Betsy F. Fink

The Viability of Autologous Fat Grafts Harvested With the LipiVage System : A Comparative Study

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 × 106 /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.

Two Lyophilized Polymer Matrix Recombinant Human Bone Morphogenetic Protein-2 Carriers in Rabbit Calvarial Defects

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.

Effect of a freeze-dried CMC/PLGA microsphere matrix of rhBMP-2 on bone healing

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.

Long-Term Preservation of Adipose Aspirates After Conventional Lipoplasty

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 degrees 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.

The Effect of Calcium Channel Blockers on Smoking-Induced Skin Flap Necrosis

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.

Analysis of nitric oxide activity in prevention of reperfusion injury.

This project was designed to determine the role of nitric oxide (NO) in the prevention of ischemia–reperfusion injury. Inferiorly based rectus abdominis muscle flaps were elevated in pigs and subjected to 6 hours of ischemia followed by 4 hours of reperfusion. Group I animals received a bolus of L-arginine before reperfusion, and a continuous infusion once flow was restored. Group II animals served as controls and received an equal volume of saline as a bolus and subsequent continuous infusion. Microdialysis was used to measure tissue NO levels, and these were correlated with muscle survival determined by vital staining with nitroblue tetrazolium. The results demonstrated a significant increase in tissue NO levels in L-arginine-supplemented animals (p < 0.05), which in turn correlated with a significant increase in muscle survival (p = 0.0051). These results suggest that administration of supplemental L-arginine to ischemic skeletal muscle during reperfusion results in increased NO production and decreased tissue damage.

Cryopreservation of Adipose Tissues: The Role of Trehalose

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 fate of cryopreserved adipose aspirates after in vivo transplantation

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 patients 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.

Skin contraction with pulsed CO2 and erbium:YAG laser.

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 constant length. Second, change in length of a sample under no tension was measured during laser treatment. These two disparate analyses represent the two possible extremes of the clinical situation in which skin exists under some tension with some laxity to allow for decrease in length. A custom apparatus with digital interface for skin tension measurements was used to produce single sample tracings of change in skin tension with laser treatment. Length change was measured for individual samples by continuous sonomicrometer readings. Individual sample data were then plotted in a time versus tension/length graph. Skin contracts immediately to a peak level and then relaxes to a sustained plateau level for both CO2 and erbium:YAG lasers. Increased contraction was noted when the beam penetrated into the dermis. Greater peak and plateau contraction is observed after the beam has penetrated into the dermis. Skin contraction varies directly with energy for CO2 and erbium:YAG laser. Findings were similar when skin tension was measured with the sample held at constant length and when length change was measured with the sample under no tension. Char left on the skin after a pass with CO2 laser substantially decreases skin contraction. High-density settings with CO2 laser yield pulse stacking, which effectively irradiates the same portion of tissue with char on it. Skin contraction varies inversely with computer pattern density settings for CO2 laser due to this pulse stacking effect. Density has little effect on skin contraction for the erbium:YAG laser because little char is generated. Histologic analysis identified a zone of coagulated dermis that correlates linearly with skin contraction.

Cryopreservation of Composite Tissue Flaps: Experimental Studies in the Rat

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°C, and stored in liquid nitrogen for 2 weeks. Ten fresh and 10 cryopreserved/thawed flaps were examined histologically with hematoxylin & eosin 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, hematoxylin & eosin, 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.