mRNA at 6 h in fetal rat calvarial cells and MC3T3-E1 osteoblastic cells after stimulation with FGF-S. Actinomycin D inhibited these increases in VEGF mRNA, whereas cycloheximide did not. The stability of VEGF mRNA was not increased after FGF-2 treatment. Furthermore, FGF-2 induced dose-dependent increases in VEGF protein levels (P < 0.01). Although in MC3T3-E1 cells, TGF beta 1 stimulates a 6-fold peak increase in VEGF mRNA after 3 h of stimulation, we found that both TGF beta 2 and TGF beta 3 yielded 2- to 8-fold peak increases in VEGF mRNA levels noted after 6 h of stimulation. Similarly, both TGF beta 2 and TCF beta 3 dose dependently increased VEGF protein production. To determine whether FGF-2-induced increases in VEGF mRNA may have occurred independently of TGF beta, we disrupted TGF beta signal transduction (using adenovirus encoding a truncated form of TGF beta receptor II), which attenuated TGF beta 1 induction of VEGF mRNA, but did not impede FGF-2 induction of VEGF mRNA. In summary, FGF-2-induced VEGF expression by osteoblastic cells is a dose-dependent event that may be independent of concomitant FGF-2-induced modulation of TGF beta activity
— id: 54567, year: 2000, vol: 141, page: 2075, stat: Journal Article,

Practice standards in carpal tunnel syndrome electrodiagnosis
Sander, H W; Saadeh, P B
2000 May;25(3):589-590, Journal of hand surgery (American volume)
— id: 112143, year: 2000, vol: 25, page: 589, stat: Journal Article,

A molecular analysis of the isolated rat posterior frontal and sagittal sutures: differences in gene expression [In Process Citation]
Spector JA; Mehrara BJ; Greenwald JA; Saadeh PB; Steinbrech DS; Smith LP; Longaker MT
2000 Sep;106(4):852-861, Plastic & reconstructive surgery
Although it is one of the most commonly occurring craniofacial congenital disabilities, craniosynostosis (the premature fusion of cranial sutures) is nearly impossible to prevent because the molecular mechanisms that regulate the process of cranial suture fusion remain largely unknown. Recent studies have implicated the dura mater in determining the fate of the overlying cranial suture; however, the molecular biology within the suture itself has not been sufficiently investigated. In the murine model of cranial suture fusion, the posterior frontal suture is programmed to begin fusing by postnatal day 12 in rats (day 25 in mice), reliably completing bony union by postnatal day 22 (day 45 in mice). In contrast, the sagittal suture remains patent throughout the life of the animal. Using this model, this study sought to examine for the first time what differences in gene expression--if any--exist between the two sutures with opposite fates. For each series of experiments, 35 to 40 posterior frontal and sagittal suture complexes were isolated from 6-day-old Sprague-Dawley rat pups. Suture-derived cell cultures were established, and ribonuicleic acid was derived from snap-frozen, isolated suture tissue. Results demonstrated that molecular differences between the posterior frontal and sagittal suture complexes were readily identified in vivo, although these distinctions were lost once the cells comprising the suture complex were cultured in vitro. Hypothetically, this change in gene expression resulted from the loss of the influence of the underlying dura mater. Significant differences in the expression of genes encoding extracellular matrix proteins existed in vivo between the posterior frontal and sagittal sutures. However, the production of the critical, regulatory cytokine transforming growth factor beta-1 was equal between the two suture complexes, lending further support to the hypothesis that dura mater regulates the fate of the overlying cranial suture
— id: 11464, year: 2000, vol: 106, page: 852, stat: Journal Article,

Expression of adenovirally delivered gene products in healing osseous tissues
Spector JA; Mehrara BJ; Luchs JS; Greenwald JA; Fagenholz PJ; Saadeh PB; Steinbrech DS; Longaker MT
2000 May;44(5):522-528, Annals of plastic surgery
Gene therapy has moved from the promise of laboratory investigation to the reality of clinical practice in just the last decade. Various methods for delivery of genes to host cells have been developed and utilized both in vitro and in vivo. From the perspective of the plastic surgeon, gene therapy holds the promise to augment healing in clinical situations that remain difficult to treat, such as chronic wounds, osteoradionecrosis, or possibly to expedite current clinical practices, such as distraction osteogenesis. The authors chose to investigate the potential for gene therapy in osseous tissues using a replication-deficient adenovirus vector to deliver the marker transgene beta-galactosidase. An adenovirus vector is ideal for use in situations in which transgene expression is desired for only a relatively short period of time, such as wound and fracture healing. Utilizing a rat mandibular osteotomy model, they demonstrated that, using an adenoviral vector, foreign genes can be delivered in a simple fashion and can be expressed in a reliable manner within and around the osteotomy site for at least 10 days. Furthermore, there was no evidence of transfection of distant tissues associated with local application of the adenovirus vector. With this information, clinicians may now attempt to deliver osteogenic and angiogenic genes in a site-specific fashion to improve and expedite osseous healing
— id: 11710, year: 2000, vol: 44, page: 522, stat: Journal Article,

Gene expression of TGF-beta, TGF-beta receptor, and extracellular matrix proteins during membranous bone healing in rats
Steinbrech DS; Mehrara BJ; Rowe NM; Dudziak ME; Luchs JS; Saadeh PB; Gittes GK; Longaker MT
2000 May;105(6):2028-2038, Plastic & reconstructive surgery
Poorly healing mandibular fractures and osteotomies can be troublesome complications of craniomaxillofacial trauma and reconstructive surgery. Gene therapy may offer ways of enhancing bone formation by altering the expression of desired growth factors and extracellular matrix molecules. The elucidation of suitable candidate genes for therapeutic intervention necessitates investigation of the endogenously expressed patterns of growth factors during normal (i.e., successful) fracture repair. Transforming growth factor beta1 (TGF-beta1), its receptor (Tbeta-RII), and the extracellular matrix proteins osteocalcin and type I collagen are thought to be important in long-bone (endochondral) formation, fracture healing, and osteoblast proliferation. However, the spatial and temporal expression patterns of these molecules during membranous bone repair remain unknown. In this study, 24 adult rats underwent mandibular osteotomy with rigid external fixation. In addition, four identically treated rats that underwent sham operation (i.e., no osteotomy) were used as controls. Four experimental animals were then killed at each time point (3, 5, 7, 9, 23, and 37 days after the procedure) to examine gene expression of TGF-beta1 and Tbeta-RII, osteocalcin, and type I collagen. Northern blot analysis was used to compare gene expression of these molecules in experimental animals with that in control animals (i.e., nonosteotomized; n = 4). In addition, TGF-beta1 and T-RII proteins were immunolocalized in an additional group of nine animals killed on postoperative days 3, 7, and 37. The results of Northern blot analysis demonstrated a moderate increase (1.7 times) in TGF-beta1 expression 7 days postoperatively; TGF-beta1 expression returned thereafter to near baseline levels. Tbeta-RII mRNA expression was downregulated shortly after osteotomy but then increased, reaching a peak of 1.8 times the baseline level on postoperative day 9. Osteocalcin mRNA expression was dramatically downregulated shortly after osteotomy and remained low during the early phases of fracture repair. Osteocalcin expression trended slowly upward as healing continued, reaching peak expression by day 37 (1.7 times the control level). In contrast, collagen type IalphaI mRNA expression was acutely downregulated shortly after osteotomy, peaked on postoperative days 5, and then decreased at later time points. Histologic samples from animals killed 3 days after osteotomy demonstrated TGF-beta1 protein localized to inflammatory cells and extracellular matrix within the fracture gap, periosteum, and peripheral soft tissues. On postoperative day 7, TGF-beta1 staining was predominantly localized to the osteotomized bone edges, periosteum, surrounding soft tissues, and residual inflammatory cells. By postoperative day 37, complete bony healing was observed, and TGF-beta1 staining was localized to the newly formed bone matrix and areas of remodeling. On postoperative day 3, Tbeta-RII immunostaining localized to inflammatory cells within the fracture gap, periosteal cells, and surrounding soft tissues. By day 7, Tbeta-RII staining localized to osteoblasts of the fracture gap but was most intense within osteoblasts and mesenchymal cells of the osteotomized bone edges. On postoperative day 37, Tbeta-RII protein was seen in osteocytes, osteoblasts, and the newly formed periosteum in the remodeling bone. These observations agree with those of previous in vivo studies of endochondral bone formation, growth, and healing. In addition, these results implicate TGF-beta1 biological activity in the regulation of osteoblast migration, differentiation, and proliferation during mandibular fracture repair. Furthermore, comparison of these data with gene expression during mandibular distraction osteogenesis may provide useful insights into the treatment of poorly healing fractures because distraction osteogenesis has been shown to be effective in the management of these difficult clinical cases
— id: 11668, year: 2000, vol: 105, page: 2028, stat: Journal Article,

Hypoxia increases insulinlike growth factor gene expression in rat osteoblasts
Steinbrech DS; Mehrara BJ; Saadeh PB; Greenwald JA; Spector JA; Gittes GK; Longaker MT
2000 May;44(5):529-534, Annals of plastic surgery
Vascular disruption secondary to fracture leads to a hypoxic zone of injury where the oxygen tension at the center of the wound is quite low. In this dynamic microenvironment, a number of growth factors are elaborated to stimulate the synthetic processes of fracture repair. Previously the authors have shown the hypoxia-induced increase of vascular endothelial growth factor expression in osteoblasts. The purpose of these experiments was to examine osteoblast expression of insulinlike growth factors (IGF) I and II--cytokines believed to play a role in increased collagen synthesis, chemotaxis, and proliferation of osteoblasts in response to hypoxia. Primary cell cultures of osteoblasts isolated from neonatal rat calvaria were subjected to hypoxia (PO2 = 35 mmHg) for 0, 3, 6, 24, and 48 hours. Northern blot analysis of ribonucleic acid (RNA) from resulting cultures demonstrated a more than 60% increase in IGF-II messenger RNA (mRNA) expression after 3 hours of hypoxia. IGF-II mRNA expression continued to increase through later time points to 200% and 260% of baseline at 24 and 48 hours respectively. In contrast, IGF-I demonstrated no significant change in mRNA expression compared with baseline control (normoxia) cultures. In these experiments the authors have demonstrated a hypoxia-induced increase in IGF-II but not IGF-I in primary osteoblasts. The differential expression of these two growth factors may underscore important differences in the behavior of osteoblasts in the hypoxic fracture microenvironment. Taken together, these data add additional support to the theory that hypoxia induces gene-specific changes in expression of molecules important to extracellular matrix formation for successful bone healing
— id: 11709, year: 2000, vol: 44, page: 529, stat: Journal Article,

VEGF expression in an osteoblast-like cell line is regulated by a hypoxia response mechanism
Steinbrech DS; Mehrara BJ; Saadeh PB; Greenwald JA; Spector JA; Gittes GK; Longaker MT
2000 Apr;278(4):C853-C860, American journal of physiology. Cell physiology
Angiogenesis is essential for the increased delivery of oxygen and nutrients required for the reparative processes of bone healing. Vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, has been implicated in this process. We have previously shown that hypoxia specifically and potently regulates the expression of VEGF by osteoblasts. However, the molecular mechanisms governing this interaction remain unknown. In this study, we hypothesized that the hypoxic regulation of VEGF expression by osteoblasts occurs via an oxygen-sensing mechanism similar to the regulation of the erythropoietin gene (EPO). To test this hypothesis, we examined the kinetics of oxygen concentration on osteoblast VEGF expression. In addition, we analyzed the effects of nickel and cobalt on the expression of VEGF in osteoblastic cells because these metallic ions mimic hypoxia by binding to the heme portion of oxygen-sensing molecules. Our results indicated that hypoxia potently stimulates VEGF mRNA expression. In addition, we found that nickel and cobalt both stimulate VEGF gene expression in a similar time- and dose-dependent manner, suggesting the presence of a hemelike oxygen-sensing mechanism similar to that of the EPO gene. Moreover, actinomycin D, cycloheximide, dexamethasone, and mRNA stabilization studies collectively established that this regulation is predominantly transcriptional, does not require de novo protein synthesis, and is not likely mediated by the transcriptional activator AP-1. These studies demonstrate that hypoxia, nickel, and cobalt regulate VEGF expression in osteoblasts via a similar mechanism, implicating the involvement of a heme-containing oxygen-sensing molecule. This may represent an important mechanism of VEGF regulation leading to increased angiogenesis in the hypoxic microenvironment of healing bone
— id: 11774, year: 2000, vol: 278, page: C853, stat: Journal Article,

Regional differentiation of rat cranial suture-derived dural cells is dependent on association with fusing and patent cranial sutures
Mehrara BJ; Greenwald J; Chin GS; Dudziak M; Sagrioglu J; Steinbrech DS; Saadeh PB; Gittes GK; Longaker MT
1999 Sep;104(4):1003-1013, Plastic & reconstructive surgery
A significant body of literature supports a role for the dura mater underlying cranial sutures in the regulation of sutural fate. These studies have implicated regional differentiation of the dura mater based on association with fusing and patent rat cranial sutures. The purpose of these experiments was to isolate and characterize dural cells associated with fusing (posterior frontal) and patent (sagittal) rat cranial sutures. Six-day-old rats were killed, and the dura mater underlying the posterior frontal and sagittal sutures was harvested. Dural cells were briefly trypsinized and allowed to reach confluence. Two litters (10 animals per litter) were used for each set of experiments. Cells were harvested after the first and fifth passages for analysis of vimentin and desmoplakin expression (characteristic of human meningeal cells), cellular proliferation, density at confluence (a measure of cellular contact inhibition), and alkaline phosphatase production. In addition, bone nodule formation and collagen I production were analyzed in first passage cells. The results indicate that suture-derived dural cells can be established and that these cells coexpress vimentin and desmoplakin. In addition, it is demonstrated that first-passage sagittal suture-derived dural cells proliferate significantly faster and have decreased cellular contact inhibition than posterior frontal suture-derived cells (p < 0.01). Finally, it is shown that suture-derived dural cells have osteoblast-like properties, including alkaline phosphatase production, collagen I expression, and bone nodule formation in vitro. The possible mechanisms by which regional differentiation of suture-derived dural cells occur are discussed
— id: 11850, year: 1999, vol: 104, page: 1003, stat: Journal Article,

Rat mandibular distraction osteogenesis: II. Molecular analysis of transforming growth factor beta-1 and osteocalcin gene expression
Mehrara BJ; Rowe NM; Steinbrech DS; Dudziak ME; Saadeh PB; McCarthy JG; Gittes GK; Longaker MT
1999 Feb;103(2):536-547, Plastic & reconstructive surgery
Distraction osteogenesis is a powerful technique capable of generating viable osseous tissue by the gradual separation of osteotomized bone edges. Although the histologic and ultrastructural changes associated with this process have been extensively delineated, the molecular events governing these changes remain essentially unknown. We have devised a rat model of mandibular distraction osteogenesis that facilitates molecular analysis of this process. Such information has significant clinical implications because it may enable targeted therapeutic manipulations designed to accelerate osseous regeneration. In this study, we have evaluated the expression of transforming growth factor beta-1, a major regulator of osteogenesis during endochondral bone formation and development, and osteocalcin, an abundant noncollagenous extracellular matrix protein implicated in the regulation of mineralization and bone turnover. The right hemimandible of 36 adult male rats was osteotomized, and a customized distraction device was applied. Animals were allowed to recover and, after a 3-day latency period, were distracted at a rate of 0.25 mm twice daily for 6 days followed by a 2- or 4-week consolidation period. Distraction regenerate was harvested after the latency period, days 2, 4, or 6 of distraction, and after 2 or 4 weeks of consolidation and processed for Northern analysis (n = 4 at each time point) and immunohistochemical localization of TGF-beta1 (n = 2 at each time point). Six sham-operated animals (i.e., skin incision without osteotomy) were also killed (immediately postoperatively), and the mandibles were harvested and prepared in a similar fashion. Equal loading and transfer of RNA for Northern analysis was ensured by stripping and probing membranes with a probe against GAPDH (a housekeeping gene). Our results demonstrate that the spatial and temporal patterns of TGF-beta1 mRNA expression and protein production coincide with osteoblast migration, differentiation, and extracellular matrix synthesis. In addition, we demonstrate that TGF-beta1 production may be an important regulator of vasculogenesis during mandibular distraction osteogenesis. Finally, we have shown that osteocalcin gene expression coincides temporally with mineralization during rat mandibular distraction osteogenesis
— id: 7938, year: 1999, vol: 103, page: 536, stat: Journal Article,

Adenovirus-mediated gene therapy of osteoblasts in vitro and in vivo
Mehrara BJ; Saadeh PB; Steinbrech DS; Dudziak M; Spector JA; Greenwald JA; Gittes GK; Longaker MT
1999 Aug;14(8):1290-1301, Journal of bone & mineral research
Modulation of biological pathways governing osteogenesis may accelerate osseous regeneration and reduce the incidence of complications associated with fracture healing. Transforming growth factor beta1 (TGF-beta1) is a potent growth factor implicated in the regulation of osteogenesis and fracture repair. The use of recombinant proteins, however, has significant disadvantages and has limited the clinical utility of these molecules. Targeted gene therapy using adenovirus vectors is a technique that may circumvent difficulties associated with growth factor delivery. In this study, we investigate the efficacy of replication-deficient adenoviruses containing the human TGF-beta1 and the bacterial lacZ genes in transfecting osteoblasts in vitro and osseous tissues in vivo. We demonstrate that adenovirus-mediated gene therapy efficiently transfects osteoblasts in vitro with the TGF-beta1 virus causing a marked up-regulation in TGF-beta1 mRNA expression even 7 days after transfection. Increased TGF-beta1 mRNA expression was efficiently translated into protein production and resulted in approximately a 46-fold increase in TGF-beta1 synthesis as compared with control cells (vehicle- or B-galactosidase-transfected). Moreover, virally produced TGF-beta1 was functionally active and regulated the expression of collagen IalphaI (5-fold increase) and the vascular endothelial growth factor (2.5-fold increase). Using an adenovirus vector encoding the Escherichia coli LacZ gene, we demonstrated that adenovirus-mediated gene transfer efficiently transfects osteoblasts and osteocytes in vivo and that transfection can be performed by a simple percutaneous injection. Finally, we show that delivery of the hTGF-beta1 gene to osseous tissues in vivo results in significant changes in the epiphyseal plate primarily as a result of increased thickness of the provisional calcification zone
— id: 6186, year: 1999, vol: 14, page: 1290, stat: Journal Article,

Expression of high-affinity receptors for TGF-beta during rat cranial suture fusion
Mehrara BJ; Steinbrech DS; Saadeh PB; Gittes GK; Longaker MT
1999 May;42(5):502-508, Annals of plastic surgery
The etiology of craniosynostosis is unknown. The elucidation of the biological pathways responsible for this disorder has been hampered by an inability to evaluate cranial sutures before, during, and after cranial suture fusion. The programmed fusion of the rat posterofrontal (PF) suture postnatally provides an excellent model to study the molecular events that occur during cranial suture fusion. Previous experiments have implicated transforming growth factor beta (TGF-beta) growth factors in the regulation of PF suture fusion. The purpose of these experiments was to localize the expression of high-affinity receptors for these growth factors during cranial suture fusion. Four rats were sacrificed on postnatal days 8, 12, 17, and 40 (N = 16). The PF and sagittal sutures were harvested and prepared for immunohistochemical localization of TGF-beta receptor 1 and receptor 2 (Tbeta-RI, Tbeta-RII) protein. Results indicate that immunostaining for Tbeta-RI and Tbeta-RII is markedly increased in the dura mater and osteoblasts of the sutural margin of the PF suture during active suture fusion (on postnatal days 12, 17, and 40) compared with the osteoblasts and dura mater underlying the patent sagittal suture. These results, in combination with the authors' previous findings as well as studies supporting a role for TGF-beta molecules in the regulation of osteogenesis, implicate TGF-beta signaling in the regulation of suture fusion. The possible mechanisms of ligand-receptor interaction are discussed
— id: 56440, year: 1999, vol: 42, page: 502, stat: Journal Article,

Human cartilage engineering: chondrocyte extraction, proliferation, and characterization for construct development
Saadeh PB; Brent B; Mehrara BJ; Steinbrech DS; Ting V; Gittes GK; Longaker MT
1999 May;42(5):509-513, Annals of plastic surgery
To date, many efforts to engineer cartilage have focused on matrix construction with the goal of producing a durable construct as cartilage replaces the resorbing matrix. However, the importance of matrix construction is at least matched by the challenge of efficient chondrocyte extraction, culture expansion, and prevention of dedifferentiation. This challenge is underscored by the large number of chondrocytes needed for a clinically significant construct such as an ear. Because human rib provides a large, readily available source of hyaline cartilage, the authors evaluated human rib chondrocyte extraction and found that maximum viable cell yield occurred after a 6-hour digestion. They also evaluated human microtic auricular remnant chondrocyte extraction and identified fibroblast contamination as a shortcoming of this potential source of chondrocytes. Initially, rib chondrocytes proliferated in vitro with a doubling time of approximately 1 week. As the cells were passaged, proliferation decreased such that the cells stopped proliferating and adopted a large, spindle-shaped morphology by passage 6. Interestingly, no increase in proliferation was noted when rib chondrocytes were stimulated with transforming growth factor beta 1, bone morphogenetic protein 2, and basic fibroblast growth factor. The major obstacles to the use of autologous rib chondrocytes in matrix construction are the low cell yield from a small piece of rib and the limited proliferation that these cells will undergo in vitro. Further investigation of culture systems and mitogenic cytokines may help resolve these limitations
— id: 12010, year: 1999, vol: 42, page: 509, stat: Journal Article,

Transforming growth factor-beta1 modulates the expression of vascular endothelial growth factor by osteoblasts
Saadeh, P B; Mehrara, B J; Steinbrech, D S; Dudziak, M E; Greenwald, J A; Luchs, J S; Spector, J A; Ueno, H; Gittes, G K; Longaker, M T
1999 Oct;277(4 Pt 1):C628-C637, American journal of physiology
Angiogenesis is essential to both normal and pathological bone physiology. Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis, whereas transforming growth factor-beta1 (TGF-beta1) modulates bone differentiation, matrix formation, and cytokine expression. The purpose of this study was to investigate the relationship between TGF-beta1 and VEGF expression in osteoblasts and osteoblast-like cells. Northern blot analysis revealed an early peak of VEGF mRNA (6-fold at 3 h) in fetal rat calvarial cells and MC3T3-E1 osteoblast-like cells after stimulation with TGF-beta1 (2.5 ng/ml). The stability of VEGF mRNA in MC3T3-E1 cells was not increased after TGF-beta1 treatment. Actinomycin D inhibited the TGF-beta1-induced peak in VEGF mRNA, whereas cycloheximide did not. Blockade of TGF-beta1 signal transduction via a dominant-negative receptor II adenovirus significantly decreased TGF-beta1 induction of VEGF mRNA. Additionally, TGF-beta1 induced a dose-dependent increase in VEGF protein expression by MC3T3-E1 cells (P < 0.01). Dexamethasone similarly inhibited VEGF protein expression. Both TGF-beta1 mRNA and VEGF mRNA were concurrently present in rat membranous bone, and both followed similar patterns of expression during rat mandibular fracture healing (mRNA and protein). In summary, TGF-beta1-induced VEGF expression by osteoblasts and osteoblast-like cells is a dose-dependent event that may be intimately related to bone development and fracture healing
— id: 133222, year: 1999, vol: 277, page: C628, stat: Journal Article,

Analysis of TGF-beta production by fusing and nonfusing mouse cranial sutures in vitro
Sagiroglu JS; Mehrara BJ; Chau D; Saadeh PB; Gittes GK; Longaker MT
1999 May;42(5):496-501, Annals of plastic surgery
The role of transforming growth factor beta (TGF-beta) in the regulation of cranial suture fusion has been studied by various qualitative techniques such as in situ hybridization and immunohistochemistry. Although the relative expression of TGF-beta isoforms has been assessed in these studies, increased expression of TGF-beta has not been demonstrated in a quantitative fashion. Therefore, the purpose of this study was to quantify TGF-beta production by fusing (posterofrontal [PF]) and nonfusing (sagittal) mouse sutures using two different quantitative TGF-beta assays. The PF and sagittal sutures of 25-day-old mice were harvested and cultured separately in vitro. Culture media conditioned for 48 hours were collected after 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 days of culture, and total TGF-beta production was assessed using a TGF-beta bioassay. For a quantitative TGF-beta1 immunoassay, media conditioned for 48 hours were collected after 3, 5, 7, 9, 14, 22, and 28 days of culture. The TGF-beta bioassay revealed large amounts of total TGF-beta activity in both PF and sagittal sutures during the first week of culture, with decreasing amounts thereafter. Absolute TGF-beta activity in conditioned media collected from PF sutures at several early time points was higher than those obtained from sagittal sutures; however, these differences were not statistically significant. The results of the TGF-beta1 immunoassay (enzyme-linked immunosorbent assay) were similar to the bioassay in that the highest TGF-beta1 levels were noted during the first week of culture period and decreased thereafter. Analysis of variance of these samples, however, revealed significantly more TGF-beta1 protein in samples collected from the PF suture compared with the sagittal suture on days 3 and 5 of culture (p < 0.05). TGF-beta1 levels in the conditioned media obtained from PF sutures remained elevated compared with the sagittal suture on days 7 and 9; however, these differences were not statistically significant. Increased production of TGF-beta in the conditioned media of fusing PF sutures is the first such quantitative demonstration of growth factor upregulation during suture fusion and supports the hypothesis that TGF-beta expression may be important in cranial suture fusion
— id: 56439, year: 1999, vol: 42, page: 496, stat: Journal Article,

Median and ulnar palm-wrist studies
Sander, H W; Quinto, C; Saadeh, P B; Chokroverty, S
1999 Aug;110(8):1462-1465, Clinical neurophysiology
OBJECTIVES: Routine carpal tunnel electrodiagnosis frequently includes median (MPW) and ulnar (UPW) palm-wrist mixed nerve conduction latency determinations over 8 cm. Despite widespread use, normative palmar latency difference (PLD) and UPW values, and the relative utility of onset latency (OL) or peak latency (PL) measurements are controversial. The current study was conducted to determine normative values for these parameters. METHODS: MPW and UPW studies were performed unilaterally in 33 normal controls. The PLD-OL and PLD-PL were calculated. The mean, range, standard deviation, and upper limits of normal were determined. 74 hands (50 patients) with both clinical and electrophysiologic median neuropathy were also studied. RESULTS: The abnormal MPW and UPW cut-offs were both 1.8 ms (OL), and 2.3 ms (PL). The abnormal PLD cut-offs were 0.5 ms (OL and PL). Using either OL or PL, PLD parameters were similar within controls, and also within CTS patients. Using either OL or PL, UPW parameters were similar between controls and CTS patients. CONCLUSIONS: An abnormal PLD cut-off of 0.5 is recommended. This is slightly higher than some prior recommendations, however it should minimize the likelihood of false positive studies. Onset and peak latency measurements are likely to have similar clinical utility
— id: 112147, year: 1999, vol: 110, page: 1462, stat: Journal Article,

Sensitive median-ulnar motor comparative techniques in carpal tunnel syndrome
Sander, H W; Quinto, C; Saadeh, P B; Chokroverty, S
1999 Jan;22(1):88-98, Muscle & nerve
We describe two modified methods for median-to-ulnar motor conduction comparison in the diagnosis of median neuropathy at the wrist: the median-thenar to ulnar-thenar latency difference (TTLD), and the median-thenar to ulnar-hypothenar latency difference (THLD). We also describe an F-wave ulnar-to-median comparative test, the F-wave latency difference (FWLD). The abnormal cutoffs based upon 34 normal controls are: TTLD, 0.8 ms; THLD, 1.2 ms; FWLD, 0.6 ms. In 50 patients (79 hands) with clinically defined carpal tunnel syndrome and electrophysiological evidence of median neuropathy at the wrist (based upon a prolonged median nerve palm-wrist latency), the diagnostic sensitivities were: 95-98%, 85-88%, and 75-78%, respectively. These tests are therefore highly sensitive. They are easily performed and require minimal additional effort to incorporate into commonly used clinical electrodiagnostic routines. They may be advantageous when a concomitant polyneuropathy is present, and they may also help avoid technical pitfalls and aid in identification of anatomic variants
— id: 112152, year: 1999, vol: 22, page: 88, stat: Journal Article,

Diaphragmatic denervation in intensive care unit patients
Sander, H W; Saadeh, P B; Chandswang, N; Greenbaum, D; Chokroverty, S
1999 Jan-Feb;39(1):3-5, Electromyography & clinical neurophysiology
The causes of prolonged requirement for mechanical ventilation in the intensive care unit (ICU) are currently a subject of investigation. Critical illness polyneuropathy (CIP), an axonal polyneuropathy that frequently occurs with prolonged sepsis and multi-organ failure, has been cited as a frequent cause of difficulty with weaning from a ventilator. The relative contribution of diaphragmatic denervation in ICU patients with and without CIP has not been definitively determined. We reviewed 102 ventilator dependent intensive care unit (ICU) patients. Critical illness polyneuropathy (CIP) was diagnosed based upon electrodiagnostic criteria. Electrodiagnostic studies included diaphragmatic needle electromyography (EMG) to evaluate for diaphragmatic denervation. The medical charts of the patients with diaphragmatic denervation were reviewed for etiologies other than CIP for the diaphragmatic denervation. Our results suggest: 1) Respiratory impairment in ICU patients may often be unrelated to either CIP or diaphragmatic denervation; 2) Only about half of ventilator dependent CIP patients have diaphragmatic denervation; 3) Diaphragmatic denervation in ICU patients frequently may be attributable to causes other than CIP
— id: 112151, year: 1999, vol: 39, page: 3, stat: Journal Article,

Trapezius CMAP amplitude asymmetry in accessory neuropathy
Sander, H W; Saadeh, P B; D'Alessandri, C J; Chokroverty, S
1999 Oct-Dec;39(7):411-414, Electromyography & clinical neurophysiology
In accessory neuropathy electrodiagnosis, upper trapezius compound muscle action potential (CMAP) latencies and amplitudes are commonly measured. The few prior reports describing middle and lower trapezius recording have traditionally emphasized latency value determination. The utility of amplitude measurement with middle and lower trapezius recording has not, to our knowledge, been previously described in individual patients with accessory neuropathy. We report three patients (A-C) who developed unilateral accessory neuropathy following surgical procedures. Accessory nerve conduction studies were performed with surface recording over the upper, middle, and lower trapezius muscles. Latency values were normal except for a prolonged lower trapezius latency value in patient B. Side-side trapezius amplitude comparisons revealed striking asymmetries from all three recording sites in patients A and B (71-95% CMAP amplitude decrements) and in the lower trapezius recording of patient C. Middle and lower trapezius side-side CMAP amplitude comparisons may increase the sensitivity of accessory neuropathy electrodiagnosis
— id: 112145, year: 1999, vol: 39, page: 411, stat: Journal Article,

Fibroblast response to hypoxia: the relationship between angiogenesis and matrix regulation
Steinbrech DS; Longaker MT; Mehrara BJ; Saadeh PB; Chin GS; Gerrets RP; Chau DC; Rowe NM; Gittes GK
1999 Jun 15;84(2):127-133, Journal of surgical research
A number of studies have demonstrated the critical role of angiogenesis for successful wound repair in the surgical patient. Vascular disruption from tissue injury due to trauma or surgery leads to a hypoxic zone in the healing wound. In this dynamic process, angiogenesis is vital for the delivery of oxygen, nutrients, and growth factors necessary to initiate the synthetic processes of wound healing. Fibroblasts, invading the wound early in the healing process, are involved in extracellular matrix (ECM) deposition as well as wound contraction. However, the exact mechanisms by which important genes are regulated remain unknown. In order to examine these processes, we studied the effects of hypoxia on fibroblasts for the expression of VEGF, type IalphaI collagen, and matrix-metalloproteinase-3, three genes essential for the regulation of angiogenesis, ECM deposition, and ECM degradation in wound healing. Primary cell cultures of normal human dermal fibroblasts (NHDFs) were placed in hypoxia for varying periods of time. Northern blot hybridization was performed with [alpha32P]dCTP-labeled cDNA probes for VEGF, type IalphaI collagen, and MMP-3. The results demonstrated a time-dependent VEGF mRNA upregulation (470% of baseline) under hypoxia. Type IalphaI collagen increased (170% of baseline) at 24 h, but was then abruptly downregulated to 3.8% of baseline at 48 h. MMP-3 was incrementally downregulated to 2.2% of baseline at 48 h. These experiments focused on the effect of hypoxia on genes thought to play a role in wound repair. VEGF upregulation in the hypoxic microenvironment of the early wound may serve to stimulate angiogenesis. Type IalphaI collagen, though upregulated early on, was abruptly downregulated at 48 h. This downregulation may reflect the in vivo requirement for angiogenesis to deliver oxygen for successful hydroxylation and collagen synthesis in the wound. MMP-3, also downregulated at 48 h, may also implicate the need for angiogenesis. These data support the theory that hypoxia-driven angiogenesis is critical for ECM formation and remodeling in successful soft tissue repair. Furthermore, they may represent the role of hypoxia as an important regulator to efficiently balance these complex processes in the healing wound
— id: 57543, year: 1999, vol: 84, page: 127, stat: Journal Article,

Hypoxia upregulates VEGF production in keloid fibroblasts
Steinbrech DS; Mehrara BJ; Chau D; Rowe NM; Chin G; Lee T; Saadeh PB; Gittes GK; Longaker MT
1999 May;42(5):514-519, Annals of plastic surgery
The etiology of keloid formation is diverse. They are characterized grossly as thick scar tissue that extends beyond the boundaries of the original wound. Histologically, keloids are composed of excessive collagen with an abnormally large number of partially or totally occluded microvessels. This occlusion of keloid microvessels has been hypothesized to contribute to a hypoxic microenvironment within these pathological scars. Vascular endothelial growth factor (VEGF), a potent endothelial cell mitogen, and proangiogenic cytokine have been implicated in normal and pathological wound healing. The purpose of this study was to evaluate the amount of VEGF protein production by fibroblast cell lines derived from keloids and normal human dermal skin in hypoxic compared with normoxic culture conditions. By enzyme-linked immunosorbent protein assay, VEGF was increased in both keloid and normal human dermal fibroblasts in hypoxia over normoxic controls. There was not, however, a significant difference between upregulation of VEGF protein when comparing the keloid and normal fibroblast groups. As the result of the data, alternative hypotheses for hypoxia-induced keloid formation were explored: (1) downstream modulation or signal transduction of VEGF, (2) VEGF production from cells other than fibroblasts, (3) the importance of matrix accumulation stimulated by hypoxia, or (4) increased migration of cells (other than fibroblasts) specific to keloid biology. These hypotheses may help explain the possible role of hypoxia in the pathogenesis of keloid formation. Future studies involving in situ hybridization or immunohistochemical analysis may offer greater insight into the mechanisms underlying keloid formation. Ultimately, our therapeutic goal is the utilization of biomolecular approaches for the suppression of keloid formation
— id: 12009, year: 1999, vol: 42, page: 514, stat: Journal Article,

Gene expression of insulin-like growth factors I and II in rat membranous osteotomy healing
Steinbrech DS; Mehrara BJ; Rowe NM; Dudziak ME; Saadeh PB; Gittes GK; Longaker MT
1999 May;42(5):481-487, Annals of plastic surgery
Poorly healing mandibular osteotomies can be a difficult problem in reconstructive surgery. Many therapies have been attempted to augment the healing of mandibular fractures, defects, or osteotomies, but these methods have substantial drawbacks or have been ineffective. The difficulty in treating poorly healing bony defects has led to the exploration of gene therapy as a possible approach to supplement or accelerate mandibular fracture healing. To understand at what point the introduction of a suitable gene candidate might be of benefit in mandibular healing, it is imperative to examine the temporal expression of bone growth factors in a model of membranous bone healing. Insulinlike growth factors (IGFs) I and II are two such bone growth factor candidates because of their known potent in vitro as well as in vivo effects on bone formation. In this study the authors demonstrate the temporal pattern of IGF I and IGF II gene expression during mandibular osteotomy healing using a rat model. Their data reveal that IGF I and IGF II were elevated 7 days after a mandibular osteotomy that was held in external fixation. The upregulation of IGF I and IGF II during mandibular bone healing underscores the importance of these growth factors in bone repair. Gene therapy utilizing recombinant viral constructs containing IGFs I and II may be of benefit during mandibular bone healing in an effort to augment clinical scenarios of poor or retarded bony repair
— id: 12011, year: 1999, vol: 42, page: 481, stat: Journal Article,

Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro
Steinbrech DS; Mehrara BJ; Saadeh PB; Chin G; Dudziak ME; Gerrets RP; Gittes GK; Longaker MT
1999 Sep;104(3):738-747, Plastic & reconstructive surgery
Numerous studies have demonstrated the critical role of angiogenesis for successful osteogenesis during endochondral ossification and fracture repair. Vascular endothelial growth factor (VEGF), a potent endothelial cell-specific cytokine, has been shown to be mitogenic and chemotactic for endothelial cells in vitro and angiogenic in many in vivo models. Based on previous work that (1) VEGF is up-regulated during membranous fracture healing, (2) the fracture site contains a hypoxic gradient, (3) VEGF is up-regulated in a variety of cells in response to hypoxia, and (4) VEGF is expressed by isolated osteoblasts in vitro stimulated by other fracture cytokines, the hypothesis that hypoxia may regulate the expression of VEGF by osteoblasts was formulated. This hypothesis was tested in a series of in vitro studies in which VEGF mRNA and protein expression was assessed after exposure of osteoblast-like cells to hypoxic stimuli. In addition, the effects of a hypoxic microenvironment on osteoblast proliferation and differentiation in vitro was analyzed. These results demonstrate that hypoxia does, indeed, regulate expression of VEGF in osteoblast-like cells in a dose-dependent fashion. In addition, it is demonstrated that hypoxia results in decreased cellular proliferation, decreased expression of proliferating cell nuclear antigen, and increased alkaline phosphatase (a marker of osteoblast differentiation). Taken together, these data suggest that osteoblasts, through the expression of VEGF, may be in part responsible for angiogenesis and the resultant increased blood flow to fractured bone segments. In addition, these data provide evidence that osteoblasts have oxygen-sensing mechanisms and that decreased oxygen tension can regulate gene expression, cellular proliferation, and cellular differentiation
— id: 6180, year: 1999, vol: 104, page: 738, stat: Journal Article,

Transforming growth factor beta superfamily members in cartilage repair
Frenkel S; Saadeh P; Mehrara B; Steinbrech D; Gittes G; Longaker MT
1998 ;49:516-518, Surgical forum
— id: 105479, year: 1998, vol: 49, page: 516, stat: Journal Article,

Phrenic nerve conduction studies in the intensive care unit
Saadeh, P B; Sander, H W
1996 Aug;19(8):1057-1058, Muscle & nerve
— id: 112166, year: 1996, vol: 19, page: 1057, stat: Journal Article,