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.

Intraneural Ganglion of the Peroneal Nerve-A Rare Cause of Pediatric Peroneal Nerve Palsy: A Case Report.

Intraneural ganglia are benign mucinous cysts located within the epineurium of a peripheral nerve. The pathogenesis and formation of intraneural ganglia are controversial. The main theories described in the literature are of degenerative, synovial or de novo occurrence. We present the case of a 14-year-old boy who presented in our outpatient clinic with a complaint of interdigital neuralgia between hallux and second toe, as well as left foot drop. MRI examination showed a hyperintense cystic distension of the common peroneal nerve measuring 130 mm × 5 mm extending from the poplitea to the anterior compartment of the leg. We performed microscopic decompression and neurolysis surgery. The cyst showed a sac-like distension at its distal end with connection to the tibiofibular joint and was resected. After 8 weeks, postoperatively, the boy claimed to be pain-free and slight recovery of the superficial peroneal nerve was noticed. At 6 months postoperative, the patient showed a continuous improvement of motor function, demonstrating foot eversion with 3/5 muscle strength and foot extension with 2/5 muscle strength. Intraneural ganglia reported for pediatric patients represent a very rare entity. To the best of our knowledge, less than 15 cases have been described within the English-speaking literature.

[Functional reconstruction in traumatic paralysis]. / Funktionelle Rekonstruktionen bei traumatischen Lähmungen.

BACKGROUND: Traumatic nerve injuries are associated with a high morbidity and long rehabilitation times. The extent of a nerve lesion and the related regeneration potential can often only be estimated during the course, whereby the time window for successful surgical interventions is limited. OBJECTIVE: The incidence and distribution of traumatic nerve lesions are reported. Algorithms for treatment decisions are presented. MATERIAL AND METHODS: Statistics from the German TraumaRegister DGU® as well as international registers were evaluated. The results of basic research and expert recommendations for diagnostics and treatment are discussed. RESULTS AND CONCLUSION: A strategic approach to conservative and surgical treatment of traumatic nerve injuries depending on the extent of injury and resulting regeneration potential is recommended. In conjunction with the clinical course, electrophysiology and imaging diagnostics, e.g. nerve sonography, can help to differentiate between neurapraxia, axonotmesis and neurotmesis.

Electroporation: A Sustainable and Cell Biology Preserving Cell Labeling Method for Adipogenous Mesenchymal Stem Cells.

Human mesenchymal stem cells derived from adipose tissue (AD-hMSCs) represent a promising source for tissue engineering and are already widely used in cell therapeutic clinical trials. Until today, an efficient and sustainable cell labeling system for cell tracking does not exist. We evaluated transient transfection through electroporation for cell labeling and compared it with lentiviral transduction for AD-hMSCs. In addition, we tested whether nonsense DNA or a reporter gene such as enhanced green fluorescent protein (EGFP) is the more suitable label for AD-hMSCs. Using electroporation, the transfection efficiency reached a maximal level of 44.6 ± 1.1% EGFP-positive cells after selective and expansive cultivation of the mixed MSC population, and was 44.5 ± 1.4% after gene transfer with Cyanin3-marked nonsense-label DNA, which remained stable during 2 weeks of nonselective cultivation (37.2 ± 4.7% positive AD-hMSCs). Electroporation with both nonsense DNA and pEGFP-N1 led to a slight growth retardation of 45.2% and 59.1%, respectively. EGFP-transfected or transduced AD-hMSCs showed a limited adipogenic and osteogenic differentiation capacity, whereas it was almost unaffected in cells electroporated with the nonsense-label DNA. The nonsense DNA was detectable through quantitative real-time polymerase chain reaction for at least 5 weeks/10 passages and in differentiated AD-hMSCs. EGFP-labeled cells were trackable for 24 h in vitro and served as testing cells with new materials for dental implants for 7 days. In contrast, lentivirally transduced AD-hMSCs showed an altered natural immune phenotype of the AD-hMSCs with lowered expression of two cell type defining surface markers (CD44 and CD73) and a relevantly decreased cell growth by 71.8% as assessed by the number of colony-forming units. We suggest electroporation with nonsense DNA as an efficient and long-lasting labeling method for AD-hMSCs with the comparably lowest negative impact on the phenotype or the differentiation capacity of the cells, which may, therefore, be suitable for tissue engineering. In contrast, EGFP transfection by electroporation is efficient but may be more suitable for cell tracking within cell therapies without MSC differentiation procedures. Since current protocols of lentiviral gene transduction include the risk of cell biological alterations, electroporation seems advantageous and sustainable enough for hMSC labeling.

Three-Dimensional Modelling inside a Differential Pressure Laminar Flow Bioreactor Filled with Porous Media.

A three-dimensional computational fluid dynamics- (CFD-) model based on a differential pressure laminar flow bioreactor prototype was developed to further examine performance under changing culture conditions. Cell growth inside scaffolds was simulated by decreasing intrinsic permeability values and led to pressure build-up in the upper culture chamber. Pressure release by an integrated bypass system allowed continuation of culture. The specific shape of the bioreactor culture vessel supported a homogenous flow profile and mass flux at the scaffold level at various scaffold permeabilities. Experimental data showed an increase in oxygen concentration measured inside a collagen scaffold seeded with human mesenchymal stem cells when cultured in the perfusion bioreactor after 24 h compared to static culture in a Petri dish (dynamic: 11% O2 versus static: 3% O2). Computational fluid simulation can support design of bioreactor systems for tissue engineering application.

Noninvasive Oxygen Monitoring in Three-Dimensional Tissue Cultures Under Static and Dynamic Culture Conditions.

We present a new method for noninvasive real-time oxygen measurement inside three-dimensional tissue-engineered cell constructs in static and dynamic culture settings in a laminar flow bioreactor. The OPAL system (optical oxygen measurement system) determines the oxygen-dependent phosphorescence lifetime of spherical microprobes and uses a two-frequency phase-modulation technique, which fades out the interference of background fluorescence from the cell carrier and culture medium. Higher cell densities in the centrum of the scaffolds correlated with lower values of oxygen concentration obtained with the OPAL system. When scaffolds were placed in the bioreactor, higher oxygen values were measured compared to statically cultured scaffolds in a Petri dish, which were significantly different at day 1-3 of culture. This technique allows the use of signal-weak microprobes in biological environments and monitors the culture process inside a bioreactor.

Alignment-Annotator web server: rendering and annotating sequence alignments.

UNLABELLED: Alignment-Annotator is a novel web service designed to generate interactive views of annotated nucleotide and amino acid sequence alignments (i) de novo and (ii) embedded in other software. All computations are performed at server side. Interactivity is implemented in HTML5, a language native to web browsers. The alignment is initially displayed using default settings and can be modified with the graphical user interfaces. For example, individual sequences can be reordered or deleted using drag and drop, amino acid color code schemes can be applied and annotations can be added. Annotations can be made manually or imported (BioDAS servers, the UniProt, the Catalytic Site Atlas and the PDB). Some edits take immediate effect while others require server interaction and may take a few seconds to execute. The final alignment document can be downloaded as a zip-archive containing the HTML files. Because of the use of HTML the resulting interactive alignment can be viewed on any platform including Windows, Mac OS X, Linux, Android and iOS in any standard web browser. Importantly, no plugins nor Java are required and therefore Alignment-Anotator represents the first interactive browser-based alignment visualization. AVAILABILITY: http://www.bioinformatics.org/strap/aa/ and http://strap.charite.de/aa/.

Altered VEGF-A and receptor mRNA expression profiles, and identification of VEGF144 in foetal rat calvaria cells, in coculture with microvascular endothelial cells.

Cellular proliferation and differentiation during angiogenesis and osteogenesis require the communication of different cell types through growth factors and their receptors. Vascular endothelial growth factor (VEGF-A) plays an important role in osteoblast and endothelial cell intercommunication. We have investigated the effect of monocultures and indirect coculture of foetal rat calvarial (FRC) osteoblasts and microvascular endothelial cells (ECs) on nodule formation, proliferation, and mRNA-expression of VEGF-A and its receptors during culturing. Despite increased nodule formation in the presence of dexamethasone (Dex) in monocultures, the number of nodules and alkaline phosphatise activity were decreased in cocultured FRCs. VEGF mRNA expression over the differentiation period showed the expression of most Vegf isoforms is biphasic in both FRCs and ECs, whereas receptor expression was quite variable; however, that of Np-2 in FRCs increased steadily and significantly from 8 h to 14 days after an initial drop in expression. Significant changes in the proportion of Vegfa by Day 14 were noted mainly in the matrix-bound variants Vegf144 and Vegf188 in ECs and osteoblasts, respectively. Less striking results were seen in the expression of the soluble isoforms in either cell type. These results have identified expression of Vegf144 in osteoblasts, suggesting a possible autocrine and/or paracrine role that is affecting osteoblast mineralisation along with Vegf188, as well as possible early roles of these isoforms in initial cell attachment. Further study of VEGF expression in coculture and Vegf144 will lead to better understanding of its role in cell-cell communication and bone development.

Rehabilitation of burn patients: an underestimated socio-economic burden.

PURPOSE: Patients with burns utilise intensive medical care and rehabilitation. Deep dermal burns lead to scar contractures. Virtually no published data exists on costs for treatment of acute burns in comparison to burn sequelae. Our purpose was to collect financial data on burn therapy to estimate the socio-economic burden of thermal injuries. METHODS: German-DRG for in-patient treatment of burns was collected from our burn center. DRG-related T95.- coding served as a search tool for burn associated sequelae. To include rehabilitation costs, data from the largest health care insurance and a workmen compensation fund were acquired. FINDINGS: Acute burn treatment comprised 92% of costs for intensive care with approximately 4.600 EUR per percent total burned surface area (TBSA). Expenses for non-intensive care patients were significantly lower than for burn sequelae. Rehabilitation expenses were 4.4-fold higher than costs for acute burns including 59% for manual therapy and 37% for auxiliary material. CONCLUSIONS: TBSA multiplied by factor 4600 could serve for cost calculation of severely burned patients. Approximately 0.3 billion EUR in total or 270.000 EUR per patient/year were spent on burn sequelae. Early admission to specialized burn centers is advocated with state-of-the-art treatment to minimize burn sequelae and health care expenses.

Potential for osteogenic and chondrogenic differentiation of MSC.

The introduction of mesenchymal stem cells (MSC) into the field of tissue engineering for bone and cartilage repair is a promising development, since these cells can be expanded ex vivo to clinically relevant numbers and, after expansion, retain their ability to differentiate into different cell lineages. Mesenchymal stem cells isolated from various tissues have been intensively studied and characterized by many research groups. To obtain functionally active differentiated tissue, tissue engineered constructs are cultivated in vitro statically or dynamically in bioreactors under controlled conditions. These conditions include special cell culture media, addition of signalling molecules, various physical and chemical factors and the application of different mechanical stimuli. Oxygen concentration in the culture environment is also a significant factor which influences MSC proliferation, stemness and differentiation capacity. Knowledge of the different aspects which affect MSC differentiation in vivo and in vitro will help researchers to achieve directed cell fate without the addition of differentiation agents in concentrations above the physiological range.

Potential of mesenchymal stem cell applications in plastic and reconstructive surgery.

: Novel therapy with mesenchymal stem cells from bone marrow, adipose tissue, or other sources has raised high hopes for treatment of a variety of diseases. For plastic and reconstructive surgery, first pilot studies and clinical trials using stem cells for treatment of chronic wounds, radiation injury, or soft tissue augmentation have furnished encouraging results compared with the limitations of standard therapy, for example autologous fat grafting. Further research must be conducted to reveal the complex physiological interactions between activated stem cells and the host environment. Long-term effects and safety aspects of these novel treatment options also require randomized controlled studies. For future clinical applications, guidelines and standardized procedures for stem cell isolation and preparation, and techniques for application must be established.

A differential pressure laminar flow reactor supports osteogenic differentiation and extracellular matrix formation from adipose mesenchymal stem cells in a macroporous ceramic scaffold.

We present a laminar flow reactor for bone tissue engineering that was developed based on a computational fluid dynamics model. The bioreactor design permits a laminar flow field through its specific internal shape. An integrated bypass system that prevents pressure build-up through bypass openings for pressure release allows for a constant pressure environment during the changing of permeability values that are caused by cellular growth within a porous scaffold. A macroporous ceramic scaffold, composed of zirconium dioxide, was used as a test biomaterial that studies adipose stem cell behavior within a controlled three-dimensional (3D) flow and pressure environment. The topographic structure of the material provided a basis for stem cell proliferation and differentiation toward the osteogenic lineage. Dynamic culture conditions in the bioreactor supported cell viability during long-term culture and induced cell cluster formation and extra-cellular matrix deposition within the porous scaffold, though no complete closure of the pores with new-formed tissue was observed. We postulate that our system is suitable for studying fluid shear stress effects on stem cell proliferation and differentiation toward bone formation in tissue-engineered 3D constructs.

Stem cell differentiation depending on different surfaces.

Mesenchymal stem cells and 3D biomaterials are a potent assembly in tissue engineering. Today, a sizable number of biomaterials has been characterized for special tissue engineering applications. However, diverse material properties, such as soft or hard biomaterials, have a specific influence on cell behavior. Not only the cell attachment and proliferation, but also differentiation is controlled by the microenvironment. Material characteristics such as pore size, stiffness, roughness, and geometry affect not only the cell attachment and proliferation, but also the differentiation behavior of mesenchymal stem cells. Optimization of these features might enable direct differentiation without adjustment of the culture medium by applying expensive growth or differentiation factors. Future aspects include the design of multilayered biomaterials, where each zone fulfills a distinct function. Moreover, the embedding of growth and differentiation factors into the matrix with a controlled release rate might be advantageous to direct differentiation.

Isolation, characterization, differentiation, and application of adipose-derived stem cells.

While bone marrow-derived mesenchymal stem cells are known and have been investigated for a long time, mesenchymal stem cells derived from the adipose tissue were identified as such by Zuk et al. in 2001. However, as subcutaneous fat tissue is a rich source which is much more easily accessible than bone marrow and thus can be reached by less invasive procedures, adipose-derived stem cells have moved into the research spotlight over the last 8 years.Isolation of stromal cell fractions involves centrifugation, digestion, and filtration, resulting in an adherent cell population containing mesenchymal stem cells; these can be subdivided by cell sorting and cultured under common conditions.They seem to have comparable properties to bone marrow-derived mesenchymal stem cells in their differentiation abilities as well as a favorable angiogenic and anti-inflammatory cytokine secretion profile and therefore have become widely used in tissue engineering and clinical regenerative medicine.

Cultured keratinocytes in fibrin with decellularised dermis close porcine full-thickness wounds in a single step.

The purpose of this study was to assess the feasibility of combined keratinocyte and dermal scaffold transplantation performed in a single step for treatment of full-thickness wounds. Cultured autologous keratinocytes were suspended in fibrin and grafted together with decellularised human dermis (Alloderm) in a porcine animal model, involving 10 animals over 4 weeks. Wound healing was evaluated by planimetry. Histology included morphological analysis as well as immunohistochemistry at regular intervals (1, 2 and 4 weeks). The results showed both successful histo-integration of the in vivo composite grafts and reduced wound contraction, compared with the control group (plain epithelial grafts). Histologically a neo-epithelium originated from the grafted cells on top of the decellularised dermis, as well as a reconstituted basement membrane. After 4 weeks cellular ingrowth into the dermal matrix could be observed. The successful combination of a keratinocyte-fibrin suspension and acellular dermis applied in a single step onto full-thickness wounds resulted in closure.

Bone challenges for the hand surgeon: from basic bone biology to future clinical applications.

Bone is a complex tissue composed of a calcified extracellular matrix with specialized cells that produce, maintain, and resorb the bone. Bone also has a rich vascular and neural supply. Bone has a great capability of regeneration, healing, and remodelling that is influenced by external factors, such as stress forces, and internal regulators that include hormones, vitamins, and growth factors. These factors dictate bone biology, and variations result in pathophysiologic conditions that have clinical implications in hand surgery. Solutions to the challenges in hand surgery rely on a thorough understanding of the biology of bone.