A Microbiological and Ultrastructural Comparison of Aseptic versus Sterile Acellular Dermal Matrix as a Reconstructive Material and a Scaffold for Stem Cell Ingrowth.

BACKGROUND: Recent data suggest an increased risk for infection when acellular dermal matrix is used in breast reconstruction. This may be because some acellular dermal matrices are actually not terminally sterilized but are instead "aseptically processed." This study evaluates aseptic and sterile matrices for evidence of bacterial contamination and whether or not terminal sterilization affects matrix collagen architecture and stem cell ingrowth. METHODS: Five separate samples of 14 different matrices were analyzed by fluorescent in situ hybridization using a bacterial DNA probe to detect bacterial DNA on the matrices. Separate samples were incubated for bacteria, acid-fast bacilli, and fungi for 2 to 6 weeks to detect living organisms. The impact of terminal sterilization on the collagen network and stem cell ingrowth on the matrices was then assessed. RESULTS: Traces of bacterial DNA were encountered on all matrices, with more bacteria in the aseptic group compared with the sterile group (3.4 versus 1.6; p = 0.003). The number of positive cultures was the same between groups (3.8 percent). Electron microscopy demonstrated decreased collagen organization in the sterile group. Stem cell seeding on the matrices displayed a wide variation of cellular ingrowth between matrices, with no difference between aseptic and sterile groups (p = 0.2). CONCLUSIONS: Although there was more evidence of prior bacterial contamination on aseptically processed matrices compared with sterile matrices; clinical cultures did not differ between groups. Terminal sterilization does not appear to affect stem cell ingrowth but may come at the cost of damaging the collagen network. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Defining the Salvage Time Window for the Use of Ischemic Postconditioning in Skeletal Muscle Ischemia Reperfusion Injury.

BACKGROUND: The aim of this study was to determine the optimal salvage time window within which ischemic postconditioning can be used to ameliorate ischemia/reperfusion (I/R) injury in skeletal muscle. METHODS: A total of 48 Sprague-Dawley rats were divided into two groups: I/R only (control) and I/R with postconditioning. Subgroups were divided by duration of ischemia (2, 4, 6, and 8 hours). A pedicled gracilis muscle model was used. The postconditioning protocol consisted of six cycles of 15 seconds of reperfusion followed by 15 seconds of ischemia (total time = 3 minutes). Muscles were harvested 24 hours after I/R injury to examine tissue viability, histology, myeloperoxidase activity, and protective gene expression. RESULTS: Postconditioning groups showed improved muscle viability after 4 and 6 hours of ischemia time as compared with controls (p < 0.05). Higher expression of mitochondrial complexes I, II, III, endothelial nitric oxide synthase, inducible nitric oxide synthase, and Bcl-2 were observed in the postconditioning group after 4 and 6 hours of ischemia (p < 0.05). Lower expression of tumor necrosis factor-α and caspase 3 was observed in the postconditioning group at 4 hours (p < 0.05). Myeloperoxidase activity was similar in both groups at all-time points except 8 hours ischemia, where the control group had higher activity (p < 0.05). CONCLUSION: Results of this study demonstrate that the effective time window within which postconditioning is most effective for the salvage of skeletal muscle is between 4 and 6 hours of ischemia. Postconditioning offered improved mitochondrial and vascular function with decreased inflammation and cell death. This may be clinically useful as a postinjury salvage technique to attenuate I/R injury after 4 to 6 hours of ischemia.

Transplantation of the LGR6+ epithelial stem cell into full-thickness cutaneous wounds results in enhanced healing, nascent hair follicle development, and augmentation of angiogenic analytes.

BACKGROUND: The recently discovered leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6+) epithelial stem cell located within the follicular bulge of the adnexal compartment is capable of producing all cellular lineages of the skin. In this study, the authors sought to determine whether these cells can be transplanted for use as a type of cellular therapy for the repair of full-thickness wounds in which the native stem cell niche has been obliterated. METHODS: Full-thickness murine skin was harvested and LGR6(+GFP) epithelial stem cells were isolated using fluorescence-activated cell sorting. This enriched epithelial stem cell population was then transplanted by means of local injection into wound beds on the dorsum of nude mice. Viability, migration, healing, the development of nascent hair follicles, and gene and proteomic expression studies were performed to determine whether the engraftment of LGR6(+GFP) epithelial stem cells enhanced healing when compared with controls. RESULTS: Wound beds receiving LGR6(+GFP) epithelial stem cells showed enhanced healing; nascent follicle growth; and augmentation of the Wnt, vascular endothelial growth factor, epidermal growth factor, and platelet-derived growth factor pathways when compared with controls. CONCLUSIONS: The LGR6+ epithelial stem cells appear to hold great promise for the development of a clinically useful stem cell­based therapy for the repair of full-thickness wounds and hair regeneration. These results indicate that transplantation of LGR6+ epithelial stem cells promotes epithelialization, hair growth, and angiogenesis in tissues destined for scar formation.

Prevention of age-related dysregulation of calcium dynamics by estrogen in neurons.

To determine the impact of aging and 17beta-estradiol on neuronal Ca2+ homeostasis, intracellular Fura-2 Ca2+-imaging was conducted during 20-pulses of glutamate in hippocampal neurons cultured from embryonic (E18), middle-age (10 months) and old (24 months) rat brain. Marked age-related differences in intracellular Ca2+ ([Ca2+]i) homeostasis and striking regulation by 17beta-estradiol were seen. Embryonic neurons exhibited the greatest capacity to regulate Ca2+ homeostasis followed by middle-age neurons. In old neurons, the first peak [Ca2+]i was substantially greater than at other ages and the return to baseline Ca2+ rapidly dysregulated with an inability to restore [Ca2+]i following the first glutamate pulse which persisted throughout the 20 pulses. 17beta-Estradiol pretreatment of old neurons profoundly attenuated the peak [Ca2+]i rise and delayed the age-associated dysregulation of baseline [Ca2+]i, normalizing responses to those of middle-age neurons treated with estradiol. The efficacy of 17beta-estradiol extended below 10 pg/ml with full protection against toxicity from glutamate and Abeta (1-40). These results demonstrate age-associated dysregulation of [Ca2+]i homeostasis which was largely prevented by 17beta-estradiol with implications for estrogen/hormone therapy.