Polypeptide resurfacing method improves fibroblast's adhesion to hyaluronan strands.

Hyaluronic acid (hyaluronan, HyA) is a matrix component that takes part in cell adhesion and growth in normal and repaired tissues. Since it is soluble in water, HyA has been of limited use in tissue engineering of artificial matrices. Recent studies demonstrate that polypeptides have the twin advantages of reducing solubility of HyA and improving cellular attachment via cell surface adhesion molecule receptors. This paper describes a new approach of using a polypeptide resurfacing method to enhance the attachment of cells to HyA strands. HyA strands were crosslinked by glutaraldehyde and then resurfaced with poly-D-lysine, poly-L-lysine, glycine, or glutamine. After inoculation with fibroblasts in vitro, modified HyA was evaluated with histological and immunohistochemical staining methods for cell adhesion and proliferation. Modified HyA with fibroblast cells also were implanted in vivo. The results show that (1) both polylysines enhanced fibroblast adhesion to crosslinked HyA strands; (2) HyA strands were able to be crosslinked well by 3 days of treatment in glutaraldehyde, and as a biomaterial they could resist biodegradation; (3) modified HyA has good biocompatibility, both in vitro and in vivo. The results demonstrate that HyA material resurfaced by polypeptides has positive advantages for cellular adhesion. Resurfaced HyA has much potential as an improved biomaterial for clinical usage.     

ECM molecules mediate both Schwann cell proliferation and activation to enhance neurite outgrowth

Tissue engineering using a combination of biomaterials and cells represents a new approach to nerve repair. We have investigated the effect that extracellular matrix (ECM) molecules have on Schwann cell (SC) attachment and proliferation on the nerve conduit material poly-3-hydroxybutyrate (PHB), and SC influence on neurite outgrowth in vitro. Initial SC attachment to PHB mats was unaffected by ECM molecules but proliferation increased (laminin > fibronectin > collagen). SCs seeded onto ECM-coated culture inserts suspended above a monolayer of NG108-15 cells determined the effect of released diffusible factors. The effect of direct contact between the two cell types on ECM molecules was also investigated. In both systems SCs enhanced neurite number per cell and percentage of NG108-15 cells sprouting neurites. NG108-15 cells grown in direct contact with SCs had significantly longer neurites than those exposed to diffusible factors when seeded on laminin or fibronectin. Diffusible factors released from SCs cultured on ECM molecules appear to initiate neurite outgrowth, whereas SC-neuron contact promotes neurite elongation. SC proliferation was maximal on poly-D-lysine-coated surfaces, but these cells did not influence neurite outgrowth to the levels of laminin or fibronectin. This suggests that ECM molecules enhance cell number and activate SCs to release neurite promoting factors. Addition of ECM molecules to PHB nerve conduits containing SCs is likely to provide benefits for the treatment of nerve injuries.     

Surface modification and characterization of chitosan or PLGA membrane with laminin by chemical and oxygen plasma treatment for neural regeneration

Attachment to and proliferation on the substrate are deemed important considerations when Schwann cells (SCs) are to be seeded in synthetic nerve grafts. Good attachment is a prerequisite for the SCs to survive. Fast proliferation will yield large numbers of SCs in a short time, which appears to be promising for stimulating peripheral nerve regeneration. However, surface properties are the dominating factor in influencing the interactions between cells and synthetic nerve grafts. The aim of this study was to investigate the surface effects of laminin modified PLGA and chitosan membranes after chemical method and plasma treatment. Laminin, the extracellular matrix protein, is a permissive protein for SCs adhesion used in neural regeneration. The surface properties of laminin modified membranes were assayed by BCA, FTIR and XPS analysis. Results showed that laminin was covalently bonded onto the surface of both PLGA and chitosan membranes either by chemical method or by oxygen plasma treatment. The cell affinity of the laminin modified membranes was verified by Schwann cells culturing. Our results also indicate that oxygen plasma is indeed a better method to incorporate laminin onto the surface of membrane. Laminin-modified chitosan membrane significantly increases SCs attachment and affinity for directing peripheral nerve regeneration.     

Adhesion and proliferation of human Schwann cells on adhesive coatings

Attachment to and proliferation on the substrate are deemed important considerations when Schwann cells (SCs) are to be seeded in synthetic nerve grafts. Attachment is a prerequisite for the SCs to survive and fast proliferation will yield large numbers of SCs in a short time, which appears promising for stimulation of peripheral nerve regeneration. The aim of the present study was to compare the adhesion and proliferation of human Schwann cells (HSCs) on different substrates. The following were selected for their suitability as an internal coating of synthetic nerve grafts; the extracellular matrix proteins fibronectin, laminin and collagen type I and the poly-electrolytes poly(d-lysine) (PDL) and poly(ethylene-imine) (PEI). On all coatings, attachment of HSCs was satisfactory and comparable, indicating that this factor is not a major consideration in choosing a suitable coating. Proliferation was best on fibronectin, laminin and PDL, and worst on collagen type I and PEI. Since nerve regeneration is enhanced by laminin and/or fibronectin, these are preferred as coatings for synthetic nerve grafts seeded with SCs.     

A novel use of alginate hydrogel as Schwann cell matrix.

The use of bioresorbable conduits supplemented with Schwann cells (SCs) is a promising tissue engineering technique to replace nerve grafting. Alginate hydrogel (AH), as a SC tissue engineering matrix, has many advantages over previously used matrices but has not been evaluated for this purpose. In this study, the viability and proliferation of SCs together with SC function in AH was evaluated in vitro. AlamarBlue cell assay was used to monitor the viability of SCs in AH and compared to SC viability in collagen gel, fibrin glue, hyaluronic acid, Matrigel, and standard culture plate over 5 days in culture. The results showed that the viability and growth of SCs in different matrices over the culture period did not significantly differ to culture plate culture. SC function when suspended in AH was monitored using chick embryo dorsal root ganglia (CDRG) growth assay. Growth of CDRG in AH with or without SCs was compared to CDRG growth without AH matrix. After 3 days in culture, the mean length of neurite sprouting was measured. The results showed that there was neurite growth in AH but was reduced to 43% of control. The neurite growth in AH was, however, enhanced by 170% when SCs were suspended in the gel. In conclusion, AH supported SC viability and function in vitro and may be useful in peripheral nerve tissue engineering in reconstructive procedures.     

Effects of the in vivo predegenerated nerve graft on early Schwann cell migration: Quantitative analysis using S100-GFP mice

In peripheral nerve transection injury, continuity of axons as well as that of the basal lamina is disconnected. In such case, migrating Schwann cells (SCs) would be the only axonal guidance at an early stage of regeneration. However, it takes a few days for the dedifferentiated SCs to start migration, while axonal growth begins a few hours after injury. Consequently, the axons without guidance extensively branch out and wander off at the lesion, resulting in aberrant reinnervation. Therefore, enhancing SCs migration could be an attractive therapeutic strategy. In this study, we investigated the effects of the in vivo nerve predegeneration on SC migration and the time course of these changes. In our analysis, we established a novel animal model by nerve transplantation from S100-GFP mice (in which SCs constitutively express green fluorescent protein driven by the S100B promoter), by which SC migration could be exclusively visualized. Our results showed that SCs acquire the maximal migration ability with 14-day predegeneration, but subsequently it gradually decreased. There was a correlation between the time course of the changes in SC migration and the number of activated macrophages. These findings suggest that using predegenerated nerve grafts in repairing the transected nerves could facilitate SC migration into the recipient nerve stump. This technique could be beneficial for early establishment of axonal guidance and possible functional improvement after transection injury.     

Pulsed magnetic field promotes proliferation and neurotrophic genes expression in Schwann cells in vitro

As one of the most classic supportive cells, Schwann cells (SCs) have been considered as potential candidates for nerve regeneration. However, SCs cultured in vitro are found with attenuated biological activities, which limits their application. Pulsed magnetic field (PMF) has been demonstrated to be safe and efficient to regulate several cells activities. However, it is still unclear the effect of PMF on proliferation and expression of neurotrophic factors in SCs. Therefore, the present study was designed to examine such possible effects. The tolerance of SCs to PMF was examined by flow cytometry and scanning electron microscopy (SEM). The proliferation of cells was detected by an EdU labeling assay and a Prestoblue assay. The expression and secretion of neurotrophic factors in SCs was assayed by RT-PCR and ELISA. We found that 2.0 mT was the optimal intensity that caused relatively little apoptosis with profound proliferation in SCs. The gene expression and protein level of brain-derived neurotrophic factor (BDNF), glial cell derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF) were up-regulated following PMF stimulation, additionally, the gene expression and protein level of neurotrophin-3 (NT-3) was not enhanced by PMF. Our results suggested that PMF could improve SC proliferation and biological function, which might shed a light on the potential utilization of PMF in nerve regeneration via SC activation.     

The effect of synthetic oxygen carrier-enriched fibrin hydrogel on Schwann cells under hypoxia condition in vitro

Schwann cell (SC), which plays a key role in peripheral nerve regeneration, is one of the most classic supportive cells in neural tissue engineering. However, the biological activity of SCs seeded in nerve scaffolds decays subsequently due to local hypoxia induced by ischemia. Thus, we aimed to investigate whether a synthetic oxygen carrier-enriched fibrin gel would provide a sustained oxygen release to cultured SCs in vitro for overcoming a temporary (48 h) oxygen deprivation. In this study, perfluorotributylamine (PFTBA)-based oxygen carrying fibrin gel was prepared to provide oxygen for SCs under normoxic or hypoxic conditions. The dissolved oxygen within the culture media was measured by a blood-gas analyzer to quantify the time course of oxygen release from the PFTBA-enriched fibrin gel. SCs were cultured in the presence or absence of PFTBA-enriched fibrin gel under normoxic or hypoxic conditions. The tolerance of SCs to hypoxia was examined by a cell apoptosis assay. The growth of cells was characterized using S-100 staining and a CCK-8 assay. The migration of cells was examined using a Transwell chamber. The mRNA of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell derived neurotrophic factor (GDNF), neural cell adhesion molecule (N-CAM) and vascular endothelial growth factor (VEGF) in SCs were assayed by RT-PCR. In addition, SCs cultured in 3D PFTBA-enriched hydrogel were characterized by Live/Dead staining and the mRNA levels of BDNF, NGF, GDNF, N-CAM and VEGF were assayed by RT-PCR. The results showed that the PFTBA-enriched fibrin hydrogel was able to promote cell adhesion, migration, and proliferation under hypoxic conditions. Interestingly, PFTBA applied through the fibrin hydrogel dramatically enhanced the mRNA of BDNF, NGF, GDNF, N-CAM and VEGF under hypoxic condition. These findings highlight the possibility of enhancing nerve regeneration in cellular nerve grafts through PFTBA increased neurotropic secretion in SCs.     

复合转化生长因子-β的组织工程化周围神经修复坐骨神经缺损

探讨了转化生长因子-β(Transforming growth factor-β,TGF-β)在周围神经缺损修复中的作用。将50 ng·ml-1 TGF-β加入体外培养的雪旺细胞(Sehwann cells,SC)中,MTT和流式细胞仪观测到TGF-β能够明显促进SC增殖;ELISA方法检测到TGF-β组上清神经生长因子(NGF)含量高于对照组(P<0．05);将牛去细胞基质(Bovine acellular matrix,BAM)、SC、血清和培养基按一定的比例混合,注入聚乳酸羟基乙酸共聚物(PLGA)神经导管去修复15 mm坐骨神经缺损。30只SD大鼠分为3组,实验组:PLGA导管 SC TGF-β;空白组PLGA导管 SC;和自体神经移植组。16周后通过电生理、透射电镜等检测方法显示各组坐骨神经均得到再生,修复效果实验组与自体神经移植组无显著性差异,均优于空白组。TGF-β一方面可以明显促进SC的增殖,另一方面可以增强SC分泌NGF的功能。因此周围神经修复过程中使用外源性的TGF-β对修复周围神经缺损有较好的疗效。     

Evidence for endothelial nitric oxide as a negative regulator of Schwann cell dedifferentiation after peripheral nerve injury

The loss of intimate contact with axons triggers Schwann cells (SCs) to switch from a myelin-producing phenotype to a dedifferentiated, proliferating non-myelin-forming state after nerve injury. SC dedifferentiation is required for effective nerve regeneration. Negative regulators of SC dedifferentiation are promising targets to accelerate function recovery in acquired peripheral neuropathies. We recently reported that nitric oxide (NO) synthesized by endothelial NO synthase (eNOS) slows down functional recovery and axon regeneration after XIIth nerve crushing. This harmful action could be effected by a NO-delaying action on SC dedifferentiation. Adenoviral vectors directing the expression of a dominant negative mutant for eNOS (AVV-TeNOS) or the enhanced green fluorescent protein (AVV-eGFP) were individually injected into the distal stump just after XIIth nerve crushing. Growth-associated protein 43 (GAP-43), strongly over-expressed in dedifferentiated SCs and regenerating axons, was up-regulated in AVV-TeNOS-transduced nerves relative to AVV-eGFP-treated nerves. AVV-TeNOS increased the number of GAP-43-positive cells and bands of Bungner but did not alter the number of Hoechst-positive nuclei relative to AVV-eGFP. These results signal endothelial NO as a negative regulator of the SC dedifferentiation process, but not of SC proliferation rate, after nerve injury. Vascular-derived factors should be taken into account as feasible extrinsic regulators of SC plasticity.     

Bioactive poly(L-lactic acid) conduits seeded with Schwann cells for peripheral nerve regeneration.

This study attempted to enhance the efficacy of peripheral nerve regeneration using our previously tested poly(L-lactic acid) (PLLA) conduits by incorporating them with allogeneic Schwann cells (SCs). The SCs were harvested, cultured to obtain confluent monolayers and two concentrations (1 x 10(4) and 1 x 10(6) SC/ml) were combined with a collagen matrix (Vitrogen) and injected into the PLLA conduits. The conduits were then implanted into a 12 mm right sciatic nerve defect in rats. Three control groups were used: isografts, PLLA conduits filled with collagen alone and empty silicone tubes. The sciatic functional index (SFI) was calculated monthly through four months. At the end of second and fourth months, the gastrocnemius muscle was harvested and weighed for comparison and the graft conduit and distal nerve were harvested for histomorphologic analysis. The mean SFI demonstrated no group differences from isograft control. By four months, there was no significant difference in gastrocnemius muscle weight between the experimental groups compared to isograft controls. At four months, the distal nerve demonstrated a statistically lower number of axons mm2 for the high and low SC density groups and collagen control. The nerve fiber density was significantly lower in all of the groups compared to isograft controls by four months. The development of a "bioactive" nerve conduit using tissue engineering to replace autogenous nerve grafts offers a potential approach to improved patient care. Although equivalent nerve regeneration to autografts was not achieved, this study provides promising results for further investigation.     

Biomimetic materials replicating Schwann cell topography enhance neuronal adhesion and neurite alignment in vitro

It is well established that Schwann cells (SCs) promote and enhance axon guidance and nerve regeneration by providing multiple cues, including extracellular matrix, cell surface molecules, neurotrophic factors and cellular topography. Which of the elements of the complex environment associated with SCs provides the essential information for directed nerve growth is unclear, because, until now, it has been impossible to investigate their contributions individually. Our development of biomimetic materials that replicate the micro- and nanoscale topography of SCs has allowed us to investigate for the first time the role of cellular topography in directing nerve growth. Dorsal root ganglion (DRG) neurons were cultured on flat poly(dimethyl siloxane) (PDMS) and on PDMS replicas with protruding SC topography. Image analysis showed that more neurons adhered to the replicas than to the flat substrates, and that neurite growth on the replicas followed the underlying SC pattern. Neuronal alignment was dependent on cell density. Live SCs derived from the DRG also grew along the replica SC pattern. These results suggest that the combination of micro- and nanoscale topographical cues provided by SCs can influence nerve growth and point toward design parameters for future nerve guidance channels.     

Biomimetic materials replicating Schwann cell topography enhance neuronal adhesion and neurite alignment in vitro

It is well established that Schwann cells (SCs) promote and enhance axon guidance and nerve regeneration by providing multiple cues, including extracellular matrix, cell surface molecules, neurotrophic factors and cellular topography. Which of the elements of the complex environment associated with SCs provides the essential information for directed nerve growth is unclear, because, until now, it has been impossible to investigate their contributions individually. Our development of biomimetic materials that replicate the micro- and nanoscale topography of SCs has allowed us to investigate for the first time the role of cellular topography in directing nerve growth. Dorsal root ganglion (DRG) neurons were cultured on flat poly(dimethyl siloxane) (PDMS) and on PDMS replicas with protruding SC topography. Image analysis showed that more neurons adhered to the replicas than to the flat substrates, and that neurite growth on the replicas followed the underlying SC pattern. Neuronal alignment was dependent on cell density. Live SCs derived from the DRG also grew along the replica SC pattern. These results suggest that the combination of micro- and nanoscale topographical cues provided by SCs can influence nerve growth and point toward design parameters for future nerve guidance channels.     

Effects of an avidin-biotin binding system on Schwann cells attachment, proliferation, and gene expressions onto electrospun scaffolds

Effective Schwann cells (SCs) attachment is a prerequisite for the successful construction of tissue-engineered nerve. The present study aimed to investigate the role of an avidin-biotin binding system (ABBS) for neural tissue engineering. The attachment, proliferation, and morphology of biotinylated SCs on avidin-treated scaffolds were examined, and the effects of avidin, biotin, and the avidin-biotin binding system on SCs gene expressions were also studied. The results indicated that the attachment of biotinylated SCs onto avidin-treated scaffolds was promoted obviously within a short time (10 min). Meanwhile, there were no great differences in terms of proliferation and morphology of SCs between the two groups after cultivation for 14 days. The gene expressions of S100, GDNF, BDNF, NGF, CNTF, and PMP22 were up-regulated significantly by biotin rather than aligned scaffolds or avidin. The present study demonstrated that ABBS enhanced the attachment and maturation of SCs onto the electrospun scaffolds without adverse effects on the proliferation of SCs in the long term, suggesting the potential application of ABBS in the neural tissue engineering.     

Supplementation of acellular nerve grafts with skin derived precursor cells promotes peripheral nerve regeneration

Introduction of autologous stem cells into the site of a nerve injury presents a promising therapy to promote axonal regeneration and remyelination following peripheral nerve damage. Given their documented ability to differentiate into Schwann cells (SCs) in vitro, we hypothesized that skin-derived precursor cells (SKPs) could represent a clinically-relevant source of transplantable cells that would enhance nerve regeneration following peripheral nerve injury. In this study, we examined the potential for SKP-derived Schwann cells (SKP–SCs) or nerve-derived SCs to improve nerve regeneration across a 12 mm gap created in the sciatic nerve of Lewis rats bridged by a freeze-thawed nerve graft. Immunohistology after 4 weeks showed survival of both cell types and early regeneration in SKP seeded grafts was comparable to those seeded with SCs. Histomorphometrical and electrophysiological measurements of cell-treated nerve segments after 8 weeks survival all showed significant improvement as compared to diluent controls. A possible mechanistic explanation for the observed results of improved regenerative outcomes lies in SKP–SCs' ability to secrete bioactive neurotrophins. We therefore conclude that SKPs represent an easily accessible, autologous source of stem cells for transplantation therapies which act as functional Schwann cells and show great promise in improving regeneration following nerve injury.     

Effects of cerebrolysin on rat Schwann cells in vitro

Although the peripheral nervous system (PNS) is capable of regeneration, these processes are limited. As a potential means to augment PNS regeneration, the effects of cerebrolysin (CL), a proteolytic peptide fraction, were tested in vitro on Schwann cell (SC) proliferation, stress resistance, phagocytic and cluster-forming capacity. Primary SC/fibrocyte co-cultures were prepared from dorsal root ganglia of 5–7-day-old rats. SCs were subjected to mechanical stress by media change and metabolic stress by serum glucose deprivation (SGD). Cell survival was assessed using MTT test. SC proliferation was determined by counting BrdU-labeled cells. SC clustering was studied by ImageJ analysis of S100 immunostaining. Wallerian degeneration (WD) was evaluated by measuring acetylcholine-esterase staining within sciatic nerves in vitro. It was found that CL caused no effect on MTT turnover in the tested doses. CL inhibited SC proliferation in a dose-dependent manner. Media change and additional SGD stress inhibited SC clustering. CL enhanced the reorganization of SC clusters and was able to counteract SGD-induced cluster defects. Moreover, CL accelerated WD in vitro. CL was able to enhance the functions of SCs that are relevant to nerve regeneration. Thus, our findings suggest that CL may be suitable for therapeutic usage to enhance PNS regeneration/reconstruction.     

Studies on nerve cell affinity of chitosan-derived materials

Reparation of the central nervous system (CNS) is important because when it is impaired its recovery is difficult and concomitant malfunction of other parts of body occurs. In our previous studies, chitosan was found to be a good material supporting nerve repair. The purpose of this article was to study the ability of chitosan and some chitosan-derived materials to facilitate the growth of nerve cells. Those materials were chitosan, glutaraldehyde-crosslinked chitosan, glutaraldehyde-crosslinked chitosan-gelatin conjugate, a chitosan-gelatin mixture, chitosan coated with polylysine (CAP), and a chitosan-polylysine mixture (CPL). Gelatin and polylysine were used as controls. After nerve cells (gliosarcoma cells and normal cerebral cells) were grown on those materials, their attachment, spread, and growth were observed. The adsorption of some extracellular matrix molecules such as laminin and fibronectin on the materials and the role the molecules play in nerve cell attachment and spreading were also studied by enzyme-linked immunosorbent assay and MTT method. We found that both CAP and CPL have excellent nerve cell affinity, defined as the ability to promote nerve cell to grow and function normally. Those two materials may be promising for the repair of the nervous system. Materials precoated with laminin, fibronectin, and serum were analyzed for their nerve cell affinity. Results suggest that after being precoated with laminin and fibronectin solution or serum, all material have better nerve cell affinity.     

Glial cell plasticity in sensory ganglia induced by nerve damage

Numerous studies have been done on the effect of nerve injury on neurons of sensory ganglia but little is known about the contribution of satellite glial cells (SCs) in these ganglia to post-injury events. We investigated cell-to-cell coupling and ultrastructure of SCs in mouse dorsal root ganglia after nerve injury (axotomy). Under control conditions SCs were mutually coupled, but mainly to other SCs around a given neuron. After axotomy SCs became extensively coupled to SCs that enveloped other neurons, apparently by gap junctions. Serial section electron microscopy showed that after axotomy SC sheaths enveloping neighboring neurons formed connections with each other. Such connections were absent in control ganglia. The number of gap junctions between SCs increased 6.5-fold after axotomy. We propose that axotomy induces growth of perineuronal SC sheaths, leading to contacts between SCs enveloping adjacent neurons and to formation of new gap junctions between SCs. These changes may be an important mode of glial plasticity and can contribute to neuropathic pain.     

Addition of fibronectin to alginate matrix improves peripheral nerve regeneration in tissue-engineered conduits

Schwann cell (SC) transplantation has been proposed to encourage peripheral nerve regeneration, but an optimal SC-carrying matrix would be needed. The aim of this study was to characterize how the addition of fibronectin to alginate would affect the outcome of nerve regeneration promoted by Schwann cells embedded in this matrix. Genetically labeled rat SCs were obtained by lacZ gene transduction. SCs were suspended in alginate hydrogel matrix with/without addition of liquid fibronectin, and their viability and growth in the different types of matrices were assessed in vitro by AlamarBlue assay. In vivo assessment of SC transplantation in the matrix was carried out with poly-3-hydroxybutyrate (PHB) conduits to bridge a sciatic nerve gap. The grafted conduits were harvested at 2, 3, and 6 weeks and assessed for the presence of labeled SCs in relation to regrowing axons. The amount and rate of axonal regeneration were assessed by quantitative immunohistochemistry. Addition of fibronectin to alginate hydrogel improved SC viability and growth profile in vitro. X-Gal staining confirmed that SCs transplanted in PHB conduits were viable throughout the time course, and that the labeled SCs were clearly associated with regenerating axons. The regeneration rate was enhanced when liquid fibronectin was added to the alginate matrix. Furthermore, the presence of SCs also enhanced regeneration and there was an additive effect when both SCs and fibronectin were combined with alginate. In conclusion, the addition of fibronectin to alginate hydrogel matrix contributed to improve nerve regeneration, supporting SC viability and augmenting their effect on axonal growth when transplanted in a bioengineered nerve conduit.