Cryopreservation of human adipose tissues.

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<0.0011) of adipose aspirates was found in Group 3 compared with Group 2. Group 3 had only a marginally lower integrated viable adipocyte count compared with Group 1 (2.06+/-0.54 x 10(6)mL(-1) vs. 2.57+/-0.56 x 10(6)mL(-1), p=0.083). Histologically, more tissue shrinkage was evident in Group 2 compared with Group 3. In the in vivo study, various degrees of absorption of injected fat grafts were seen in all 3 groups. However, Group 3 had significantly more retained weight and volume of the injected fat grafts than Group 2 (both p<0.0001) but had significantly less retained weight and volume than Group 3 (weight, p=0.009178; volume, p=0.007836). Histologically, a large amount of tissue fibrosis was seen in Group 2, and reasonably well maintained fatty tissue with only a small amount of tissue fibrosis was seen in Group 3. The results from the present in vitro and in vivo studies, for the first time, demonstrate that our preferred cryopreservation method, the combination of 0.5M Me(2)SO and 0.2M trehalose as CPA in addition to the controlled slow cooling and fast rewarming protocol, appears to provide the maximum recovered results in cryopreservation of human adipose tissues and may become a real option after further refinements for cryopreservation of human adipose aspirates in a clinical setting.

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.

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 use in bone repair.

Enhancement of bone growth by sustained delivery of recombinant human bone morphogenetic protein-2 in a polymeric matrix.

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. METHODS: BMP-2 was incorporated in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres to obtain different release rates. Two sustained and an immediate release implants were produced by suspending the BMP-2 loaded PLGA microspheres in aqueous sodium carboxymethylcellulose (CMC), lyophilizing, and cutting the dried materials to the size of the animal bone defects. The local in vivo release at the implantation site in rat calvarial defects was determined by gamma scintigraphy using radiolabeled BMP-2. The local bone induction in the critical size of rabbit calvarial defects was evaluated six weeks post implantation. RESULTS: The immediate release implant showed about 65% initial drug release within 24 h and the remaining BMP-2 quickly exhausted from the implantation site within 7 days. The sustained release implants, showing 45-55% initial release followed by a prolonged release for 21 days, released a greater amount of BMP-2 at the implantation site and maintained higher serum BMP-2 for the longer period of time compared to the immediate release implant. Significant bone growth was observed in all BMP-2 treated defects while the defects without treatment or with BMP-2-free implant showed minimal bone healing. 75-79% of rabbit calvarial defect area was healed with newly induced bone matrix by the sustained release implants in 6 weeks as compared to 45% recovery from the immediate release implant. CONCLUSION: The sustained delivery of BMP-2 based on the biodegradable PLGA microsphere system resulted in faster and more complete bone healing in the animal model.

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.

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.

Estrus detection by using vaginal cytologic examination in miniature swine.

Vaginal smears were obtained from four Yucatan miniature swine daily for 69 days and stained with hematologic stain. Epithelial cells were categorized as superficial, large intermediate, small intermediate, or parabasal. Leukocytes were also quantitated. External signs of estrus were recorded, including swelling, discharge, restlessness, or vocalization. Mean age of three of the swine was 147 days at the beginning of the study. The fourth pig was 317 days old. The three younger swine had their first observed estrus at the age of 178 days (range, 167 to 196 days). Mean cycle length was 17 to 21 days. The moving mean leukocyte count (i.e., each value was averaged with the values for the previous day and the following day) always exceeded the epithelial cell count (regardless of type), except during the 3 to 4 days when the pigs exhibited external signs of estrus. Further, epithelial cells were at their peak during estrus, decreasing markedly during diestrus, and increasing again during proestrus. The combined superficial plus large intermediate cell counts were significantly higher during estrus than during diestrus or proestrus. We conclude that daily vaginal smears can be used to determine the stage of estrus in Yucatan pigs.

Experimental rectus abdominis myocutaneous and rectus abdominis myoperitoneal flaps as urinary bladder wall substitutes in miniature swine.

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.