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Improving Senescent Wound Healing With Local and Systemic Therapies

Szpalski, Caroline; Butala, Parag; Vandegrift, Meredith T; Knobel, Denis; Allen, Robert J; Saadeh, Pierre B; Warren, Stephen M
The population is aging, and the prevalence of chronic wounds is increasing. Because neovascularization is essential for tissue repair and both local and systemic factors affect new blood vessel formation, we hypothesize that altering either pathway would reciprocally enhance wound healing in the aged. To test this hypothesis, p53 was locally suppressed and endothelial progenitor cells (EPCs) were systemically mobilized in a murine model of senescent wound healing.Bilateral 6-mm full-thickness stented wounds were made on the dorsum of Zmpste24 mice. Animals received weekly topical p53 small interfering RNA (siRNA) (n = 25), weekly topical nonsense siRNA (n = 25), daily subcutaneous AMD3100 injections (n = 25), or daily subcutaneous saline injections (n = 25). Wounds were photographically assessed and harvested for reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunostaining over 40 days. Circulating EPC levels were measured using fluorescence-activated cell sorting analysis.Local p53 siRNA significantly improved Zmpste24 wound healing (18 ± 2 vs 40 ± 3 days; P ≤ 0.0001). p53 siRNA significantly increased local provasculogenic factors (hypoxia-inducible factor 1 α, stromal cell-derived factor 1 α, and vascular endothelial growth factor; P ≤ 0.05) and decreased local proapoptotic factors (p53, PUMA, and Bax; P ≤ 0.05). Local p53 siRNA also significantly increased the number of circulating EPCs (8 ± 0.2% vs 2.6 ± 0.1%; P ≤ 0.0001). AMD3100 treatment also significantly improved wound healing (20 ± 2 vs 40 ± 3 days; P ≤ 0.0001) and increased EPCs mobilization (7.8 ± 0.4% vs 2.6 ± 0.1%; P ≤ 0.0001). In addition, systemic AMD3100 increased local provasculogenic factors (hypoxia-inducible factor 1 α, stromal cell-derived factor 1 α, and vascular endothelial growth factor; P ≤ 0.05) and decreased local proapoptotic factors (p53, PUMA, and Bax; P ≤ 0.05). Both treatments significantly increased the number of blood vessels in the wound bed (P ≤ 0.0001).The marked delay in Zmpste24 wound healing is significantly improved by local (p53 siRNA) and systemic (AMD3100) treatments. The resulting decrease in proapoptotic factors and increase in provasculogenic factors in the wound bed as well as the increased level of circulating EPCs appear to reverse age-related wound healing impairment by enhancing wound neovascularization.
PMID: 29781855
ISSN: 1536-3708
CID: 3129732

Five Year Follow-Up of Midface Distraction in Growing Children with Syndromic Craniosynostosis

Patel, Parit A; Shetye, Pradip; Warren, Stephen M; Grayson, Barry H; McCarthy, Joseph G
BACKGROUND: Maxillary position in patients with syndromic craniosynostosis after midface distraction has been shown to be stable 1 year postoperatively. The purpose of this study is to assess midfacial position in the growing child with craniosynostosis 5 years after Le Fort III advancement with a rigid external device (RED). METHODS: Seventeen consecutive patients were identified to have the diagnosis of syndromic craniosynostosis and who underwent midface advancement. There were 10 males and 7 females, 7 patients had Crouzon syndrome, 5 had Apert syndrome, and 5 had Pfeiffer syndrome. A standard subcranial Le Fort III osteotomy was performed. Cephalometric analysis was performed to assess the position of the maxilla. RESULTS: After device removal, orbitale advanced 13.67 mm along the x axis and downward 1.70 mm along the y axis. A Point advanced 15.97 mm along the x axis and downward 1.14 mm along the y axis. At 1 year post-distraction, both orbitale and A point had advanced an additional 0.47 mm and 0.24 mm along the x axis and downward 0.58 mm and 1.78 mm along the y axis respectively. At 5 years post-distraction, orbitale moved posterior 0.58 mm and A point advanced an additional 2.08 mm along the x axis. Orbitale and A point descended 3.23 mm and 5.2 mm along the y axis respectively. CONCLUSIONS: After Le Fort III advancement with distraction, the maxillary position remains stable and continues to advance minimally along the x axis and demonstrates more growth along the y axis over the long term.
PMID: 28820838
ISSN: 1529-4242
CID: 2670672

The Effect of Processing Technique on Fat Graft Survival

Canizares, Orlando Jr; Thomson, Jennifer E; Allen, Robert J Jr; Davidson, Edward H; Tutela, John P; Saadeh, Pierre B; Warren, Stephen M; Hazen, Alexes
BACKGROUND: Wide variations in fat graft survival have been reported. The authors hypothesize that treating the adipose tissue on Telfa gauze creates a processed lipoaspirate with a more functional adipokine profile that improves fat graft survival. METHODS: Suction-assisted lipoaspirate was harvested from humans and was either processed by centrifugation, rolled on Telfa gauze, or left unprocessed. Progenitor cell populations were quantified and characterized by flow cytometry. Glycerol-3-phosphate dehydrogenase assay was used to measure the functional adipocytes. The lipoaspirates were grafted into (n = 45) wild-type mice and harvested to assess fat graft persistence. Vascular endothelial growth factor and platelet-derived growth factor-BB secretions were measured by enzyme-linked immunosorbent assay technique. RESULTS: Centrifuged lipoaspirate had a greater number of progenitor cells per gram of tissue than Telfa-processed and unprocessed lipoaspirate. However, Telfa-processed lipoaspirate had a greater number of functional adipocytes (0.104 U/ml) than centrifuged (0.080 U/ml) and unprocessed lipoaspirate (0.083 U/ml) on glycerol-3-phosphate dehydrogenase assay (p < 0.05). After 10 weeks of grafting, it had greater fat graft persistence (70.9 +/- 6.2 percent) than centrifuged (56.7 +/- 5.5 percent) and unprocessed lipoaspirate (42.2 +/- 2.7 percent) (p < 0.05). It also maintained a greater secretion of vascular endothelial growth factor and platelet-derived growth factor-BB at weeks 1 and 2 than centrifuged and unprocessed lipoaspirate. Furthermore, CD31 staining demonstrated an increase in vascular density of the Telfa-processed lipoaspirate at week 2 compared with the centrifuged lipoaspirate (37 +/- 1 percent and 14 +/- 4 percent per high-power field; p < 0.05). CONCLUSIONS: Lipoaspirate processing technique has a significant impact on fat graft survival rate. Increasing the number of functional adipocytes by processing the fat on Telfa gauze may augment the secretion of angiogenic and mitogenic adipokines within the graft, thereby improving its survivability. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.
PMID: 29068928
ISSN: 1529-4242
CID: 2756582

Discussion on: Nasoalveolar Molding Therapy for the Treatment of Unilateral Cleft Lip and Palate Improves Nasal Symmetry and Maxillary Alveolar Dimensions

Alperovich, Michael; Brecht, Lawrence E; Warren, Stephen M
PMID: 28005738
ISSN: 1536-3732
CID: 2374512

Design and validation of a dynamic cell-culture system for bone biology research and exogenous tissue-engineering applications

Allori, Alexander C; Davidson, Edward H; Reformat, Derek D; Sailon, Alexander M; Freeman, James; Vaughan, Adam; Wootton, David; Clark, Elizabeth; Ricci, John L; Warren, Stephen M
Bone lacunocanalicular fluid flow ensures chemotransportation and provides a mechanical stimulus to cells. Traditional static cell-culture methods are ill-suited to study the intricacies of bone biology because they ignore the three-dimensionality of meaningful cellular networks and the lacunocanalicular system; furthermore, reliance on diffusion alone for nutrient supply and waste product removal effectively limits scaffolds to 2-3 mm thickness. In this project, a flow-perfusion system was custom-designed to overcome these limitations: eight adaptable chambers housed cylindrical cell-seeded scaffolds measuring 12 or 24 mm in diameter and 1-10 mm in thickness. The porous scaffolds were manufactured using a three-dimensional (3D) periodic microprinting process and were composed of hydroxyapatite/tricalcium phosphate with variable thicknesses, strut sizes, pore sizes and structural configurations. A multi-channel peristaltic pump drew medium from parallel reservoirs and perfused it through each scaffold at a programmable rate. Hermetically sealed valves permitted sampling or replacement of medium. A gas-permeable membrane allowed for gas exchange. Tubing was selected to withstand continuous perfusion for > 2 months without leakage. Computational modelling was performed to assess the adequacy of oxygen supply and the range of fluid shear stress in the bioreactor-scaffold system, using 12 x 6 mm scaffolds, and these models suggested scaffold design modifications that improved oxygen delivery while enhancing physiological shear stress. This system may prove useful in studying complex 3D bone biology and in developing strategies for engineering thick 3D bone constructs
PMID: 24027138
ISSN: 1932-6254
CID: 957902

Basal View Reference Photographs for Nasolabial Appearance Rating in Unilateral Cleft Lip and Palate

Rubin, Marcie S; Lowe, Kristen M; Clouston, Sean; Shetye, Pradip R; Warren, Stephen M; Grayson, Barry H
The Asher-McDade system is a 5-point ordinal scale frequently used to rate the components of nasolabial appearance, including nasal form and nasal symmetry, in unilateral cleft lip and palate. Although reference photographs illustrating this scale have been identified for the frontal and right profile view, no reference photographs exist for the basal view. The aim of this study was to identify reference photographs for nasal form and nasal symmetry from the basal view to illustrate this scale and facilitate its use. Four raters assessed nasolabial appearance (form and symmetry) on basal view photographs of 50 children (average age 8 years) with a repaired cleft lip. Intraclass correlation coefficients show fair to moderate inter-rater reliability. Cronbach alpha indicated strong agreement between raters (0.77 nasal form; 0.78 nasal symmetry; 0.80 overall), along with low duplicate measurement error and strong internal consistency between the measures. The photographs with the highest agreement among raters were selected to illustrate each point on the 5-point scale for nasal form and for nasal symmetry, resulting in the selection of 10 reference photographs. The basal view reference photograph set developed from this study may complement existing reference photograph sets for other views and facilitate rating tasks.
PMID: 26163840
ISSN: 1536-3732
CID: 1668602

Comparative Study of Early Secondary Nasal Revisions and Costs in Patients With Clefts Treated With and Without Nasoalveolar Molding

Patel, Parit A; Rubin, Marcie S; Clouston, Sean; Lalezaradeh, Frank; Brecht, Lawrence E; Cutting, Court B; Shetye, Pradip R; Warren, Stephen M; Grayson, Barry H
The present study aims to determine the risk of early secondary nasal revisions in patients with complete unilateral and bilateral cleft lip and palate (U/BCLP) treated with and without nasoalveolar molding (NAM) and examine the associated costs of care. A retrospective cohort study from 1990 to 1999 was performed comparing the risk of early secondary nasal revision surgery in patients with a CLP treated with NAM and surgery (cleft lip repair and primary surgical nasal reconstruction) versus surgery alone in a private practice and tertiary level clinic. The NAM treatment group consisted of 172 patients with UCLP and 71 patients with BCLP, whereas the non-NAM-prepared group consisted of 28 patients with UCLP and 5 with BCLP. The risk of secondary nasal revision for patients with UCLP was 3% in the NAM group and 21% in the non-NAM group. The risk of secondary nasal revision for patients with BCLP was 7% in the NAM group compared with 40% in the non-NAM group. Using multicenter averages, the non-NAM revision rates were calculated at 37.8% and 48.5% for U/BCLP, respectively. Applying these risks of revision, NAM treatment led to an estimated savings of between $491 and $4893 depending on the type of cleft. In conclusion, NAM can reduce the number of early secondary nasal revision surgeries and, therefore, reduce the overall cost of care.
PMID: 26080163
ISSN: 1536-3732
CID: 1632252

Unilateral Craniofacial Microsomia: Unrecognized Cause of Pediatric Obstructive Sleep Apnea

Szpalski, Caroline; Vandegrift, Meredith; Patel, Parit A; Appelboom, Geoffrey; Fisher, Mark; Marcus, Jeffrey; McCarthy, Joseph G; Shetye, Pradip R; Warren, Stephen M
Bilateral craniofacial microsomia causes obstructive sleep apnea (OSA). We hypothesize that unilateral craniofacial microsomia (UCFM) is an underappreciated cause of OSA. The records of all pediatric UCFM patients from 1990 to 2010 were reviewed; only complete records were included in the study. UCFM patients with OSA (apnea hypopnea index >1/hr) were compared to UCFM patients without OSA. Univariate and multivariate Fisher and chi tests were performed. Of the 62 UCFM patients, 7 (11.3%) had OSA. All OSA patients had Pruzansky IIB or III mandibles. OSA patients presented with snoring (71.4%), failure to thrive (FTT) (57.1%), and chronic respiratory infections (42.8%). Snoring (P < 0.001), Goldenhar syndrome (P = 0.001), and FTT (P = 0.004) were significantly associated with OSA, but race, obesity, clefts, respiratory anomalies, adenotonsillar hypertrophy, and laterality were not. The prevalence of OSA in UCFM patients is up to 10 times greater than in the general population. Snoring, Goldenhar syndrome, and FTT are significantly associated with the presence of OSA.
PMID: 26080175
ISSN: 1536-3732
CID: 1704042

Acellular dermal matrix-based gene therapy augments graft incorporation

Vandegrift, Meredith T; Szpalski, Caroline; Knobel, Denis; Weinstein, Andrew; Ham, Maria; Ezeamuzie, Obinna; Warren, Stephen M; Saadeh, Pierre B
BACKGROUND: Acellular dermal matrix (ADM) is widely used for structural or dermal replacement purposes. Given its innate biocompatibility and its potential to vascularize, we explored the possibility of ADM to function as a small interfering RNA (siRNA) delivery system. Specifically, we sought to improve ADM vascularization by siRNA-mediated inhibition of prolyl hydroxylase domain-2 (PHD2), a cytoplasmic protein that regulates hypoxia inducible factor-1alpha, and improve neovascularization. MATERIALS AND METHODS: Fluorescently labeled siRNA was used to rehydrate thin implantable ADM. Pharmacokinetic release of siRNA was determined. Twelve millimeter sections of ADM reconstituted with PHD2 siRNA (nonsense siRNA as control) and applied to dorsal wounds of 40 FVB mice. Grafts were sewn in, bolstered, and covered with occlusive dressings. Photographs were taken at 0, 7, and 14 d. Wounds were harvested at 7 and 14 d and analyzed (messenger RNA, protein, histology, and immunohistochemistry). RESULTS: Release kinetics was first-order with 80% release by 12 h. By day 14, PHD2-containing ADM appeared viable and adherent, whereas controls appeared nonviable and nonadherent. Real-time reverse transcription-polymerase chain reaction demonstrated near-complete knockdown of PHD2, whereas vascular endothelial growth factor and FGF-2 were increased 2.3- and 4.7-fold. On enzyme-linked immunosorbent assay, vascular endothelial growth factor was increased more than fourfold and stromal cell-derived factor doubled. Histology demonstrated improved graft incorporation in treated groups. Immunohistochemical demonstrated increased vascularity measured by CD31 staining and increased new cell proliferation by denser proliferating cell nuclear antigen staining in treated versus controls. CONCLUSIONS: We concluded that ADM is an effective matrix for local delivery of siRNA. Strategies to improve the matrix and/or genetically alter the local tissue environment can be envisioned.
PMID: 25676463
ISSN: 1095-8673
CID: 1604522

Assessment of Presurgical Clefts and Predicted Surgical Outcome in Patients Treated With and Without Nasoalveolar Molding

Rubin, Marcie S; Clouston, Sean; Ahmed, Mohammad M; M Lowe, Kristen; Shetye, Pradip R; Broder, Hillary L; Warren, Stephen M; Grayson, Barry H
Obtaining an esthetic and functional primary surgical repair in patients with complete cleft lip and palate (CLP) can be challenging because of tissue deficiencies and alveolar ridge displacement. This study aimed to describe surgeons' assessments of presurgical deformity and predicted surgical outcomes in patients with complete unilateral and bilateral CLP (UCLP and BCLP, respectively) treated with and without nasoalveolar molding (NAM). Cleft surgeon members of the American Cleft Palate-Craniofacial Association completed online surveys to evaluate 20 presurgical photograph sets (frontal and basal views) of patients with UCLP (n = 10) and BCLP (n = 10) for severity of cleft deformity, quality of predicted surgical outcome, and likelihood of early surgical revision. Five patients in each group (UCLP and BCLP) received NAM, and 5 patients did not receive NAM. Surgeons were masked to patient group. Twenty-four percent (176/731) of surgeons with valid e-mail addresses responded to the survey. For patients with UCLP, surgeons reported that, for NAM-prepared patients, 53.3% had minimum severity clefts, 58.9% were anticipated to be among their best surgical outcomes, and 82.9% were unlikely to need revision surgery. For patients with BCLP, these percentages were 29.8%, 38.6%, and 59.9%, respectively. Comparing NAM-prepared with non-NAM-prepared patients showed statistically significant differences (P < 0.001), favoring NAM-prepared patients. This study suggests that cleft surgeons assess NAM-prepared patients as more likely to have less severe clefts, to be among the best of their surgical outcomes, and to be less likely to need revision surgery when compared with patients not prepared with NAM.
PMID: 25534051
ISSN: 1049-2275
CID: 1415912