Preparation and Analysis of PCL Spun Chitosan Scaffolds as Guidance Channels for Peripheral Nerve Regeneration

The results of this work show that the process of oriented solidification and lyophilisation is able to produce porous chitosan scaffolds with appropriate porosity and pore size for nerve regeneration. Interesting in this context are the results of statistical analysis of image analysis from SEM micrographs of uncrosslinked and UV cross-linked samples. The average pore size and mean minimum pore diameter show only small differences if the cooling rate is varied from B = 1…5 K / min and the temperature gradient from G = 1, 1.5, 2.0 K / mm. The average pore size (cross sectional area) of these samples can be estimated with reasonable accuracy, with 2100 μm². The average minimum pore diameter is within the range of 36-38 μm. These values are in a favourable range for the cell growth of nerve regeneration

Downstream processing of high chain length polysialic acid using membrane adsorbers and clay minerals for application in tissue engineering

Polysialic acid (polySia) is a carbohydrate polymer of varying chain length. It is a promising scaffold material for tissue engineering. In this work, high chain length polySia was produced by an Escherichia coli K1 strain in a 10-L bioreactor in batch and fed-batch mode, respectively. A new downstream process for polySia is presented, based on membrane adsorber technology and use of inorganic anion exchanger. These methods enable the replacement of precipitation steps, such as acetone, cetavlon, and ethanol precipitation of the already established purification process. The purification process was simplified, while process efficiency and product qualities were improved. The overall yield of polySia from a 10-L batch cultivation process was 61% and for 10-L fed-batch cultivation process the yield was 40% with an overall purity of 98%. The endotoxin content was determined to be negligible (14 EU mg-1). The main advantage of this new downstream process is that polySia with high chain length of more than 130 degree of polymerization can be obtained. In fed-batch cultivation, chain lengths up to 160 degree of polymerization were obtained.DFG/FOR/54

In vitro evaluation of cell-seeded chitosan films for peripheral nerve tissue engineering

Natural biomaterials have attracted an increasing interest in the field of tissue-engineered nerve grafts, representing a possible alternative to autologous nerve transplantation. With the prospect of developing a novel entubulation strategy for transected nerves with cell-seeded chitosan films, we examined the biocompatibility of such films in vitro. Different types of rat Schwann cells (SCs)-immortalized, neonatal, and adult-as well as rat bone-marrow-derived mesenchymal stromal cells (BMSCs) were analyzed with regard to their cell metabolic activity, proliferation profiles, and cell morphology after different time points of mono-and cocultures on the chitosan films. Overall the results demonstrate a good cytocompatibility of the chitosan substrate. Both cell types were viable on the biomaterial and showed different metabolic activities and proliferation behavior, indicating cell-type-specific cell-biomaterial interaction. Moreover, the cell types also displayed their typical morphology. In cocultures adult SCs used the BMSCs as a feeder layer and no negative interactions between both cell types were detected. Further, the chitosan films allow neurite outgrowth from dissociated sensory neurons, which is additionally supported on film preseeded with SC-BMSC cocultures. The presented chitosan films therefore demonstrate high potential for their use in tissue-engineered nerve grafts.This work was supported by the European Community's Seventh Framework Programme (FP7-HEALTH-2011) under grant agreement No. 278612. This work was also co-funded by Programa Operacional Regional do Norte (ON.2-O Novo Norte), ao abrigo do Quadro de Referencia Estrategico Nacional (QREN), and atraves do Fundo Europeu de Desenvolvimento Regional (FEDER). The authors gratefully acknowledge the delivery of the chitosan raw material by Altakitin S.A., Portugal, and the fabrication of chitosan films by Medovent GmbH, Germany

BIOHYBRID – Biohybrid templates for peripheral nerve regeneration

[Excerpt] Peripheral nerve injuries represent a major cause for morbidity and disability in affected patients and cause substantial costs for society in a global perspective. It has been estimated that peripheral nerve injuries affect 2.8% of trauma patients,many of whom acquire life-long disability (Noble et al., 1998). With respect to an incidence of nerve injuries of 13.9/100,000 inhabitants per year (Asplund et al., 2009) and the number of inhabitants in the EU (495,000,000 inhabitants in 2007), the number of peripheral nerve injuries requiring repair and reconstruction, excluding nerve injuries by amputations, may be 70,000 annually only in EU countries. Related to peripheral nerve injuries, the costs for society are substantial and consist of direct (costs for surgery, outpatient visits and rehabilitation) and indirect (lost production) costs. Individual median and ulnar nerve injuries in the forearm have total costs of EUR 51,000 and 31,000, respectively, where around 85% of the costs consist of loss of production (Rosberg et al., 2005), still excluding costs for adjusted quality of life ( Eriksson et al., 2011) . Thus, one may estimate that the annual costs only in the EU may be as high as EUR 2.2 billion, indicating that improved treatment strategies for peripheral nerve injuries may not only improve the situation for patients, but may also significantly reduce costs for society. [...](undefined

Culture Conditions for Human Induced Pluripotent Stem Cell-Derived Schwann Cells: A Two-Centre Study

Adult human Schwann cells represent a relevant tool for studying peripheral neuropathies and developing regenerative therapies to treat nerve damage. Primary adult human Schwann cells are, however, difficult to obtain and challenging to propagate in culture. One potential solution is to generate Schwann cells from human induced pluripotent stem cells (hiPSCs). Previously published protocols, however, in our hands did not deliver sufficient viable cell numbers of hiPSC-derived Schwann cells (hiPSC-SCs). We present here, two modified protocols from two collaborating laboratories that overcome these challenges. With this, we also identified the relevant parameters to be specifically considered in any proposed differentiation protocol. Furthermore, we are, to our knowledge, the first to directly compare hiPSC-SCs to primary adult human Schwann cells using immunocytochemistry and RT-qPCR. We conclude the type of coating to be important during the differentiation process from Schwann cell precursor cells or immature Schwann cells to definitive Schwann cells, as well as the amounts of glucose in the specific differentiation medium to be crucial for increasing its efficiency and the final yield of viable hiPSC-SCs. Our hiPSC-SCs further displayed high similarity to primary adult human Schwann cells