Photochemical Tissue Passivation of Arteriovenous Grafts Prevents Long-Term Development of Intimal Hyperplasia in a Swine Model.

BACKGROUND: The autologous vein remains the standard conduit for lower extremity and coronary artery bypass grafting despite a 30%-50% 5-y failure rate, primarily attributable to intimal hyperplasia (IH) that develops in the midterm period (3-24 mo) of graft maturation. Our group discovered that externally strengthening vein grafts by cross-linking the adventitial collagen with photochemical tissue passivation (PTP) mitigates IH in an arteriovenous model at 4 wk. We now investigate whether this effect is retained in the midterm period follow-up. METHODS: Six Hanford miniature pigs received bilateral carotid artery interposition vein grafts. In each animal, the external surface of one graft was treated with PTP before grafting, whereas the opposite side served as the untreated control. The grafts were harvested after 3 mo. Ultrasound evaluation of all vein grafts was performed at the time of grafting and harvest. The grafts were also evaluated histomorphometrically and immunohistologically for markers of IH. RESULTS: All vein grafts were patent at 3 mo except one graft in the PTP-treated group because of early technical failure. The control vein grafts had significantly greater IH than PTP-treated grafts at 3 mo, as evidenced by the intimal area (2.6 ± 1.0 mm2versus 1.4 ± 1.5 mm2, respectively, P = 0.045) and medial area (5.1 ± 1.9 mm2versus 2.7 ± 2.4 mm2, respectively, P = 0.048). The control grafts had an increased presence and proliferation of mural myofibroblasts with greater smooth muscle actin and proliferating cell nuclear antigen staining. CONCLUSIONS: PTP treatment to the external surface of the vein grafts decreases IH at 3 mo after arteriovenous grafting and may prevent future graft failure.

Photochemical Tissue Passivation Prevents Contracture of Full Thickness Wounds in Mice.

BACKGROUND AND OBJECTIVES: Wound contracture formation from excessive myofibroblast activity can result in debilitating morbidities. There are currently no treatments to prevent contracture. Photochemical tissue passivation (PTP), an established, safe, and user-friendly treatment modality, crosslinks collagen by a light-activated process, thus modulating the wound healing response and scarring. We hypothesised that PTP treatment would reinforce wounds by blunting the fibrotic response thus limiting contracture. STUDY DESIGN/MATERIALS AND METHODS: Full-thickness, 1 cm × 1 cm excisional wounds were created on the dorsum of 32 C57BL/6 mice. Treated wounds were painted with photosensitizing dye and exposed to visible light. Wounds were serially photographed over 6 weeks to measure wound contracture. At 7, 14, 21, and 42 days after wound creation, mice were euthanized and wounds were harvested for histologic review by a dermatopathologist. RESULTS: By Day 7, control wounds had significantly more contracture than those treated with PTP (33.0 ± 17.1% and 19.3 ± 9.0%, respectively; P = 0.011). PTP-treated wounds maintained approximately 20% less contracture than controls from Day 14 and on (P < 0.05). By Day 42, wounds had contracted by 86.9 ± 5.5% in controls and 64.2 ± 3.2% in PTP-treated wounds (P < 0.03). Histologically, PTP wounds had earlier growth and development of dermal collagen, neovascularization, and development of skin appendages, compared with control wounds. CONCLUSIONS: PTP significantly limits contracture of full-thickness wounds and improves wound healing. PTP-treated wounds histologically demonstrate more mature structural organization than untreated wounds and closely resemble native skin. PTP treatment may be applicable not only for excisional wounds, but also for wounds with a high incidence of contracture and associated morbidity. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Photochemical Tissue Passivation Attenuates AV Fistula Intimal Hyperplasia.

OBJECTIVE: We hypothesized that decreasing vein compliance would protect the vein against stretch injury and reduce intimal hyperplasia (IH). BACKGROUND: Although arteriovenous fistulas (AVFs) are the criterion standard for vascular access, their effectiveness is limited by poor patency with 40% to 60% failing due to IH. Venous stretch injury from exposure to arterial pressure induces IH. Photochemical tissue passivation (PTP) crosslinks adventitial collagen, decreasing vein compliance to resemble that of an artery. METHODS: AVFs were created between the femoral artery and epigastric vein in rats (n = 29). PTP was performed on the vein immediately before vessel anastomosis. AVFs were harvested after four weeks. Venous diameter was measured at the initial procedure and harvest. Intimal area was measured for each segment. Ultrasound was performed at harvest to measure AVF flow. RESULTS: Following AVF construction, venous diameter increased by 10% ±â€Š18% for PTP-treated vessels and 78% ±â€Š27% for controls (P ≤ 0.0001). At one month, PTP reduced AVF dilation by 71% compared to control (69% ±â€Š29% vs 241% ±â€Š78%; P ≤ 0.0001). Both juxta-anastomotic intimal area and total intimal area were reduced in PTP-treated vessels compared to control vessels. Specifically, intimal area was 0.024 ±â€Š0.018 and 0.095 ±â€Š0.089 mm for PTP-treated juxta-anastomotic segments of AVF and control, respectively (P < 0.05). Mean total intimal area for PTP-treated and control AVF were 0.080 ±â€Š0.042 and 0.190 ±â€Š0.110 mm, respectively (P < 0.03). AVF flow was 46.9 ±â€Š35.3 and 19.1 ±â€Š10.1 mL/min for PTP-treated and control AVF, respectively (P < 0.109). CONCLUSIONS: These data demonstrate that PTP represents a promising therapy for the prevention of AVF IH, a process that might improve surgical outcomes for patients receiving hemodialysis.

Prevention of vein graft intimal hyperplasia with photochemical tissue passivation.

OBJECTIVE: Saphenous vein is the conduit of choice for bypass grafting. Saphenous vein grafts have poor long-term patency rates because of intimal hyperplasia (IH) and subsequent accelerated atherosclerosis. One of the primary triggers of IH is endothelial injury resulting from excessive dilation of the vein after exposure to arterial pressures. Photochemical tissue passivation (PTP) is a technology that cross-links adventitial collagen by a light-activated process, which limits dilation by improving vessel compliance. The objective of this study was to investigate whether PTP limits the development of IH in a rodent venous interposition graft model. METHODS: PTP is accomplished by coating venous adventitia with a photosensitizing dye and exposing it to light. To assess the degree of collagen cross-linking after PTP treatment, a biodegradation assay was performed. Venous interposition grafts were placed in the femoral artery of Sprague-Dawley rats. Rats were euthanized after 4 weeks, and intimal thickness was measured histologically. Vein dilation at the time of the initial procedure was also measured. RESULTS: Time to digestion was 63 ± 7 minutes for controls, 101 ± 2.4 minutes for rose bengal (RB), and 300 ± 0 minutes for PTP (P < .001 PTP vs control). A total of 37 animals underwent the procedure: 12 PTP, 12 RB only, and 13 untreated controls. Dilation of the graft after clamp release was 99% for control, 65% for RB only, and 19% for PTP-treated (P < .001 PTP vs control). Intimal thickness was 77 ± 59 µm in controls, 60 ± 27 µm in RB only, and 33 ± 28 µm in PTP-treated grafts. There was a statistically significant 57% reduction in intimal thickness after treatment with PTP compared with untreated controls (P = .03). CONCLUSIONS: PTP treatment of venous interposition grafts in a rat model resulted in significant collagen cross-linking, decreased vessel compliance, and significant reduction in IH.

Eradication of multidrug-resistant pseudomonas biofilm with pulsed electric fields.

Biofilm formation is a significant problem, accounting for over eighty percent of microbial infections in the body. Biofilm eradication is problematic due to increased resistance to antibiotics and antimicrobials as compared to planktonic cells. The purpose of this study was to investigate the effect of Pulsed Electric Fields (PEF) on biofilm-infected mesh. Prolene mesh was infected with bioluminescent Pseudomonas aeruginosa and treated with PEF using a concentric electrode system to derive, in a single experiment, the critical electric field strength needed to kill bacteria. The effect of the electric field strength and the number of pulses (with a fixed pulse length duration and frequency) on bacterial eradication was investigated. For all experiments, biofilm formation and disruption were confirmed with bioluminescent imaging and Scanning Electron Microscopy (SEM). Computation and statistical methods were used to analyze treatment efficiency and to compare it to existing theoretical models. In all experiments 1500 V are applied through a central electrode, with pulse duration of 50 µs, and pulse delivery frequency of 2 Hz. We found that the critical electric field strength (Ecr) needed to eradicate 100-80% of bacteria in the treated area was 121 ± 14 V/mm when 300 pulses were applied, and 235 ± 6.1 V/mm when 150 pulses were applied. The area at which 100-80% of bacteria were eradicated was 50.5 ± 9.9 mm(2) for 300 pulses, and 13.4 ± 0.65 mm(2) for 150 pulses. 80% threshold eradication was not achieved with 100 pulses. The results indicate that increased efficacy of treatment is due to increased number of pulses delivered. In addition, we that showed the bacterial death rate as a function of the electrical field follows the statistical Weibull model for 150 and 300 pulses. We hypothesize that in the clinical setting, combining systemic antibacterial therapy with PEF will yield a synergistic effect leading to improved eradication of mesh infections.

Autologous fat processing via the Revolve system: quality and quantity of fat retention evaluated in an animal model.

BACKGROUND: Currently, fat graft viability and retention cannot be reliably predicted. The reasons for this variability are not fully understood, although fat processing has been implicated. OBJECTIVES: The authors compare the in vitro quantity and in vivo fat retention from lipoaspirate processed by the Revolve system (LifeCell, Bridgewater, New Jersey) compared with centrifugation and decantation. METHODS: Ten patients were enrolled in this prospective study. Lipoaspirate from each patient was processed by each of 3 methods: decantation, centrifugation, and the Revolve system. Biochemical characteristics and free oil, adipose, and aqueous phases of the processed fats were determined. Fat grafts were implanted in nude mice; volume retention and quality of the fat grafts were evaluated after 28 days. Viability of retained fat was demonstrated by intact adipocytes and neovascularization on histology. RESULTS: Of the 10 patients, 9 were women and 1 was a man. Mean patient age was 40.7 ± 8.9 years (range, 30-55 years). Fat tissue obtained from all methods had good physiological properties with neutral pH and isotonic salt concentrations. The Revolve system yielded significantly less blood cell debris, a higher percentage of adipose tissue, and a lower percentage of free oil compared with the other 2 methods. Fat tissue retention from Revolve samples was significantly higher (73.2%) than that from decanted samples (37.5%) and similar to that from centrifuged samples (67.7%). CONCLUSIONS: The Revolve system produced physiologically compatible, preinjection fat with reduced contaminants and free oil in conjunction with high fat content. In an animal model, volume retention of Revolve-processed fat grafts was significantly greater than decanted samples. The Revolve system presents a fat-processing option that was less time-consuming, easier to use, and more efficient in this study than standard centrifugation or decantation.

The impact of liposuction cannula size on adipocyte viability.

PURPOSE: Autologous fat transfer ("fat grafting") is widely used in cosmetic and reconstructive surgery, but long-term outcomes remain inconsistent. Each step in the transfer process can cause mechanical damage to the graft tissue. In particular, liposuction breaks aspirated adipose tissue into distinct globules and subjects it to shear forces, both of which can impact subsequent fat graft viability. The optimal size of the liposuction cannula for use in fat grafting is not known. METHODS AND TECHNIQUES: Controlled lipoaspirate samples were collected from adult female patients undergoing elective liposuction of the abdomen and flanks with uniform aspiration pressure (-25 in Hg) and either a 3- or 5-mm standard blunt-tip liposuction cannula. Individual grafts of 1.00±(0.01) gram were prepared and injected into the bilateral flanks of nude mice with a 14-gauge catheter. After six weeks, these grafts were explanted and analyzed by weight and histology. RESULTS: At six weeks, fat lobules in the 5-mm group retained 25% more weight than those in the 3-mm group [mean (SD), 0.70 (0.07) vs 0.56 (0.09) g, n=24/group, P<0.01). Histologic analysis revealed more intact, nucleated adipocytes in the 5-mm group than in the 3-mm group [4.42 (0.92) vs 3.10 (0.56) on a 1-5 rating scale]. The 5-mm group exhibited both less infiltrate [1.58 (0.17) vs 3.13 (0.70)] and less fibrosis [1.67 (0.45) vs 3.13 (0.89)] than the 3-mm group. CONCLUSIONS: In this controlled model of fat grafting with either a 5- or 3-mm aspiration cannula, the use of a larger aspiration cannula led to improved graft retention and quality. This finding has important implications for clinical applications of fat grafting.

A novel murine island skin flap for ischemic preconditioning.

BACKGROUND: Ischemia reperfusion injury is a well-known phenomenon affecting skin flap viability. One method to improve flap viability is ischemic preconditioning. Previous murine flap models used random flaps. We developed a single pedicle island skin flap which allows us to create true ischemia by clamping the single pedicle. Our first aim was to describe a novel murine skin flap model with a definable, reproducible injury. Our second aim was to test the usefulness of this model by demonstrating mitigation of injury via ischemic preconditioning. MATERIALS AND METHODS: Dorsal lateral thoracic artery pedicle island skin flaps (3.5 x 1.5 cm) were elevated in 39 male C57/BL6 mice: a Control group (n = 7), 10 h of ischemia (n = 21), and Preconditioning (2 cycles of 20 min ischemia: 20 minutes reperfusion) + 10-h ischemia (n = 11). After flap elevation, a silicon sheet barrier was placed. The axial pedicles were occluded, and the flaps were inset with 6-0 prolene. In all mice, ischemia was followed by 1 wk of reperfusion. At 1 wk, percent necrosis was measured and an analysis of variance was performed. RESULTS: The percent of flap necrosis was 1.1% +/- 1.11% in controls. Animals that were subjected to 10 h of ischemia developed 33.14% +/- 7.23% necrosis. Preconditioned animals that underwent 10 h of ischemia demonstrated a 43% reduction in necrosis (18.82% +/- 5.68%). There was a statistically significant difference among all groups (P < or = 0.001). CONCLUSION: Rat models have been the standard for skin flap experiments. We have developed a novel murine single pedicle island skin flap model with reproducible injury. This model has numerous advantages, including ease of handling, low cost, appropriateness for biomedical studies, and the availability of genetically altered animals. We also confirmed this model's usefulness in a study of mitigation of ischemia reperfusion injury through ischemic preconditioning.

Photochemical Tissue Passivation Reduces Vein Graft Intimal Hyperplasia in a Swine Model of Arteriovenous Bypass Grafting.

BACKGROUND: Bypass grafting remains the standard of care for coronary artery disease and severe lower extremity ischemia. Efficacy is limited by poor long-term venous graft patency secondary to intimal hyperplasia (IH) caused by venous injury upon exposure to arterial pressure. We investigate whether photochemical tissue passivation (PTP) treatment of vein grafts modulates smooth muscle cell (SMC) proliferation and migration, and inhibits development of IH. METHODS AND RESULTS: PTP was performed at increasing fluences up to 120 J/cm(2) on porcine veins. Tensiometry performed to assess vessel elasticity/stiffness showed increased stiffness with increasing fluence until plateauing at 90 J/cm(2) (median, interquartile range [IQR]). At 90 J/cm(2), PTP-treated vessels had a 10-fold greater Young's modulus than untreated controls (954 [IQR, 2217] vs 99 kPa [IQR, 63]; P=0.03). Each pig received a PTP-treated and untreated carotid artery venous interposition graft. At 4-weeks, intimal/medial areas were assessed. PTP reduced the degree of IH by 66% and medial hypertrophy by 49%. Intimal area was 3.91 (IQR, 1.2) and 1.3 mm(2) (IQR, 0.97; P≤0.001) in untreated and PTP-treated grafts, respectively. Medial area was 9.2 (IQR, 3.2) and 4.7 mm(2) (IQR, 2.0; P≤0.001) in untreated and PTP-treated grafts, respectively. Immunohistochemistry was performed to assess alpha-smooth muscle actin (SMA) and proliferating cell nuclear antigen (PCNA). Objectively, there were less SMA-positive cells within the intima/media of PTP-treated vessels than controls. There was an increase in PCNA-positive cells within control vein grafts (18% [IQR, 5.3]) versus PTP-treated vein grafts (5% [IQR, 0.9]; P=0.02). CONCLUSIONS: By strengthening vein grafts, PTP decreases SMC proliferation and migration, thereby reducing IH.

Prevention of capsular contracture with photochemical tissue passivation.

BACKGROUND: Capsular contracture is the most common complication following the insertion of breast implants. Within a decade, half of patients will develop capsular contracture, leading to significant morbidity and need for reoperation. There is no preventative treatment available and the recurrence rate remains high. Photochemical tissue passivation is a novel tissue-stabilization technique that results in collagen cross-linking. It can rapidly link collagen fibers in situ, preserving normal tissue architecture. By using this therapy to passivate the collagenous tissues of the implant pocket, the authors hope to prevent the development of pathogenic collagen bundles and subsequent capsule contracture. METHODS: Six-cubic centimeter tissue expanders were placed below the panniculus carnosus muscle along the dorsum of New Zealand white rabbits. Fibrin glue was instilled into each implant pocket to induce contracture. Treated pockets received photochemical tissue passivation by coating them with a photosensitizing dye and exposing the area to a 532-nm light. After 8 weeks, capsule tissue was harvested for histologic evaluation. RESULTS: Implant capsule thickness is the number one prognostic factor for contracture development. The authors demonstrated a 52 percent decrease in capsule thickness in the passivated group compared with controls. Photochemical tissue passivation resulted in fewer fibrohistiocytic cells and macrophages and in reduced synovial metaplasia and smooth muscle actin deposition. CONCLUSIONS: Photochemical tissue passivation significantly decreased both capsule thickness and smooth muscle actin deposition. It is a promising technique for preventing capsular contracture that can be performed at the time of initial surgery without a significant increase in procedure time.

Comparative analysis of processing methods in fat grafting.

BACKGROUND: Centrifugation is a popular processing method, with an unclear mechanism of action. Hypotheses include fat concentration, reduced inflammatory response by removal of blood, and concentration of adipose-derived stem cells. The authors performed multiple experiments to determine the role of centrifugation and compared it with a different processing method (mesh/gauze technique). METHODS: Lipoaspirate components were quantified after centrifugation at increasing speed to determine concentration efficacy. For comparison, the authors quantified the concentration efficacy of mesh/gauze. They also compared the number of adipose-derived stem cells isolated by either method. To determine the effects of each component, they compared fat alone to fat mixed with various spinoff components in a mouse model. They also compared centrifugation to mesh/gauze. RESULTS: The adipocyte fraction remains constant above 5000 g, whereas 1200 g results in 91 percent concentrated fat. Mesh/gauze also results in 90 percent concentrated fat. The number of adipose-derived stem cells in 1 g of fat was 1603 ± 2020 and 1857 ± 1832 in the centrifuge and mesh/gauze groups, respectively (p = 0.86). Five "add-back" groups were created: fat plus oil, fat plus surgical tumescence, fat plus fresh tumescence, fat plus cell pellets and fresh tumescence, and fat plus cell pellets. The fat-only group had better retention than the groups mixed with tumescence, regardless of whether it was surgical, fresh, or had cell pellets. Oil did not affect grafts. Centrifugation at 1200 g was equivalent to mesh/gauze (0.73 ± 0.12 g and 0.72 ± 0.13 g, respectively). CONCLUSIONS: Centrifugation improves graft retention by concentration of the adipocyte fraction. The concentration efficacy of mesh/gauze is equivalent to centrifugation at 1200 g, with equivalent in vivo outcomes.

The effect of pressure and shear on autologous fat grafting.

BACKGROUND: Fat grafting has become routine in plastic surgery because of low donor-site morbidity, a low complication rate, and fast recovery time. The optimal technique, however, has yet to be defined. Two critical variables are pressure and shear, both defined as force divided by area. In this study, the authors examined the effect of pressure and shear on human fat grafts in a nude mouse model. METHODS: For negative pressure, tumescent liposuction was performed on fresh panniculectomy specimens. Suction pressure was either -15 inHg or -25 inHg. Lipoaspirate was centrifuged at 1200 g and injected into the flanks of nude mice. For positive pressure, positive pressure was applied to lipoaspirate up to 6 atm for up to 3 minutes and then injected into nude mice. For shear stress, lipoaspirate was centrifuged at 1200 g for 3 minutes and then injected with a fast flow rate (3 to 5 cc/second) or slow flow rate (0.5 to 1 cc/second). After 4 weeks, the fat grafts were analyzed for weight and histology. RESULTS: For negative pressure, there were no differences in weight or histology with high versus low suction pressures. For positive pressure, application of positive pressures up to 6 atm for up to 3 minutes did not create a significant difference in graft weight or histology at 4 weeks. For shear stress, in vivo, a slow injection pressure yielded a 38 percent increase in weight (p < 0.001) compared with fast injection. Histology was similarly affected. CONCLUSIONS: Higher aspiration pressures up to -0.83 atm did not affect fat graft viability in vivo. Positive pressure up to 6 atm also did not affect fat graft viability. The degree of shear stress, which is a function of flow rate, did significantly affect fat graft viability. Fat grafts injected slowly with low shear stress significantly outperformed fat injected with high shear stress. These data suggest that shear stress is a more important variable regarding fat graft viability than pressure.

Micro-mechanical fractional skin rejuvenation.

BACKGROUND: The most commonly performed skin rejuvenation procedure, laser resurfacing, is associated with adverse events and significant expense. The authors have developed a novel device that uses micro-coring needles to remove tissue in a fractional pattern and avoid the side effects of laser therapy. The authors compare the efficacy of these needles to standard needles in a pig model. METHODS: One swine was treated with three needle types: standard hypodermic, solid hypodermic, and the authors' novel coring needles. Thirty-two 1 × 1-inch sites per flank received either 20 or 40 percent treatment coverage. Photographs were taken and punch biopsies were performed at days 0, 7, 28, 56, and 84. Biopsy specimens were evaluated for histology and collagen content. RESULTS: All treatment sites healed quickly, with no evidence of scarring or infection. Coring sites were easily identified and contained increased fibroblast activity and newly synthesized collagen. At 1 month, the papillary dermis and epidermis of the coring sites were up to 196 percent thicker compared with controls (p < 0.001). The coring sites had enhanced undulating rete ridges-consistent with regeneration. At 3 months, a pronounced increase in collagen fibers and newly organized and augmented elastic fibers was seen. Enzyme-linked immunosorbent assay confirmed an 89 percent increase in collagen content in these coring sites (p < 0.001). CONCLUSIONS: This novel approach to skin rejuvenation was found to effectively induce the microscopic and biological endpoints of skin rejuvenation. This may provide a new modality for the safe and cost-effective treatment of age-related rhytides, skin laxity, photodamage, scarring, and striae.

A novel approach to adipocyte analysis.

BACKGROUND: Fat grafting bench research is difficult because many traditional endpoints cannot be used reliably with adipocytes. Manual cell counting with trypan blue is a common method of measuring cell viability. There are, however, multiple known limitations, including human error, inability to analyze cell size, overestimation of adipocyte viability, and labor intensity. In this study, the authors demonstrate the effectiveness of an improved method of accurate adipocyte analysis using an automated cell counter. METHODS: Human lipoaspirate was obtained, centrifuged, and digested. Samples were analyzed using a hemocytometer and an automated cell counter with two viability dyes. Results were then optimized by novel methods of preparation using carboxymethyl cellulose and formalin. RESULTS: Manual trypan blue cell counts ranged from 2,750,000 to 19,200,000 live cells/ml. Automated cell counts significantly reduced variability (3,230,000 to 4,290,000 cells/ml). Counting cells between 40 and 150 µm, which is more specific to adipocytes, yielded 1,040,000 to 1,420,000 viable cells/ml. Using a second viability dye, CellTiter Blue, cell counts ranged between 993,000 and 1,340,000 live cells/ml. Adding carboxymethyl cellulose substantially decreased sampling variability by 80 percent, and the use of formalin prevented the decrease in cell counts over 4 hours from 432,000 to 7,000 cells/ml. CONCLUSIONS: This novel method utilizing automated cell counters can more accurately identify the viable adipocyte population without the limitations of traditional cell counting. In addition, the use of carboxymethyl cellulose and formalin in the preparation process can decrease variability and stabilize cell counts over time. This is an efficient, specific, and reliable method of adipocyte analysis.

Polymer therapy: a novel treatment to improve fat graft viability.

BACKGROUND: Autologous fat grafting is currently undergoing a renaissance. However, fat grafts are limited by unpredictable survival. Poloxamers can act as tissue surfactants. These nonionic surfactants have been shown to stabilize the membranes of damaged cells and to protect against injury and apoptosis in numerous models. This study was designed to investigate the ability of poloxamers to protect harvested adipocytes and to increase fat graft survival. METHODS: Lipoaspirate was obtained from surgical patients. Samples were washed in normal saline, centrifuged at 200 g, treated with various poloxamers or poloxamer components for 30 minutes, centrifuged at 200 g, and implanted into the flanks of nude mice in 1.0-cc, 1.0-g lobules. The grafts were explanted serially for 10 days and at 6 weeks. Endpoints were weight, apoptosis, cell viability, DNA content, and histology. RESULTS: Grafts treated with poloxamers P188, F108, and F127 demonstrated increased graft survival by weight. Fat grafts treated with poloxamers L64 and P188 demonstrated improvement in cell viability, and those treated with poloxamers L64, P188, and F38 demonstrated improved histology. P188-treated grafts demonstrated a 50 percent reduction in apoptosis compared with saline-treated controls (p < 0.05) and an overall 72 percent survival by weight at 6 weeks. P188 demonstrated statistically significant improvement by weight, DNA content, histology, and cell viability (89 percent versus 33 percent). CONCLUSIONS: The authors demonstrate that poloxamers, with membrane-sealing capability, can increase graft survival. Among these poloxamers, P188 demonstrated statistically significant improvement in apoptosis, graft survival by weight, cell viability, DNA content, and histology.

Poloxamer 188 protects against ischemia-reperfusion injury in a murine hind-limb model.

BACKGROUND: Ischemia-reperfusion injury can activate pathways generating reactive oxygen species, which can injure cells by creating holes in the cell membranes. Copolymer surfactants such as poloxamer 188 are capable of sealing defects in cell membranes. The authors postulated that a single-dose administration of poloxamer 188 would decrease skeletal myocyte injury and mortality following ischemia-reperfusion injury. METHODS: Mice underwent normothermic hind-limb ischemia for 2 hours. Animals were treated with 150 microl of poloxamer 188 or dextran at three time points: (1) 10 minutes before ischemia; (2) 10 minutes before reperfusion; and (3) 2 or 4 hours after reperfusion. After 24 hours of reperfusion, tissues were analyzed for myocyte injury (histology) and metabolic dysfunction (muscle adenosine 5'-triphosphate). Additional groups of mice were followed for 7 days to assess mortality. RESULTS: When poloxamer 188 treatment was administered 10 minutes before ischemia, injury was reduced by 84 percent, from 50 percent injury in the dextran group to 8 percent injury in the poloxamer 188 group (p < 0.001). When administered 10 minutes before reperfusion, poloxamer 188 animals demonstrated a 60 percent reduction in injury compared with dextran controls (12 percent versus 29 percent). Treatment at 2 hours, but not at 4 hours, postinjury prevented substantial myocyte injury. Preservation of muscle adenosine 5'-triphosphate paralleled the decrease in myocyte injury in poloxamer 188-treated animals. Poloxamer 188 treatment significantly reduced mortality following injury (10 minutes before, 75 percent versus 25 percent survival, p = 0.0077; 2 hours after, 50 percent versus 8 percent survival, p = 0.032). CONCLUSION: Poloxamer 188 administered to animals decreased myocyte injury, preserved tissue adenosine 5'-triphosphate levels, and improved survival following hind-limb ischemia-reperfusion injury.