Saleh M. Shenaq

Increased free fat-graft survival with the long-term, local delivery of insulin, insulin-like growth factor-I, and basic fibroblast growth factor by PLGA/PEG microspheres.

&NA; The present investigation evaluates the effects of longterm, local delivery of insulin, insulin‐like growth factor‐1 (IGF‐1), and basic fibroblast growth factor (bFGF) on fatgraft survival using a poly (lactic‐co‐glycolic‐acid)‐polyethylene glycol (PLGA/PEG) microsphere delivery system. Twelve‐micrometer PLGA/PEG microspheres incorporated separately with insulin, IGF‐1, and bFGF were manufactured using a double‐emulsion solvent‐extraction technique. Inguinal fat from Sprague Dawley rats was harvested, diced, washed, and mixed with (1) insulin microspheres, (2) insulin‐like growth factor‐1 microspheres, (3) basic fibroblast growth factor microspheres, (4) a combination of the insulin and IGF‐1 microspheres, and (5) a combination of insulin, IGF‐1, and bFGF microspheres. The treated fat grafts were implanted autologously into subdermal pockets in six animals for each group. Animals receiving untreated fat grafts and fat grafts treated with blank microspheres constituted two external control groups (six animals per external control group). At 12 weeks, all fat‐graft groups were compared on the basis of weight maintenance and a histomorphometric analysis of adipocyte area percentage, indices of volume retention and cell composition, respectively. Weight maintenance was defined as the final graft weight as a percent of the implanted graft weight. All growth factor treatments significantly increased fat‐graft weight maintenance objectively, and volume maintenance grossly, in comparison with the untreated and blank microsphere‐treated controls. Treatment with insulin and IGF‐1, alone or in combination, was found to increase the adipocyte area percentage in comparison with fat grafts treated with bFGF alone or in combination with other growth factors. In conclusion, the findings of this study indicate that long‐term, local delivery of growth factors with PLGA/PEG microspheres has the potential to increase fat‐graft survival rates. Further, the type of growth factor delivered may influence the cellular/stromal composition of the grafted tissue. (Plast. Reconstr. Surg. 105: 1712, 2000.)

Targeted expression of IGF-1 transgene to skeletal muscle accelerates muscle and motor neuron regeneration

Currently, there is no known medical treatment that hastens the repair of damaged nerve and muscle. Using IGF‐1 transgenic mice that specifically express human recombinant IGF‐1 in skeletal muscle, we test the hypotheses that targeted gene expression of IGF‐1 in skeletal muscle enhances motor nerve regeneration after a nerve crush injury. The IGF‐1 transgene affects the initiation of the muscle repair process after nerve injury as shown by increased activation of SCA‐1positive myogenic stem cells. Increased satellite cell differentiation and proliferation are observed in IGF‐1 transgenic mice, shown by increased expression of Cyclin D1, MyoD, and myogenin. Expression of myogenin and nicotinic acetylcholine receptor subunits, initially increased in both wild‐type and IGF‐1 transgenic mice, are restored to normal levels at a faster rate in IGF‐1 transgenic mice, which indicates a rescue of nerve‐evoked muscle activity. Expression of the IGF‐1 transgene in skeletal muscle results in accelerated recovery of saltatory nerve conduction, increased innervation as detected by neurofilament expression, and faster recovery of muscle mass. These studies demonstrate that local expression of IGF‐1 augments the repair of injured nerve and muscle.

De novo adipose tissue generation through long-term, local delivery of insulin and insulin-like growth factor-1 by PLGA/PEG microspheres in an in vivo rat model: A novel concept and capability

&NA; This study was undertaken to characterize the duration of long‐term growth factor delivery by poly(lactic‐co‐glycolicacid)‐polyethylene glycol (PLGA/PEG) microspheres and to evaluate the potential of long‐term delivery of insulin and insulin‐like growth factor‐1 (IGF‐1) for the de novo generation of adipose tissue in vivo. PLGA/PEG microspheres containing insulin and IGF‐1, separately, were produced by a double‐emulsion solvent‐extraction technique. In the first phase of the experiment, the in vitro release kinetics of the microspheres were evaluated for the optical density and polyacrylamide gel electrophoresis of solutions incubated with insulin‐containing microspheres for four different periods of time (n = 1). The finding of increased concentrations of soluble insulin with increased incubation time confirmed continual protein release. In the second stage of the experiment, 16 rats were divided equally into four study groups (insulin, IGF‐1, insulin + IGF‐1, and blank microspheres) (n = 4). Insulin and IGF‐1 containing microspheres were administered directly to the deep muscular fascia of the rat abdominal wall to evaluate the potential for de novo adipose tissue generation via adipogenic differentiation from native nonadipocyte cell pools in vivo. Animals treated with blank microspheres served as an external control group. At the 4‐week harvest period, multiple ectopic islands of adipose tissue were observed on the abdominal wall of the animals treated with insulin, IGF‐1, and insulin + IGF‐1 microspheres. Such islands were not seen in the blank micro sphere group. Hematoxylin and eosin‐stained sections of the growth factor groups demonstrated mature adipocytes interspersed with fibrous tissue superficial to the abdominal wall musculature and continuous with the fascia. Oil‐Red‐O stained sections demonstrated that these cells contained lipid. Computer‐aided image analysis of histologic sections confirmed that there were statistically significant increases in the amount of “ectopic” adipose neotissue developed on the abdominal wall of animals treated with growth factor microspheres. In conclusion, this study confirms the long‐term release of proteins from PLGA/PEG microspheres up to 4 weeks and demonstrates the potential of long‐term local insulin and IGF‐1 to induce adipogenic differentiation to mature lipid‐containing adipocytes from nonadipocyte cell pools in vivo at 4 weeks. (Plast. Reconstr. Surg. 105: 1721, 2000.)

Local Overexpression of Thrombomodulin for In Vivo Prevention of Arterial Thrombosis in a Rabbit Model

-Endothelial thrombomodulin plays a critical role in hemostasis by binding thrombin and subsequently converting protein C to its active form, a powerful anticoagulant. Thrombomodulin thus represents a central mechanism by which patency is maintained in normal vessels. However, thrombomodulin expression decreases in perturbed endothelial cells, predisposing to thrombotic occlusion. An adenoviral construct expressing thrombomodulin (Adv/RSV-THM) was created and functionally characterized in vitro and in vivo. The impact of local overexpression of thrombomodulin on in vivo thrombus formation was subsequently examined in a stasis/injury model of arterial thrombosis. The construct prevented arterial thrombosis formation in all animals, while viral and nonviral controls typically developed occluding thrombi. By histological analysis, nonviral controls exhibited intravascular thrombus occluding a mean of 70.52+/-3.72% of available lumen, while viral controls reached 86. 85+/-2.82% thrombotic occlusion; in contrast, Adv/RSV-THM reduced thrombosis to 28.61+/-3.31% of lumen in cross section. No significant intima-to-media ratio was observed in the thrombomodulin group relative to controls. Local infiltration of granulocytes and macrophages significantly decreased in the Adv/RSV-THM group relative to controls, while neutrophilic infiltration increased in viral controls relative to nonviral controls. This construct thus offers a viable technique for promoting a locally thromboresistant small-caliber artery, without the inflammatory damage that has limited many other adenoviral applications.

Modulation of peripheral nerve regeneration: a tissue-engineering approach. The role of amnion tube nerve conduit across a 1-centimeter nerve gap.

A new type of a biodegradable nerve graft conduit material, the amnion tube, has been developed in our laboratory. To test the tube in the peripheral nerve regeneration process, it was initially applied across a 1-cm sciatic nerve gap in rats and was compared with other nerve conduit materials. We used male Sprague-Dawley rats as our animal model. The experiment included 66 rats that were randomly assigned into five groups: autograft (n = 17), amnion tube (n = 19), silicone tube (n = 20), no repair (n = 7), and sham group (n = 3). The process of peripheral nerve regeneration was evaluated at 2, 4, 10, and 17 weeks following injury and repair by using morphologic and functional assessments of the outcome of nerve regeneration in each animal. Nerve regeneration across the amnion tube nerve conduit was comparable with that seen in autograft and superior to that of the silicone group. A uniform nerve tissue was seen filling and crossing the amnion conduit, and the regenerated nerve from the proximal stump reached the distal end and was undifferentiated from the normal nerve tissues. At 4 months, the amnion tube biodegraded and no longer could be identified and differentiated from the nerve tissues. The amnion tube animal group showed a number of axons very close to that in the nerve autograft group (37,157 versus 33,054). Functional recovery at a 2- to 4-week interval was significantly statistically higher only in the amnion tube animal group (p = 0.01). However, the improvement disappeared between 10 and 17 weeks. In conclusion, the amnion tube is a potential ideal nerve conduit material secondary to its unique characteristics: it contains important neurotropic factors, is biodegradable, provokes a very weak immune response, is semiflexible, is readily available, and is easily manufactured into different sizes and diameters. (Plast. Reconstr. Surg. 105: 660, 2000.)

Thrombomodulin Overexpression to Limit Neointima Formation

BACKGROUND-These studies were initiated to confirm that high-level thrombomodulin overexpression is sufficient to limit neointima formation after mechanical overdilation injury. METHODS AND RESULTS-An adenoviral construct expressing thrombomodulin (Adv/RSV-THM) was created and functionally characterized in vitro and in vivo. The impact of local overexpression of thrombomodulin on neointima formation 28 days after mechanical overdilation injury was evaluated. New Zealand White rabbit common femoral arteries were treated with buffer, viral control, or Adv/RSV-THM and subjected to mechanical overdilation injury. The treated vessels (n=4 per treatment) were harvested after 28 days and evaluated to determine intima-to-media (I/M) ratios. Additional experiments were performed to determine early (7-day) changes in extracellular elastin and collagen content; local macrophage, T-cell, and neutrophil infiltration; and local thrombus formation as potential contributors to the observed impact on 28-day neointima formation. The construct significantly decreased neointima formation after mechanical dilation injury in this model. By histological analysis, buffer controls exhibited mean I/M ratios of 0.76+/-0.06%, whereas viral controls reached 0.77+/-0.08%; in contrast, Adv/RSV-THM reduced I/M ratios to 0.47+/-0.06%. Local inflammatory infiltrate decreased in the Adv/RSV-THM group relative to controls, whereas matrix remained relatively preserved. Rates of early thrombus formation also decreased in Adv/RSV-THM animals. CONCLUSIONS-This construct thus offers a viable technique for promoting a locally neointima-resistant small-caliber artery via decreased thrombus bulk, normal matrix preservation, and decreased local inflammation without the inflammatory damage that has limited many other adenoviral applications.

Study of the delay phenomenon in the rat TRAM flap model.

Partial flap necrosis is frequently associated with the pedicled TRAM flap in the clinical setting. We created a TRAM flap model in rats (one control group and four delay groups; n = 8 in each group) and used a variety of flap delay techniques and vascular manipulations in an effort to increase the surviving area of the flap. The areas of surviving skin paddles in the five groups ranged from 4.4 cm2 in the control group to 9.9 cm2 in one of the delay groups. Analysis of variance and Tukeys pairwise comparisons indicated that delay groups II, III, and IV differed significantly from the control group (ANOVA F = 15.99, p < 0.001; Tukey experimental ER = 0.05, individual ER = 0.0068; critical value = 4.07). Ligation of the superficial inferior epigastric vessels and deep superior epigastric vessels (dominant pedicle) 1 week before TRAM flap surgery increased the area of survival of the skin paddle significantly in the rat model. The procedure is relatively easy to perform, and our laboratory findings should be readily reproducible in clinical practice.

Prefabricated flaps : experimental and clinical review

Prefabricated flaps are a useful tool for the reconstructive surgeon and present a number of advantages: 1. Specific preferred tissue composites, regardless of their native vascular origin, can be transferred as free or pedicled flaps. 2. Larger flaps of specialized tissue may be transferred safely. 3. Donor-site morbidity is reduced. 4. The functional outcome for the patients may be more satisfactory. The various methods of flap prefabrication include vascular induction through stage transfer; pretransfer delay, expansion, and grafting; the use of alloplastic materials; and tissue bioengineering. We have reviewed both the experimental and clinical research on flap prefabrication, describing the theory, technique, and advantages of each method.

The effect of the Accreditation Council for Graduate Medical Education Duty Hours Policy on plastic surgery resident education and patient care: an outcomes study.

The Accreditation Council for Graduate Medical Education (ACGME) Work-Hours Duty Policy became effective on July 1, 2003, mandating the reduction of resident duty work hours. The Baylor College of Medicine Multi-Institutional Integrated Plastic Surgery Program instituted a resident duty work-hours policy on July 1, 2002 (1 year ahead of the national mandate). Outcomes data are needed to facilitate continuous improvements in plastic surgical residency training while maintaining high-quality patient care. To assess the effect of this policy intervention on plastic surgery resident education as measured through the six core competencies and patient/resident safety, the investigators surveyed all categorical plastic surgery residents 6 months after implementation of the policy. This work represents the first empiric study investigating the effect of duty hours reduction on plastic surgery training and education. The categorical plastic surgery residents at the Baylor College of Medicine Multi-Institutional Integrated Plastic Surgery Program completed a 68-item survey on a five-point Likert scale (1 = strongly disagree to 5 = strongly agree). Residents were asked to rate multiple parameters based on the ACGME six core competencies, including statements on patient care and clinical/operative duties, resident education, resident quality of life, and resident perceptions on this policy. All surveys were completed anonymously. The sample size was n = 12 (program year 3 through program year 6), with a 100 percent response rate. Univariate and bivariate statistical analysis was conducted with SPSS version 10.0 statistical software. Specifically, interquartile deviations were used to find consensus among resident responses to each statement. Descriptive statistics indicated higher percentages of agreement on a majority of statements in three categories, including patient care and clinical/operative duties, academic duties, and resident quality of life. Using interquartile deviation, the highest levels of consensus among the residents were found in positive statements addressing resident alertness (both in and out of the operative environment), time to read/prepare for cases/conferences, efficacy of the didactic curriculum, and overall satisfaction with this policy for surgery resident education. Residents also felt that their patients favored this work hours policy. In addition, there was high consensus that this policy improved overall patient care. The majority of residents identified a negative effect of this policy through an increase in cross-coverage responsibilities, however, and half of the residents perceived that faculty negatively viewed their unavailability postcall. In addition, no consensus among the residents was achieved regarding perceptions on overall weekly operative experience. Plastic surgery residents perceived that the reduction of resident work hours through adherence to the ACGME guidelines has beneficial effects on patient care and clinical/operative duties, academic duties, and resident quality of life. Residents felt, however, that these benefits may increase cross-coverage workloads. Furthermore, residents were concerned about faculty perception of their changes in postcall duties. In contrast to previously published findings in the general surgery literature, the current results indicate that residents do not believe that this policy negatively affects continuity of patient care. In fact, the current findings suggest that adherence to this policy improves patient care on multiple levels. The effect on the operative experience remains to be elucidated. Further large-scale and longitudinal research design and analysis is warranted to better assess the results of the ACGME resident duty work-hours policy in plastic surgery resident education.

Increased axonal regeneration through a biodegradable amnionic tube nerve conduit: effect of local delivery and incorporation of nerve growth factor/hyaluronic acid media.

&NA; The authors emphasize the possible pharmacological enhancement of axonal regeneration using a specific growth factor/extracellular media incorporated in a biodegradable nonneural nerve conduit material. They investigated the early effects on nerve regeneration of continuous local delivery of nerve growth factor (NGF) and the local incorporation of hyaluronic acid (HA) inside a newly manufactured nerve conduit material from fresh human amnionic membrane. Human amnionic membrane contains important biochemical factors that play a major neurotrophic role in the nerve regeneration process. The process of manufacturing a nerve conduit from fresh human amnionic membrane is described. This nerve conduit system was used in rabbits to bridge a 25‐mm nerve gap over 3 months. NGF was released locally, over 28 days, at the distal end of the tube via a system of slow release, and HA was incorporated inside the lumen of the tube at the time of surgery. NGF/HA treatment promoted axonal regeneration across the amnionic tube nerve conduit (8,962 ± 383 myelinated axons) 45% better than the nontreated amnionic tube group (6,180 ± 353 myelinated axons). The authors demonstrate that NGF/HA media enhances additional axonal regeneration in the amnionic tube nerve conduit. This result is secondary to the effect of the amnion promoting biochemical factors, in combination with the NGF/HA effect on facilitating early events in the nerve regeneration process. Mohammad JA, Warnke PH, Pan Y‐C, Shenaq S. Increased axonal regeneration through a biodegradable amnionic tube nerve conduit: effect of local delivery and incorporation of nerve growth factor/hyaluronic acid media. Ann Plast Surg 2000;44;59‐64