Organization of Hannover Skin Bank: Sterile culture and procurement protocols for viable cryopreserved allogeneic skin grafts of living donors.

Preserved allogeneic donor skin still represents one of the gold standard therapies in temporary wound coverage in severely burned patients or chronic wounds. Allogeneic skin grafts are currently commercially available as cryo- or glycerol-preserved allografts through skin tissue banks all over the world. Most of the skin tissue banks rely on human cadaveric skin donations. Due to the chronic shortage of human allogeneic transplants, such as skin, and increasing costs in the procurement of allografts from other skin tissue banks, Hannover Medical School has been building up its own skin tissue bank based on allogeneic skin grafts from living donors who underwent surgical treatment (i.e., body-contouring procedures, such as abdominioplasties). This article presents procedures and protocols for the procurement and processing of allogeneic skin grafts according to national legislation and European regulations and guidelines. Beside protocols, initial microbiological data regarding the sterility of the harvested grafts are presented. The results currently form the basis for further investigations as well as clinical applications. In summary, a microbiological testing and acceptance procedure is presented that ensures adequate patient safety and skin viability.

Spider silk erectile nerve reconstruction in robot-assisted radical prostatectomy: a first-in-men feasibility analysis.

PURPOSE: To investigate the safety and feasibility of spider silk interposition for erectile nerve reconstruction in patients undergoing robotic radical prostatectomy (RARP). METHODS: The major-ampullate-dragline from Nephila edulis was used for spider silk nerve reconstruction (SSNR). After removal of the prostate with either uni- or bilateral nerve-sparing, the spider silk was laid out on the site of the neurovascular bundles. Data analysis included inflammatory markers and patient reported outcomes. RESULTS: Six patients underwent RARP with SSNR. In 50% of the cases, only a unilateral nerve-sparing was performed, bilateral nerve-sparing could be performed in three patients. Placement of the spider silk conduit was uneventful, contact of the spider silk with the surrounding tissue was mostly sufficient for a stable connection with the proximal and distal ends of the dissected bundles. Inflammatory markers peaked until postoperative day 1 but stabilized until discharge without any need for antibiotic treatment throughout the hospital stay. One patient was readmitted due to a urinary tract infection. Three patients reported about erections sufficient for penetration after three months with a continuous improvement of erectile function both after bi- and unilateral nerve-sparing with SSNR up to the last follow-up after 18 months. CONCLUSION: In this analysis of the first RARP with SSNR, a simple intraoperative handling without major complications was demonstrated. While the series provides evidence that SSNR is safe and feasible, a prospective randomized trial with long-term follow-up is needed to identify further improvement in postoperative erectile function due to the spider silk-directed nerve regeneration.

Cell culture media notably influence properties of human mesenchymal stroma/stem-like cells from different tissues.

BACKGROUND AIMS: Mesenchymal stroma/stem-like cells (MSCs) are a popular cell source and hold huge therapeutic promise for a broad range of possible clinical applications. However, to harness their full potential, current limitations in harvesting, expansion and characterization have to be overcome. These limitations are related to the heterogeneity of MSCs in general as well as to inconsistent experimental protocols. Here we aim to compare in vitro methods to facilitate comparison of MSCs generated from various tissues. METHODS: MSCs from 3 different tissues (bone marrow, dental pulp, adipose tissue), exemplified by cells from 3 randomly chosen donors per tissue, were systematically compared with respect to their in vitro properties after propagation in specific in-house standard media, as established in the individual laboratories, or in the same commercially available medium. RESULTS: Large differences were documented with respect to the expression of cell surface antigens, population doubling times, basal expression levels of 5 selected genes and osteogenic differentiation. The commercial medium reduced differences in these parameters with respect to individual human donors within tissue and between tissues. The extent, size and tetraspanin composition of extracellular vesicles were also affected. CONCLUSIONS: The results clearly demonstrate the extreme heterogeneity of MSCs, which confirms the problem of reproducibility of results, even when harmonizing experimental conditions, and questions the significance of common parameters for MSCs from different tissues in vitro.

Impact of Soil Microbial Amendments on Tomato Rhizosphere Microbiome and Plant Growth in Field Soil.

There is increased interest by the agricultural industry in microbial amendments that leverage natural beneficial interactions between plants and soil microbes to improve crop production. However, translating fundamental knowledge from laboratory experiments into efficient field application often has mixed results, and there is less clarity about the interaction between added microbes and the native microbial community, where microorganisms belonging to the same phylogenic clades often reside. In this study, four commercially available microbial amendments were examined in two greenhouse experiments using field soil to assess their impact on tomato plant growth and the native soil microbial communities. The amendments contained different formulations of plant growth-promoting bacteria (Lactobacilli, Rhizobia, etc.), yeasts, and mycorrhizal fungi. The application of the tested amendments in greenhouse conditions resulted in no significant impact on plant growth. A deeper statistical analysis detected variations in the microbial communities that accounted only for 0.25% of the total species, particularly in native taxa not related to the inoculated species and represented less than 1% of the total variance. This suggests that under commercial field conditions, additional confounding variables may play a role in the efficacy of soil microbial amendments. This study confirms the necessity of more in-depth validation requirements for the formulations of soil microbial amendments before delivery to the agricultural market in order to leverage their benefits for the producers, the consumers, and the environment.

Large-Volume Vascularized Muscle Grafts Engineered From Groin Adipose Tissue in Perfusion Bioreactor Culture.

BACKGROUND: Muscle tissue engineering still remains a major challenge. An axial vascular pedicle and a perfusion bioreactor are necessary for the development and maintenance of a large-volume engineered muscle tissue to provide circulation within the construct. This study aimed to determine whether large-volume vascularized muscle-like constructs could be made from rat groin adipose tissue in a perfusion bioreactor. METHODS: Epigastric adipofascial flaps based on the inferior superficial epigastric vessels were elevated bilaterally in male Lewis rats and connected to the bioreactor. The system was run using a cable pump and filled with myogenic differentiation medium in the perfusion bioreactor for 1, 3, 5, or 7 weeks. The resulting tissue constructs were characterized with respect to the morphology and muscle-related expression of genes and proteins. RESULTS: The histological examination demonstrated intact muscle-like tissue fibers; myogenesis was verified by the expression of myosin, MADS box transcription enhancer factor 2 D, desmin-a disintegrin and metalloproteinase domain (ADAM) 12-and M-cadherin using reverse transcription-polymerase chain reaction. Western blot analysis for desmin, MyoD1, N-cadherin, and ADAM12 was performed to verify the myogenic phenotype of the extracted differentiated tissue and prove the formation of muscle-like constructs. CONCLUSIONS: A large-volume vascularized muscle tissue could be engineered in a perfusion bioreactor. The resulting tissue had muscle-like histological features and expressed muscle-related genes and proteins, indicating that the trans-differentiation of adipose tissue into muscle tissue occurred.

Down-Regulation of MHC Class I Expression in Human Keratinocytes Using Viral Vectors Containing US11 Gene of Human Cytomegalovirus and Cultivation on Bovine Collagen-Elastin Matrix (Matriderm®): Potential Approach for an Immune-Privileged Skin Substitute.

Skin transplantation, especially in burn patients, is still challenging because surgeons are faced with limited disposability of autologous donor side material. The in vitro culture of keratinocytes has become an important reconstructive option. However, only non-immunogenic allogenic keratinocytes offer the opportunity to develop a skin graft that can overcome rejection. The purpose of the study was to develop targeted gene modification of keratinocytes in order to reduce immunogenicity for the use as allogenic transplantable skin graft by decreasing the expression of MHC class I. To reduce MHC class I expression, viral vectors containing the US11 gene of human cytomegalovirus were generated and tested on their functionality using Western blotting, indirect immunofluorescence staining, and flow cytometry. Transfected keratinocytes were seeded on commercially available bovine collagen-elastin matrices and further cultured for histological and cell survival assays. Results showed transient down-regulation of MHC class I after 24 h post-transfection, with recovery of MHC class I expression after 48 h. Histological assessments showed long-term cell survival as well as histological patterns comparable to epidermal layers of healthy human skin. The data postulates the potential application of US11 transfected keratinocytes as an approach towards an immune-privileged skin substitute. Nevertheless, further studies and data are needed.

Positive in vitro wound healing effects of functional inclusion bodies of a lipoxygenase from the Mexican axolotl.

BACKGROUND: AmbLOXe is a lipoxygenase, which is up-regulated during limb-redevelopment in the Mexican axolotl, Ambystoma mexicanum, an animal with remarkable regeneration capacity. Previous studies have shown that mammalian cells transformed with the gene of this epidermal lipoxygenase display faster migration and wound closure rate during in vitro wound healing experiments. RESULTS: In this study, the gene of AmbLOXe was codon-optimized for expression in Escherichia coli and was produced in the insoluble fraction as protein aggregates. These inclusion bodies or nanopills were shown to be reservoirs containing functional protein during in vitro wound healing assays. For this purpose, functional inclusion bodies were used to coat cell culture surfaces prior cell seeding or were added directly to the medium after cells reached confluence. In both scenarios, AmbLOXe inclusion bodies led to faster migration rate and wound closure, in comparison to controls containing either no AmbLOXe or GFP inclusion bodies. CONCLUSIONS: Our results demonstrate that AmbLOXe inclusion bodies are functional and may serve as stable reservoirs of this enzyme. Nevertheless, further studies with soluble enzyme are also necessary in order to start elucidating the exact molecular substrates of AmbLOXe and the biochemical pathways involved in the wound healing effect.

Brassica seed meal soil amendments transform the rhizosphere microbiome and improve apple production through resistance to pathogen reinfestation.

Brassicaceae seed meal (SM) formulations were compared with preplant 1,3-dichloropropene/chloropicrin (Telone-C17) soil fumigation for the ability to control apple replant disease and to suppress pathogen or parasite reinfestation of organic orchard soils at two sites in Washington State. Preplant soil fumigation and an SM formulation consisting of either Brassica juncea-Sinapis alba or B. juncea-B. napus each provided similar levels of disease control during the initial growing season. Although tree growth was similar in fumigated and SM-amended soil during the initial growing season, tree performance in terms of growth and yield was commonly superior in B. juncea-S. alba SM-amended soil relative to that in fumigated soil at the end of four growing seasons. SM-amended soils were resistant to reinfestation by Pratylenchus penetrans and Pythium spp. relative to fumigated soils and corresponded with enhanced tree performance. Phytotoxic symptoms were observed in response to SM amendment at one of two orchard sites, were dependent upon season of application, and occurred in an SM formulation-specific manner. After 2 years, the rhizosphere microbiome in fumigated soils had reverted to one that was indistinguishable from the no-treatment control. In contrast, rhizosphere soils from the SM treatment possessed unique bacterial and fungal profiles, including specific microbial elements previously associated with suppression of plant-pathogenic fungi, oomycetes, and nematodes. Overall diversity of the microbiome was reduced in the SM treatment rhizosphere, suggesting that enhanced "biodiversity" was not instrumental in achieving system resistance or pathogen suppression.

Impact of repeated fumigant applications on soil properties, crop yield, and microbial communities in a plastic-mulched tomato production system.

Pre-plant soil fumigation is widely applied to control nematodes, soil-borne fungal pathogens, and weeds in vegetable crops. However, most of the research evaluating the effect of fumigants on crop yield and soil microbial communities has been done on single compounds despite growers mainly applying fumigant combinations. We studied the effect of different fumigant combinations (chloropicrin, 1,3-dichloropropene, and metam potassium) on soil properties, crop yield, and the soil bacterial and fungal microbiome for two consecutive years in a plastic-mulched tomato production system in Florida (United States). While combinations of fumigants did not improve plant productivity more than the individual application of these products, application of fumigants with >60 % chloropicrin did significantly increase yield. Fumigant combinations had no significant effect on bacterial diversity, but fumigants with >35 % chloropicrin reduced soil fungal diversity and induced temporary changes in the soil bacterial and fungal community composition. These changes included short-term increases in the relative abundance of Firmicutes and Ascomycota, as well as decreases in other bacterial and fungal taxa. Repeated fumigation reduced network complexity and the relative abundance of several predicted bacterial functions and fungal guilds, particularly after fumigation and at end of harvest (3-months post fumigation). A structural equation model (SEM) showed fumigants not only directly impact crop yield, but they can also indirectly determine variations in plant productivity through effects on the soil microbiome. Overall, this study increases our understanding of the environmental and agricultural impacts of fumigants in a plastic-mulched tomato production system.

Nitrogen-fixing bacterial communities differ between perennial agroecosystem crops.

Biocrusts, common in natural ecosystems, are specific assemblages of microorganisms at or on the soil surface with associated microorganisms extending into the top centimeter of soil. Agroecosystem biocrusts have similar rates of nitrogen (N) fixation as those in natural ecosystems, but it is unclear how agricultural management influences their composition and function. This study examined the total bacterial and diazotrophic communities of biocrusts in a citrus orchard and a vineyard that shared a similar climate and soil type but differed in management. To contrast climate and soil type, these biocrusts were also compared with those from an apple orchard. Unlike natural ecosystem biocrusts, these agroecosystem biocrusts were dominated by proteobacteria and had a lower abundance of cyanobacteria. All of the examined agroecosystem biocrust diazotroph communities were dominated by N-fixing cyanobacteria from the Nostocales order, similar to natural ecosystem cyanobacterial biocrusts. Lower irrigation and fertilizer in the vineyard compared with the citrus orchard could have contributed to biocrust microbial composition, whereas soil type and climate could have differentiated the apple orchard biocrust. Season did not influence the bacterial and diazotrophic community composition of any of these agroecosystem biocrusts. Overall, agricultural management and climatic and edaphic factors potentially influenced the community composition and function of these biocrusts.