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Background: Calcium channel blockers have been shown experimentally to reverse many of the effects of nicotine. The purpose of this study was to assess the effect of calcium channel blockers on smoking-induced skin flap necrosis. Methods: Forty male albino Wistar rats were divided into four groups. Groups A, B, and C were treated in a controlled smoking chamber for 20 minutes daily for 21 days. On day 14, caudally based dorsal skin flaps (3 × 10 cm) were created. On days 14 through 21, group B animals received verapamil (20 mg/kg/day) by gavage. Group C received nifedipine (10 mg/kg/day). On day 21, standardized photographs were taken and flap survival areas determined. Urine cotinine concentrations were measured on days 14 and 21. Results: The mean cotinine level at surgery was 161 ng/ml in group A (smoking), 149 ng/ml in group B (verapamil), and 168 ng/ml in group C (nifedipine). These differences were not statistically significant. Cotinine concentration at surgery for group D (no smoking) was less than 10 ng/ml. The mean flap survival in group D was 79.1 percent, compared with 63.7 percent in group A (p= 0.003). The mean flap survival in group B (verapamil) was 72.8 percent, compared with 73.7 percent in group C (nifedipine). Both values were significantly greater than in group A (p = 0.04 and p = 0.008, respectively). Conclusions: In this study, enteral calcium channel blockers were associated with a statistically significant improvement in flap survival compared with untreated animals with an equivalent smoke exposure. Calcium channel blockers may reduce perioperative risk in active smokers who require skin flap surgery.

Scientific studies on cryopreservation of adipose tissues have seldom been performed. The purpose of our present study is conducted both in vitro and in vivo to develop a novel cryopreservation method that can be used successfully for long-term preservation of human adipose tissues for possible future clinical application. In this study, samples of adipose aspirates were obtained from 36 adult white female patients after liposuction and collected from the middle layer after centrifugation. In the in vitro study, suitable cryoprotectant agents (CPAs) and their concentrations and possible combinations were selected from our preliminary experiment. A combination of dimethyl sulfoxide (Me(2)SO) and trehalose as CPA with the optimal concentration (0.5M Me(2)SO and 0.2M trehalose) was chosen and then used throughout the study. In addition, maximal recovery of adipose tissues was achieved after cryopreservation using slow cooling without seeding (1-2 degrees C/min to -30 degrees C, followed by plunging to -196 degrees C for storage) and fast warming (in 40 degrees C water bath, averaging 35 degrees C/min). Fresh adipose aspirates (Group 1), cryopreserved adipose aspirates without CPAs (Group 2), or cryopreserved adipose aspirates with CPAs (Group 3) were evaluated by integrated adipocyte counts and histology. In the in vivo study, fresh adipose aspirates (Group 1), cryopreserved adipose aspirates without CPAs (Group 2), or cryopreserved adipose aspirates with CPAs (Group 3) were injected into a nude mouse. The retained adipose aspirates (fat grafts) were harvested in each animal at 4 months and their weight, volume, and histology was assessed. In the in vitro study, significantly higher integrated viable adipocyte count (2.06+/-0.54 x 10(6)mL(-1) vs. 1.07+/-0.41 x 10(6)mL(-1), p

The purpose of this study was to test the authors' hypothesis that previously cryopreserved adipose aspirates collected from conventional liposuction could still be a reliable source of human processed lipoaspirate cells.Adipose aspirates were collected from 12 adult female patients after conventional liposuction of the abdomen and were then preserved by an optimal cryopreservation method with added cryoprotective agents (0.5 M dimethyl sulfoxide and 0.2 M trehalose). Cryopreservation of the adipose tissues was subsequently conducted with controlled slow cooling and then stored in liquid nitrogen (-196 degrees C). One gram of fresh or cryopreserved (after fast rewarming) adipose aspirates was processed in vitro and the resulting cell pellet, consisting of processed lipoaspirate cells, was cultured separately. The length of time until processed lipoaspirate cells became adherent to the culture plate was recorded and the number of processed lipoaspirate cells after a 2-week culture was counted.Flat, spindle-shape processed lipoaspirate cells from the cryopreserved group became adherent to the plate within 48 to 72 hours after initial culture compared with the fresh group, where the cells became adherent by 24 hours. After a 2-week culture, the cryopreserved aspirates yielded an average of 3.7 +/- 1.4 x 10(5) processed lipoaspirate cells per milliliter, equal to 90 percent of the yielded number of cells obtained from the fresh aspirates (4.1 +/- 1.4 x 10(5) cells/ml).The authors' results indicate that although there is a latency of cell growth after an optimal cryopreservation, cryopreserved adipose aspirates can yield a significant number of processed lipoaspirate cells compared with fresh aspirates and may be a reliable source of human processed lipoaspirate cells because they can still be processed later after long-term preservation.

Background Optimal cryopreservation permits the long-term storage of living cells or tissues that may have potential clinical applications. Unfortunately, there are no successful studies on the long-term preservation of adipose aspirates for possible autologous fat grafting. Objective The purpose of the current study was (1) to test our hypothesis that adipose aspirates obtained from conventional lipoplasty could be preserved and stored at low temperature (below −85°C) by means of an optimal cryopreservation technique and (2) to develop a novel approach to effectively preserve adipose aspirates for future applications. Methods The middle layer of adipose aspirates obtained from conventional lipoplasty was collected after centrifugation and each specimen was then randomized into 3 groups: the control group, fresh adipose aspirates without preservation; experimental group 1, simple cryopreservation with liquid nitrogen only; and experimental group 2, optimal cryopreservation with cryoprotective agents consisting of a combination of dimethyl sulfoxide (DMSO) and trehalose. Cryopreservation of adipose aspirates was conducted with controlled slow cooling and fast rewarming rates. Fresh or cryopreserved adipose aspirates in each group were evaluated by viable adipocyte counts, glycerol-3-phosphate dehydrogenase (G3PDH) assay, and routine histology. Results Significantly more viable adipocytes and better cellular function of adipose aspirates were found in the experimental group 2 compared to the results in the experimental group 1. Conclusions Our results indicated that an optimal cryopreservation approach that utilizes a combination of DMSO and trehalose as cryoprotective agents appears to provide good long-term preservation of adipose aspirates obtained from conventional lipoplasty, albeit not as ideal as fresh specimens. An in vivo study will be conducted to confirm the results from our present in vitro study.

Purpose. The purpose of this study was to develop a polymeric sustained delivery system for recombinant human bone morphogenetic protein-2 (BMP-2) and to evaluate local bone growth induced by the sustained release of BMP-2 in an animal model.

The purpose of this study was to assess the physical response of skin to laser resurfacing in a real-time, quantitative fashion. The study was designed to assess skin contraction from two opposite standpoints. First, change in tension was measured during laser application while samples were held at

The efficacy of calcium channel blockers in reducing the effects of ischemia/reperfusion (I/R) injury in animals subjected to cigarette smoke was examined. In this study, 40 rats were divided into 4 groups. Groups A, B, and C received a controlled cigarette smoke exposure for 14 days, following which all animals underwent a standardized sciatic nerve I/R procedure. One sciatic nerve was isolated, and the femoral artery was occluded for 3 hours followed by reperfusion. Group B received verapamil (20 mg/kg/d). Group C received nifedipine (10 mg/kg/d). Mean sciatic function index (SFI) was significantly higher in nonsmoking than smoking animals, and the sciatic function index of group B (verapamil) and group C (nifedipine) was significantly greater than group A (smoking). Mean malondialdehyde at day 28 in group A was 0.96 ± 0.14 compared with 0.74 ± 0.11 in the nonsmoking group (P = 0.03), and the mean malondialdehyde in the nifedipine group was significantly greater than in group A (P = 0.05). Histologic injury scores were not significantly different among groups exposed to smoke. Smoking was associated with slower recovery following peripheral nerve I/R injury, but calcium channel blockers were shown to ameliorate these effects.

This study was designed to determine if cigarette smoking adversely affects functional recovery following ischemia/reperfusion (I/R) injury in peripheral nerves. Forty Wistar rats were divided evenly among four groups. Animals in groups A and B were exposed to cigarette smoke via a controlled smoking chamber for 20 minutes daily. On study day 14, all animals underwent a controlled I/R injury to one sciatic nerve. Recovery was assessed with walking track assessments, malondialdehyde (MDA) assay, and histology. Walking track results on study day 21 did not differ significantly between the smoking and nonsmoking animals. However, by study day 28, the nonsmoking animals showed a greater degree of functional recovery (SFI = -18.0 and -22.8, respectively, P = 0.03). MDA concentration in the smoking group was significantly higher than the nonsmoking group at the 28 day time point (P = 0.04). Exposure to cigarette smoke was associated with a slower functional recovery following peripheral nerve I/R injury.

This study evaluates the viability of adipose aspirates harvested with the LipiVage system (Genesis Biosystems Inc, Lewisville, TX), a newly developed fat harvesting device, and determines a potentially preferred method for possible large-quantity fat graft harvesting. Adipose aspirates were harvested with the LipiVage system from the abdomen of 16 female patients (group 1, n = 8) according to the instruction by the manufacturer and with conventional liposuction (group 2, n = 8). Samples from conventional liposuction were spun at 50 g for 10 minutes and the resulting middle layer of fat was collected. All fat graft samples were evaluated with trypan blue vital staining for viable adipocyte count, glycerol-3-phosphatase dehydrogenase (G3PDH) assay for intracellular enzyme activity, and histology. In this study, group 1 had significantly higher viable adipocyte count than group 2 had (3.7 +/- 0.64 versus 2.37 +/- 0.56 x 10(6) /mL, P = 0.0021). G3PDH assay showed a marked increase of intracellular enzyme activity in group 1 compared with in group 2 (0.61 +/- 0.10 versus 0.34 +/- 0.13 U/mL, P = 0.00045). Histology revealed normal structures of fragmental fatty tissues in both groups. While adipose aspirates by both modalities maintain normal structure, the LipiVage system yields a greater number of viable adipocytes and sustains a higher level of intracellular enzyme activity within fat grafts and can potentially be a preferred method of choice for large-quantity fat graft harvesting.

Cryopreservation has the potential to improve availability of donor parts for composite tissue allotransplantation and may reduce their antigenicity. This study investigates whether the component tissues of composite flaps remain viable after cryopreservation. Forty-one epigastric flaps were harvested from Lewis rats. Twenty-one flaps were perfused with DMSO/trehalose, frozen by controlled cooling to -140 degrees C, and stored in liquid nitrogen for 2 weeks. Ten fresh and 10 cryopreserved/thawed flaps were examined histologically with hematoxylineosin and factor VIII staining. An epithelial viability index was calculated for 10 fresh and 11 cryopreserved flaps using the MTT assay. In all cryopreserved samples, hematoxylineosin, and factor VIII staining revealed a well-preserved cellular architecture, which was indistinguishable from fresh specimens. The viability index for the cryopreserved samples was 10.90 +/- 2.09 compared with 12.15 +/- 1.32 for fresh flaps (P = 0.123). Results suggest that the skin, adipose, and vascular endothelial cells of composite tissue flaps retain their viability after cryopreservation and thawing.

Composite tissue allotransplantation holds great promise for upper extremity reconstruction but is limited by donor part availability. Cryopreservation may increase the availability of donor parts and even reduce antigenicity. The purpose of the study was to evaluate the viability of cryopreserved composite tissues and to demonstrate the feasibility of microvascular isotransplantation of cryopreserved composite flaps. Twenty epigastric flaps were harvested from Lewis rats. Ten flaps were analyzed fresh. Ten flaps were perfused with dimethyl sulfoxide (DMSO)/trehelose cryoprotectant agent (CPA), frozen by controlled cooling to −140°C, and stored for 2 weeks. Flaps were evaluated by factor VIII endothelial staining and MTT tetrazolium salt assay. For the in vivo phase, 30 flaps were harvested. Ten were transplanted fresh to isogenetic recipient animals, ten were perfused with CPA and transplanted, and ten were cryopreserved for 2 weeks, thawed, and transplanted. All cryopreserved samples displayed intact vascular endothelia on factor VIII staining. On MTT analysis, the epithelial viability index for the cryopreserved samples was not significantly different from fresh controls ( p=0.12). All freshly transplanted flaps (10/10) were viable at 60 days. Nine of ten flaps in the perfused/transplanted group were viable at 60 days. Survival of cryopreserved/transplanted flaps ranged from 5 to 60 days. The skin and vascular endothelial components of composite tissue flaps appear to retain their viability after cryopreservation. The in vivo studies demonstrate that the long-term survival of cryopreserved composite tissue transplants is feasible and support an indirect injury, rather than direct injury from freezing or cryoprotectant agents, as the mechanism of flap loss.

Despite advances in cryobiology, the reliable cryopreservation of complex tissues has not yet been achieved. This study evaluates the viability of cryopreserved composite flaps and demonstrates the feasibility of their transplantation. Epigastric flaps were harvested from male Lewis rats. 1.5M dimethyl sulfoxide (Me(2)SO) was used as the initial cryoprotectant agent (CPA). Samples were frozen at controlled rate to -140 degrees C and transferred to liquid nitrogen for at least two weeks. Hematoxylin and eosin (H/E) staining, MTT tetrazolium salt assay, and factor VIII immunostaining were used to evaluate the overall histology, epithelial viability, and vascular endothelial integrity, respectively, of cryopreserved flaps. For the in vivo phase, flaps were isotransplanted to 35 recipient animals, divided into three groups: fresh (n=10), perfused (n=8), and cryopreserved (n=17). Blood vessel patency was assessed via Doppler at 1, 7, and 60 days post-transplantation. For in vitro studies, cryopreserved samples (10/10) retained normal cell architecture and vascular endothelial integrity upon H/E and factor VIII staining. The viability index of cryopreserved composite flap skin (n=10) was 11.17+/-2.01, which was not significantly different from fresh controls (n=10, 12.15+/-1.32). All transplanted flaps in the fresh and perfusion groups survived with healthy color and hair growth at 60 days after operation. Survival in the cryopreserved group ranged from 2 to 60 days, with a mean of 12 days. These results demonstrate that the long term survival of cryopreserved composite tissue transplants is possible. Further studies are needed to refine protocols for the reliable cryopreservation of composite parts.

Background: Successful long-term preservation of adipose tissues may have an important impact on future clinical application of autologous fat transplantation. Our group has recently developed an optimal cryopreservation method for possible long-term preservation of adipose aspirates. Objective: The purpose of this study was to evaluate the fate of previously cryopreserved adipose aspirates after in vivo administration in an established nude mouse model. Methods: Adipose aspirates were collected from a cosmetic lipoplasty of the patient's abdomen after centrifugation. In the fresh control group (n = 20), fresh adipose aspirates were injected into the posterior scalp of a nude mouse. In the optimal cryopreservation group (n = 20), adipose aspirates after the optimal cryopreservation were injected. In the simple cryopreservation group (n = 20), adipose aspirates after the simple cryopreservation were injected. All animals in each group were observed for gross appearance of maintained fat grafts over their posterior scalps for up to 16 weeks. The final volume and weight of maintained fat grafts and their histology were evaluated at the end of the study. Results: More maintained volume, weight, and fatty tissue structure of injected free grafts were found in the optimal cryopreservation group compared with the simple cryopreservation group, but the results were still less satisfactory than those in the fresh control group. Conclusions: Based on this in vivo study, we believe that an optimal cryopreservation method developed in our laboratory provides reasonably good long-term preservation of adipose aspirates. However, further studies may still be warranted to refine our method for optimal cryopreservation of adipose tissues.

This study was conducted to evaluate the viability of fatty tissues within adipose aspirates after conventional liposuction and to examine their potential role as a source of donor material for possible future autogenous fat grafting. Samples of adipose aspirates (group I, n = 8) were obtained from adult female patients who underwent a conventional liposuction of the abdomen. Samples of fresh fatty tissues obtained from adult female patients who underwent an abdominoplasty (group II, n = 8) were cut into small pieces and served as a control. All samples were spun at 50 x g for 10 minutes; fatty tissues were then collected from the middle layer after centrifugation for the following studies: trypan blue vital staining for viable fatty cell counts, glycerol-3-phophatase dehydrogenase (G3PDH) assay for functional evaluation of fatty tissues, and routine pathology for histology of fatty tissues. There was no significant difference of viable fatty cell counts in group I compared with group II (2.57 +/- 0.56 versus 2.74 +/- 0.59 x 10/mL, P = 0.56). G3PGH assay showed a marked decrease of the enzyme activity in group I compared with group II (0.34 +/- 0.13 versus 0.76 +/- 0.13 micro/mL, P0.0001). Histologically, the normal structure of fatty tissues was found primarily in both groups. Our results indicate that although fatty tissues within adipose aspirates after conventional liposuction maintain normal structure with near the same number of viable fatty cells compared with fresh ones, they have a less-than-optimal level of cellular function and may not survive well after they are transplanted.

Background Several studies have shown that one of the sugars, trehalose, can improve tissue survival after cryopreservation when combined with other cryoprotective agents, and thus may possibly be used in cryopreservation of adipose tissues that have been found more resistant to injury after freezing. Objectives The purpose of this study was to test our hypothesis that lipoplasty-derived adipose aspirates could be effectively cryopreserved by adding trehalose as the sole cryoprotective agent (CPA), and to develop a practical technique to effectively preserve adipose tissues for future applications. Methods The middle layer of adipose aspirates obtained from conventional lipoplasty was collected after centrifugation and each specimen was randomized into 3 groups: the control group, fresh adipose aspirates without preservation; the simple cryopreservation group (no CPA); and the optimal cryopreservation group (with trehalose as a CPA). Cryopreservation of adipose aspirates was conducted with controlled slow cooling and fast rewarming rates. Fresh or cryopreserved adipose aspirates in each group were evaluated by viable adipocyte counts, glycerol-3-phosphate dehydrogenase (G3PDH) assay, and routine histology. Results More viable adipocytes and better cellular function of adipose aspirates were found in the optimal cryopreservation group compared to the simple cryopreservation group, but these results were less ideal than results from the control group. Conclusions An optimal cryopreservation method using trehalose as a CPA appears to provide better long-term preservation of adipose aspirates than a simple cryopreservation method. Further studies are needed to refine our method for cryopreservation with trehalose as a CPA and confirm our findings in vivo.

The hypothesis of this research was that implants of poly(lactide-co-glycolide) (PLGA) microspheres loaded with bone morphogenetic protein-2 (rhBMP-2) and distributed in a freeze-dried carboxymethylcellulose (CMC) matrix would produce more new bone than would matrix implants of non-protein-loaded microspheres or matrix implants of only CMC. To test this hypothesis it was necessary to fashion microsphere-loaded CMC implants that were simple to insert, fit precisely into a defect, and would not elicit swelling. Microspheres were produced via a water-in-oil-in-water double-emulsion system and were loaded with rhBMP-2 by soaking them in a buffered solution of the protein at a concentration of 5.4 mg protein per gram of PLGA. Following recovery of the loaded microspheres by lyophilization matrices for implantation were prepared by lyophilizing a suspension of the microspheres in 2% CMC in flat-bottom tissue culture plates. Similar matrices were made with 2% CMC and with 2% CMC containing blank microspheres. A full-thickness calvarial defect model in New Zealand white rabbits was used to assess bone growth. Implants fit the defect well allowing for direct application. Six weeks postsurgery, defects were collected and processed for undecalcified histology. In vitro, 60% of the loaded rhBMP-2 released from devices or microspheres in 5 to 7 days. With the unembedded microspheres releasing faster than those embedded in CMC In vivo. the rhBMP-2 microspheres greatly enhanced bone healing, whereas nonloaded PLGA microspheres in the CMC implants had little effect. The results showed that a lyophilized device of rhBMP-2 PLGA microspheres in CMC was an effective implantable protein-delivery system for the use in bone repair.

We have developed a lyophilized bone morphogenetic protein (BMP) delivery device that can be formulated to control release over 2 to 8 weeks. Bioerodible poly (d,l lactide-co-glycolide) particles loaded with 90 micrograms recombinant human BMP-2 were suspended in either carboxymethylcellulose (CMC) or methylcellulose (MC) implants. Plain CMC and MC implants served as controls, as did a nonimplanted group. A total of 40 rabbits was evaluated histologically 2, 4, or 8 weeks after receiving circular full-thickness 15-mm calvarial defects. MC appeared to prevent prolapse of periosteum and dura into the defects and did not elicit bone growth. Addition of BMP improved the result. CMC implants appeared to encourage bone growth even in the absence of BMP. When BMP was added, new bone formed earlier. CMC may influence new bone formation because it is hydrophilic. MC is less hydrophilic and may cause undue inflammation. Either can be combined with BMP to produce unitary devices that are easy to make and use.

An experiment was performed in Yucatan miniature swine to determine the feasibility and characteristics of musculocutaneous or musculoperitoneal flaps as urinary bladder wall substitutes. In five swine, a single-pedicle skin island flap (rectus abdominis myocutaneous, RAM/C) was sutured into the bladder. In five other swine the flap was a peritoneum island (rectus abdominis myoperitoneal, RAM/P). Three swine were sham-operated controls. The patches were in place for 20 weeks, remaining viable and elastic. Inflammation, maceration, and infection were absent. Skin patch histology was unchanged. The peritoneal patches became resurfaced with uroepithelium. The sham bladder volume (ml/kg body weight) did not differ significantly from RAM/P bladder volume (p = 0.54). RAM/C bladders were slightly smaller than shams (p = 0.11) and significantly smaller than RAM/P bladders (p = 0.03). Substitution of the bladder wall with RAM patch flaps is feasible. This is an important preliminary step toward our goal of nonenteral urinary bladder wall substitution.

Breast cancer is the most common cancer among women worldwide and the second leading cause of cancer-related death in women in the United States. The etiology of the majority of human breast cancers is unknown. Elevated levels of estrogen and exposure to environmental carcinogens have been suggested to be involved in human breast cancer development. Several studies have also provided evidence of the potential role of oxidative stress and lipid peroxidation in breast cancer etiology. Identification of DNA adducts and monitoring change in DNA adduct profiles/burden in normal and cancer tissue may provide clues for risk of breast cancer. In this study, we detected and compared the levels of a wide range of polar to lipophilic adducts in tumor (n= 23) and adjacent normal tissue (n= 30) from breast cancer patients with normal tissue (n=124) from mammoplasty using a 32P-postlabeling adductomics. Adducts were classified into polar (P-1, P-2a and P2b or 8-oxodG) and lipophilic (L-1) in descending order of their polarities. Our results indicated significantly elevated total DNA adduct levels in tumor and adjacent normal tissue of breast cancer patients compared to normal tissues from mammoplasty. The levels of most polar adducts (P-1) were not significantly different among the tumor, adjacent and normal breast tissue. However, relatively lesser polar adducts, i.e., P-2a, P-2b (8-oxodG) and L-1 adducts were significantly higher in tumor and adjacent normal tissues of breast cancer patients compared with normal tissues from mammoplasty. Thus, there is a clear trend of adduct accumulation in the breast tissues of cancer patients that could have acted as a precursor event in carcinogenesis process. Therefore, elevated oxidative DNA adducts such as 8-oxodG can be a possible biomarker for early detection of breast cancer risk and subjects with high DNA adduct load may potentially be at higher risk for cancer development, and could be stratified for chemopreventive intervention. (Supported from CA-90892 and Agnes Brown Duggan Endowment) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1701.