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

Myelination in the absence of UDP-galactose:ceramide galactosyl-transferase and fatty acid 2 -hydroxylase

<p>Abstract</p> <p>Background</p> <p>The sphingolipids galactosylceramide (GalCer) and sulfatide are major myelin components and are thought to play important roles in myelin function. The importance of GalCer and sulfatide has been validated using UDP-galactose:ceramide galactosyltransferase-deficient (<it>Cgt</it>^-/-) mice, which are impaired in myelin maintenance. These mice, however, are still able to form compact myelin. Loss of GalCer and sulfatide in these mice is accompanied by up-regulation of 2-hydroxylated fatty acid containing (HFA)-glucosylceramide in myelin. This was interpreted as a partial compensation of the loss of HFA-GalCer, which may prevent a more severe myelin phenotype. In order to test this hypothesis, we have generated <it>Cgt</it>^-/-mice with an additional deletion of the fatty acid 2-hydroxylase (<it>Fa2h</it>) gene.</p> <p>Results</p> <p><it>Fa2h</it>^-/-/Cgt^-/-double-deficient mice lack sulfatide, GalCer, and in addition HFA-GlcCer and sphingomyelin. Interestingly, compared to <it>Cgt</it>^-/-mice the amount of GlcCer in CNS myelin was strongly reduced in <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-mice by more than 80%. This was accompanied by a significant increase in sphingomyelin, which was the predominant sphingolipid in <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-mice. Despite these significant changes in myelin sphingolipids, compact myelin was formed in <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-mice, and g-ratios of myelinated axons in the spinal cord of 4-week-old <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-mice did not differ significantly from that of <it>Cgt</it>^-/-mice, and there was no obvious phenotypic difference between <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-and <it>Cgt</it>^-/-mice</p> <p>Conclusions</p> <p>These data show that compact myelin can be formed with non-hydroxylated sphingomyelin as the predominant sphingolipid and suggest that the presence of HFA-GlcCer and HFA-sphingomyelin in <it>Cgt</it>^-/-mice does not functionally compensate the loss of HFA-GalCer.</p

Peripheral nerve regeneration through hydrogel enriched chitosan conduits containing engineered Schwann cells for drug delivery

Critical length nerve defects in the rat sciatic nerve model were reconstructed with chitosan nerve guides filled with Schwann cells (SCs) containing hydrogel. The transplanted SCs were naive or had been genetically modified to overexpress neurotrophic factors, thus providing a cellular neurotrophic factor delivery system. Prior to the assessment in vivo, in vitro studies evaluating the properties of engineered SCs overexpressing glial cell line-derived neurotrophic factor (GDNF) or fibroblast growth factor 2 (FGF-218kDa) demonstrated their neurite outgrowth inductive bioactivity for sympathetic PC-12 cells as well as for dissociated dorsal root ganglion cell drop cultures. SCs within NVR-hydrogel, which is mainly composed of hyaluronic acid and laminin, were delivered into the lumen of chitosan hollow conduits with a 5% degree of acetylation. The viability and neurotrophic factor production by engineered SCs within NVR-Gel inside the chitosan nerve guides was further demonstrated in vitro. In vivo we studied the outcome of peripheral nerve regeneration after reconstruction of 15-mm nerve gaps with either chitosan/NVR-Gel/SCs composite nerve guides or autologous nerve grafts (ANGs). While ANGs did guarantee for functional sensory and motor regeneration in 100% of the animals, delivery of NVR-Gel into the chitosan nerve guides obviously impaired sufficient axonal outgrowth. This obstacle was overcome to a remarkable extent when the NVR-Gel was enriched with FGF-218kDa overexpressing SCs

A dual-omics approach on the effects of fibroblast growth factor-2 (FGF-2) on ventral tegmental area dopaminergic neurons in response to alcohol consumption in mice

Harmful alcohol consumption is a major socioeconomic burden to the health system, as it can be the cause of mortality of heavy alcohol drinkers. The dopaminergic (DAergic) system is thought to play an important role in the pathogenesis of alcohol drinking behaviour; however, its exact role remains elusive. Fibroblast growth factor 2 (FGF-2), a neurotrophic factor, associated with both the DAergic system and alcohol consumption, may play an important role in DAergic neuroadaptations during alcohol abuse. Within this study, we aimed to clarify the role of endogenous FGF-2 on the DAergic system and whether there is a possible link to alcohol consumption. We found that lack of FGF-2 reduces the alcohol intake of mice. Transcriptome analysis of DAergic neurons revealed that FGF-2 knockout (FGF-2 KO) shifts the molecular fingerprint of midbrain dopaminergic (mDA) neurons to DA subtypes of the ventral tegmental area (VTA). In line with this, proteomic changes predominantly appear also in the VTA. Interestingly, these changes led to an altered regulation of the FGF-2 signalling cascades and DAergic pathways in a region-specific manner, which was only marginally affected by voluntary alcohol consumption. Thus, lack of FGF-2 not only affects the gene expression but also the proteome of specific brain regions of mDA neurons. Our study provides new insights into the neuroadaptations of the DAergic system during alcohol abuse and, therefore, comprises novel targets for future pharmacological interventions

Correction to “In Vitro Assessment of Synthetic Nano Engineered Graft Designed for Further Clinical Study in Nerve Regeneration”

In the article entitled “In vitro assessment of synthetic nano engineered graft designed for further clinical study in nerve regeneration” which published in International Clinical Neuroscience Journal 2018;5(3)86-91, Figure 6 contained some errors, in which panels A, C, and D have been reported mistakenly. This mistake happened inadvertently during the data processing and data collection. The corrected Figure 6 and corresponding caption is presented in the present correction, in which the correct SEM images replaces in panels A, C, and D. The reader should note that this error does not affect the scientific quality of the work and no other section of the manuscript is affected by this error. In addition, the correction does not affect the conclusions of that paper. We would like to apologise for any inconvenience caused

In Vitro Assessment of Synthetic Nano Engineered Graft Designed for Further Clinical Study in Nerve Regeneration

Background: Electrospun nanofibrous scaffolds are considered as promising candidates in neural tissue regeneration due to their ability to support neural cell attachment, spreading and proliferation.Methods: In this paper, various type of nanofibers scaffold based on polycaprolactone) (PCL) were fabricated using electrospinning. The main drawback of PCL scaffolds is their low bioactivity of scaffold surface. To overcome this surface and composition modification was used to enhanced hydrophilicity and bioactivity of scaffold.Results: The scanning electron microscopy (SEM) results indicate that fiber diameter entirely depends on the solvent system and added component of gelatin and chitosan which by adding gelatin and chitosan fiber diameter decreased. In vitro studies using PC12 cells revealed that the plasma surface modified and blended scaffold with chitosan and gelatin nanofibrous scaffold supports cell attachment, spreading and indicate a significant increase in proliferation of PC12 in the presence of chitosan. The results demonstrated that gelatin and chitosan caused a significant enhancement in the bioactivity of the scaffold, which confirmed by MTT assay and improved the cell spreading and proliferation of neural cell on the scaffolds.Conclusion: Based on the experimental results, the PCL/chitosan/PPy conductive substrate could be used as a potential scaffold for clinical research in the field of neural regeneration and healing

Schwann Cells Overexpressing FGF-2 Alone or Combined with Manual Stimulation Do Not Promote Functional Recovery after Facial Nerve Injury

Purpose. To determine whether transplantation of Schwann cells (SCs) overexpressing different isoforms of fibroblast growth factor 2 (FGF-2) combined with manual stimulation (MS) of vibrissal muscles improves recovery after facial nerve transection in adult rat. Procedures. Transected facial nerves were entubulated with collagen alone or collagen plus naïve SCs or transfected SCs. Half of the rats received daily MS. Collateral branching was quantified from motoneuron counts after retrograde labeling from 3 facial nerve branches. Quality assessment of endplate reinnervation was combined with video-based vibrissal function analysis. Results. There was no difference in the extent of collateral axonal branching. The proportion of polyinnervated motor endplates for either naïve SCs or FGF-2 over-expressing SCs was identical. Postoperative MS also failed to improve recovery. Conclusions. Neither FGF-2 isoform changed the extent of collateral branching or polyinnervation of motor endplates; furthermore, this motoneuron response could not be overridden by MS

Severe early onset preeclampsia: short and long term clinical, psychosocial and biochemical aspects

Preeclampsia is a pregnancy specific disorder commonly defined as de novo hypertension and proteinuria after 20 weeks gestational age. It occurs in approximately 3-5% of pregnancies and it is still a major cause of both foetal and maternal morbidity and mortality worldwide1. As extensive research has not yet elucidated the aetiology of preeclampsia, there are no rational preventive or therapeutic interventions available. The only rational treatment is delivery, which benefits the mother but is not in the interest of the foetus, if remote from term. Early onset preeclampsia (<32 weeks’ gestational age) occurs in less than 1% of pregnancies. It is, however often associated with maternal morbidity as the risk of progression to severe maternal disease is inversely related with gestational age at onset2. Resulting prematurity is therefore the main cause of neonatal mortality and morbidity in patients with severe preeclampsia3. Although the discussion is ongoing, perinatal survival is suggested to be increased in patients with preterm preeclampsia by expectant, non-interventional management. This temporising treatment option to lengthen pregnancy includes the use of antihypertensive medication to control hypertension, magnesium sulphate to prevent eclampsia and corticosteroids to enhance foetal lung maturity4. With optimal maternal haemodynamic status and reassuring foetal condition this results on average in an extension of 2 weeks. Prolongation of these pregnancies is a great challenge for clinicians to balance between potential maternal risks on one the eve hand and possible foetal benefits on the other. Clinical controversies regarding prolongation of preterm preeclamptic pregnancies still exist – also taking into account that preeclampsia is the leading cause of maternal mortality in the Netherlands5 - a debate which is even more pronounced in very preterm pregnancies with questionable foetal viability6-9. Do maternal risks of prolongation of these very early pregnancies outweigh the chances of neonatal survival? Counselling of women with very early onset preeclampsia not only comprises of knowledge of the outcome of those particular pregnancies, but also knowledge of outcomes of future pregnancies of these women is of major clinical importance. This thesis opens with a review of the literature on identifiable risk factors of preeclampsia