Proliferation of endothelial cells (HUVEC) on specific-modified collagen sponges loaded with different growth factors.

In general, matrices for tissue engineering must maintain structural integrity during the process of tissue formation and promote vascularization of developing tissue. Therefore, collagen sponges, manufactured by an approach that offers the potential of unidirectional pore size, were seeded with human umbilical vein endothelial cells (HUVEC) to demonstrate a positive effect on cell proliferation. In addition, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been used to promote proliferation of HUVEC on optimized collagen sponges. Growth and viability of the cells were evaluated. Potential unidirectional pore structure demonstrated an improvement of both, endothelial cell growth and viability. Supplementation of growth factors showed an additional increase of endothelial cell growth on collagen sponges, which confirmed the high potential of combining this biomaterial with growth factors. The results suggest that a collagen sponge with a potential specific pore size could be a suitable scaffold for endothelial cells and might be a promising implantable biomaterial with enhanced angiogenic capabilities for future clinical applications.

Assembled Plastid and Mitochondrial Genomes, as well as Nuclear Genes, Place the Parasite Family Cynomoriaceae in the Saxifragales.

Cynomoriaceae, one of the last unplaced families of flowering plants, comprise one or two species or subspecies of root parasites that occur from the Mediterranean to the Gobi Desert. Using Illumina sequencing, we assembled the mitochondrial and plastid genomes as well as some nuclear genes of a Cynomorium specimen from Italy. Selected genes were also obtained by Sanger sequencing from individuals collected in China and Iran, resulting in matrices of 33 mitochondrial, 6 nuclear, and 14 plastid genes and rDNAs enlarged to include a representative angiosperm taxon sampling based on data available in GenBank. We also compiled a new geographic map to discern possible discontinuities in the parasites' occurrence. Cynomorium has large genomes of 13.70-13.61 (Italy) to 13.95-13.76 pg (China). Its mitochondrial genome consists of up to 49 circular subgenomes and has an overall gene content similar to that of photosynthetic angiosperms, while its plastome retains only 27 of the normally 116 genes. Nuclear, plastid and mitochondrial phylogenies place Cynomoriaceae in Saxifragales, and we found evidence for several horizontal gene transfers from different hosts, as well as intracellular gene transfers.

Embryonic stem cell-derived motor neurons form neuromuscular junctions in vitro and enhance motor functional recovery in vivo.

BACKGROUND: Transplantation of embryonic stem cell-derived motor neurons may support the biological integrity of denervated muscle by forming new neuromuscular junctions and up-regulating specific growth factors. The authors examined the functional properties of embryonic stem cell-derived motor neurons in vitro and the effect of these cells transplanted in vivo. METHODS: Murine GFP/HB9 embryonic stem cells were differentiated into motor neurons. Co-cultures of motor neurons and myotubes were prepared to confirm the formation of neuromuscular junctions with synaptic markers. Athymic mice (n = 59) were assigned randomly to one of three experimental groups. A tibial nerve transection was performed without nerve repair, and motor neurons were transplanted into the gastrocnemius muscles immediately after transection (n = 24) or 3 weeks after denervation (n = 24). Quantitative and histologic assessments of gastrocnemius muscle were performed at days 7 and 21 after cell transplantation. Additional experimental groups (n = 11), where the tibial nerve underwent repair after transplantation, were formed. The effect of the transplants on motor recovery following nerve repair was investigated. RESULTS: Co-culture experiments showed the formation of neuromuscular junctions. In the experiment with nerve transection without nerve repair, the muscles transplanted with motor neurons were less atrophied than control phosphate-buffered saline-injected muscles at days 7 and 21. Those muscles receiving cells transplanted 3 weeks after denervation were not preserved. The motor recovery after nerve repair with cell transplantation was significantly enhanced compared with the control group. CONCLUSIONS: Transplantation of motor neurons prevented denervation atrophy but was not capable of rescuing already atrophied muscle. After nerve repair, motor neuron transplantation improved functional recovery.

The extent of thermal injury affects fractions of mononuclear cells.

BACKGROUND: The mononuclear cell (MNC) fraction contains a variety of cell types, including stem cells such as endothelial progenitor cells (EPCs). EPC can rapidly revascularise ischaemic areas, but their role in burns is unclear. AIM: This study investigates how thermal injury to the skin might influence mononuclear cells, CD34(+) cells and circulating EPC. METHODS: The study group comprised 17 people with burns and 17 age-matched controls. Blood samples were collected at five different time points during the first 5 days of hospitalisation. Clinical parameters and scores were documented as well as cell counts for MNC, CD34(+) cells and EPC. Counts were quantified by fluorescence-activated cell sorting. Serum was tested for vascular endothelial growth factor VEGF(165) by ELISA. RESULTS: All cell populations displayed significant, differing changes in counts and percentages after burn. These effects varied markedly over time and expressed different patterns if clinical scores were subjected to significance testing. EPC counts were significantly lowered in cases with fatal outcome. CONCLUSION: Burn affects the numbers of circulating MNC, CD34(+) and EPC. These time-dependent changes imply involvement of these cell groups in the trauma. EPC counts seem to be a predictive factor for outcome of cases of severe burn.

The diversity of carbon monoxide intoxication: medical courses can differ extremely-a case report.

Intoxications of carbon monoxide are frequent and may affect systems of lung, heart, and brain, leading to coma or death in severe cases. In this case report, we present two adults who were exposed to the same source of carbon monoxide for a nearly equal period of time. The first patient, a 28-yr-old female, developed massive symptoms including loss of consciousness, respiratory insufficiency, and lung complications resulting in severe lung edema. She was intubated and ventilated for 43 h before she recovered and could be extubated. The other patient, a 22-yr-old male, recovered immediately and was fully orientated after applying an oxygen mask at the scene of incident. After admission to the intensive care unit, both patients showed an equally high serum level of COHb and received hyperbaric oxygen therapy. The male patient was discharged from hospital the following day, whereas the female remained in intensive care for 4 days. A satisfactory explanation could not be found for the difference in the clinical progression in these two cases. However, this case report shows that, in spite of almost equal serum levels of carboxyhemoglobin (COHb), the individual symptoms can vary extremely. Therefore, a detailed medical history, physical examination, supporting diagnostic measures, and the continuous monitoring of vital parameters in a specialized clinic are essential.

Evaluation of functional nerve recovery with Visual-SSI--a novel computerized approach for the assessment of the static sciatic index (SSI).

Complete nerve transection (neurotmesis) of the rat sciatic nerve is a well-established animal model. The most frequently used behavioural for evaluation of neurotmesis-induced deficits is the walking track analysis with calculation of the sciatic functional index (SFI). More recently, the static sciatic index (SSI) has been developed, which shows a good correlation with the SFI. However, despite all advantages (high accessibility, easy handling, high accuracy, cost-effectiveness), the SSI is still not widely used. We, therefore, developed a novel programme ("Visual-SSI"), which will be made freely available for the assessment of the SSI. As gold-standard for the treatment of neurotmesis-induced nerve gaps, autologous nerve transplantation studies in the rat sciatic nerve model (n=16 [6 weeks], n=8 [12 weeks]) were carried out to test the effectiveness and feasibility of the Visual-SSI software. We observed a significant recovery starting from the pre-operative condition over the 3rd, 6th, 9th weeks until the 12th week after surgery (p<0.05). Theoretically, the SSI can be calculated from both rearing and normal standing position of the rats and we investigated whether the SSI is affected differentially by these positions. We observed no significant differences between animals in a rearing and normal standing stance (p>0.05). The present method combines efficiency (simplicity of use, rapid and economical setup) with accurate and precise quantification of the functional regeneration in the sciatic nerve lesion model of the rat.

Assessment of stem cell/biomaterial combinations for stem cell-based tissue engineering.

Biomaterials are used in tissue engineering with the aim to repair or reconstruct tissues and organs. Frequently, the identification and development of biomaterials is an iterative process with biomaterials being designed and then individually tested for their properties in combination with one specific cell type. However, recent efforts have been devoted to systematic, combinatorial and parallel approaches to identify biomaterials, suitable for specific applications. Embryonic and adult stem cells represent an ideal cell source for tissue engineering. Since stem cells can be readily isolated, expanded and transplanted, their application in cell-based therapies has become a major focus of research. Biomaterials can potentially influence e.g. stem cell proliferation and differentiation in both, positive or negative ways and biomaterial characteristics have been applied to repel or attract stem cells in a niche-like microenvironment. Our consortium has now established a grid-based platform to investigate stem cell/biomaterial interactions. So far, we have assessed 140 combinations of seven different stem cell types and 19 different polymers performing systematic screening assays to analyse parameters such as morphology, vitality, cytotoxicity, apoptosis, and proliferation. We thus can suggest and advise for and against special combinations for stem cell-based tissue engineering.

Cortisol in severely burned patients: investigations on disturbance of the hypothalamic-pituitary-adrenal axis.

Thermal injuries of more than 20% of body surface area lead to conditions resembling a severe systemic inflammatory response syndrome, such as in septic shock. It has been shown that septic shock may lead to disturbances in cortisol metabolism and balance of the hypothalamic-pituitary-adrenal axis. To investigate whether such a disturbance also occurs in the very early stages of systemic inflammatory response syndrome in burned patients, we performed 20 corticotropin-releasing hormone tests on day 1 after admission to our unit. In 7 of 20 patients, a disturbance of cortisol secretion could be demonstrated. Four patients developed adrenal insufficiency. The correlation between the abbreviated burn severity index and the risk of developing adrenal insufficiency was significant (P = 0.008). We observed a higher mortality rate in adrenally insufficient patients; however, because of the small patient number, we were not able to prove this observation with a statistical significant correlation (P = 0.11). Our findings indicate that temporary adrenal insufficiency occurs in the early stages of severe injury. Further investigations will have to be performed to clarify whether such patients benefit from cortisol replacement.

Fibromatosis-like myofibroblastic tumour of forearm: case report and interdisciplinary management.

We present a rare case of a fibromatosis-like myofibroblastic tumour of the forearm with infiltration of muscular, neural, and vascular structures. This is a rare and transitional type of myofibroblastic tumour, and we emphasise important aspects of diagnosis, clinical features, interdisciplinary management, and differential diagnoses.

Plasticity of human adipose stem cells to perform adipogenic and endothelial differentiation.

Recent research findings postulate that adipocytes and endothelial cells (EC) may share a common progenitor. However, the interlinking pathways between adipose tissue and endothelium, and the differentiation potential of cells to convert from one tissue into the other via progenitor cells have not been elucidated and are therefore the focus of this study. Stromal vascular fraction (SVF) cells were isolated from liposuction aspirates or excised adipose tissue and separated into CD31+ and CD31- populations by magnet-assisted cell sorting. Differentiation to fat tissue was induced in both CD31 fractions after expansion by insulin, dexamethasone, isobutylmethylxanthine, triiodothyronine, pioglitazone, and transferrin. Differentiation was assayed enzymatically and by cell counting. Maturation to endothelium was performed with vascular endothelial growth factor (VEGF), insulin-like growth factor-1 plus 2% fetal calf serum, and confirmed by flow cytometry and tube formation assays on Matrigel. Our results show that the SVF contains a CD31-, S100+ cell type that can differentiate into adipocytes and EC. The SVF also comprises CD31+ cells that, although they have an endothelial phenotype, can be converted into mature adipocytes. These findings demonstrate the potency of SVF cells to perform both adipogenic and endothelial differentiation. Further, they reveal the plasticity of mature cells of mesenchymal origin to undergo conversion from endothelium to adipose tissue and vice versa.

The morphology and biomechanical characteristics of subcutaneously implanted tissue-engineered human septal cartilage.

The purpose of the study was to examine the morphology and biomechanical characteristics of in vivo cultured tissue-engineered human septal cartilage as a prospective autogenous transplant material for subcutaneous implantation in reconstructive procedures. Chondrocytes were enzymatically isolated from human septal cartilage biopsies. The cell number was expanded in monolayer culture. Chondrocytes were then fixed on a non-woven poly-lactide-poly-glycolide (PGLA) polymer scaffold by means of fibrin glue. The PGLA-polymer construct was implanted subcutaneously on the back of athymic mice and allowed to mature for 6 or 12 weeks. After killing the mice, the formed cartilage was tested on a material testing machine with a highly standardized reproducible setting. Biomechanical testing consisted of an indentation test, which revealed the failure load and compressive modulus of the neocartilage. The failure load shows the upper limit of supported stress. The compressive modulus is a measure of the templates' stiffness. After testing, the templates were histologically stained. Native human septal cartilage served as a control group. Histological and macroscopic examination showed cartilage formation of a hyaline-like morphology. Histological staining revealed the synthesis of abundant mucopolysaccharid matrix. The biomechanical characteristics of neocartilage proved to be of no statistical difference compared to native human septal cartilage. The failure load and compressive modulus were initially somewhat lower and reached the control group's results after 12 weeks in-vivo. Summarizing, tissue engineered nasal cartilage matches typical mechanical characteristics of native hyaline cartilage. Its elasticity and failure load are of sufficient quality to meet the clinical requirements for reconstructive surgery.

Creating artificial perichondrium by polymer complex membrane macroencapsulation: immune protection and stabilization of subcutaneously transplanted tissue-engineered cartilage.

Functional organ or tissue failure is one of the most frequent, devastating and costly problems in modern health care. The field of tissue engineering has tremendous potential for developing new functional tissue. In reconstructive surgery, cartilage engineering could be a serious alternative to the established method of autologous cartilage transplantation. Recent studies demonstrate cartilage engineering by subcutaneous implantation of chondrocyte-seeded PGA/PLA-fibrin glue scaffolds in the backs of nude mice. In both autologous cartilage transplantation and cartilage engineering, the host immune response affects transplant integrity and cartilage morphology to an unforeseeable extent. To investigate whether polyelectrolyte complex (PEC) membranes can prevent rejection of cartilage transplants without neglecting tissue metabolism, tissue-engineered cartilage encapsulated with a PEC membrane was subcutaneously implanted in the backs of nude mice. Non-encapsulated tissue-engineered cartilage was used for the control group. Histochemistry and scanning electron microscopy were performed 4 and 12 weeks after implantation. There was no interaction between the host and the implant with an intact PEC membrane. With protection by PEC encapsulation, implanted tissue-engineered cartilage showed no signs of degeneration and had a significantly weaker cellular immune response than without it. Thus, PEC membrane encapsulation appears to be a novel approach for protecting cartilage implants from host immune response after autologous transplantation.

Immunomodulation of tissue-engineered transplants: in vivo bone generation from methylprednisolone-stimulated chondrocytes.

Subcutaneously implanted, in vitro engineered tissue is generally affected by the immune system of the host even in autogenous transplantation. The aim of this study was to investigate immunomodulation of subcutaneously implanted tissue-engineered cartilage transplants by intramuscular methylprednisolone application. Transplants consisted of auricular rabbit chondrocytes, polylactide-polyglycolide co-polymer fleeces and species-specific fibrin or agarose. The transplants were subcutaneously implanted in the ridge. Thereafter, animals were separated into two groups, one with and one without methylprednisolone treatment. The specimens were histologically investigated after 6 and 12 weeks. Fleece fiber degradation was complete after 12 weeks, and all transplants showed areas of calcification. The corticosteroid-treated group presented pronounced trabecular bone generation without fibrous tissue infiltration. The untreated group showed sporadic islets of calcification without coherent bone formation, and adjacent fibrous tissue had infiltrated the transplants. Native controls and corticoid-treated transplants did not exhibit bone generation or signs of fibrous tissue infiltration. This study found that immunomodulation by intramuscular methylprednisolone application protects tissue-engineered autogenous chondrocyte transplants from fibrous tissue infiltration and induces trabecular bone formation.

Tissue engineering of bone for mandibular augmentation in immunocompetent minipigs: preliminary study.

Large mandibular defects caused by trauma, infection or resection of a tumour are still a major problem for plastic and maxillofacial surgeons. The modern concept of tissue engineering combines the osteoinductive effects of osteogenic cells with a suitable scaffold structure to promote differentiation of osteoblasts and optimal matrix production. Critical size mandibular bone defects were therefore made to investigate the osteogenic potential of periosteal cells and a bioabsorbable polymer fleece (Ethisorb 510) in minipigs. Periosteal cells were isolated from four minipigs, expanded in vitro and seeded with fibrin glue into Ethisorb 510 fleeces. Tissue constructs were used to repair critical size mandibular defects and compared with two minipigs with untreated bone defects. Bone healing was evaluated after 90 and 180 days by radiographs and a histological scoring system. The radiographs showed increased radiodensity of defects filled with the cell-fibrin-fleece-constructs compared with the untreated control group after 90 and 180 days in vivo. The defects repaired by the cell-fibrin-scaffolds (180 days in vivo) obtained the highest histological mean score 2.9 (range 2-3), while defects filled by cell-fibrin-scaffolds (90 days in vivo) achieved a mean score of 2.1 (range 2-3). In contrast, the control group (n = 2) scored 1 and 2. The results show that a combination of periosteal cells and polymer fleeces may be a promising approach for clinical mandibular augmentation.

A tissue-engineering model for the manufacture of auricular-shaped cartilage implants.

The established surgical methods of external ear reconstruction using autogenous tissue represent the current state of the art. Because of the limited possibilities for shaping conventional harvested autogenous rib cartilage, the cosmetic results of auricular reconstruction are frequently unsatisfactory. Tissue engineering could represent an alternative technique for obtaining a precisely shaped cartilage implant that avoids donor site morbidity and unsatisfactory cosmetic results. In this study, the reliability and quality of a tissue-engineering model for the manufacture of auricular-shaped human cartilage implants was investigated, focusing on the feasibility of the manufacturing process and the in vivo and in vitro maturation of an extracellular cartilage-like matrix. Implants were molded within an auricular-shaped silicone cylinder, and human nasal septal chondrocytes crosslinked by human fibrin within bioresorbable PGLA-PLLA polymer scaffolds were used. After an in vitro incubation of up to 6 weeks, defined fragments of the prefabricated auricular-shaped construct were implanted subcutaneously on the backs of nude mice for at least 6 to 12 weeks ( n=7). Scaffolds without cell loading served as controls. Macroscopic and histochemical examination after 3 and 6 weeks in vitro showed a solid compound of homogenously distributed chondrocytes within the polymer scaffold, leading only to a limited pericellular matrix formation. Analysis after 6 and 12 weeks of in vivo maturation demonstrated a solid tissue compound and neocartilage formation with the presence of cartilage-specific matrix components. Implants obtained shape and size during the entire period of implantation. The model of cartilage implant manufacturing presented here meets all biocompatible requirements for in vitro prefabrication and in vivo maturation of autogenous, individually shaped cartilage transplants.