Comparison of the Schwann cell surface and Schwann cell extracellular matrix as promoters of neurite growth

Summary The ability of Schwann cells to influence the direction and rate of neurite growth was investigated in a tissue culture model of the bands of Büngner of injured peripheral nerve. The arrangement of this culture system allowed testing of the growth-promoting properties of the Schwann cell surface and extracellular matrix (ECM) assembled by Schwann cells rather than soluble substances secreted into conditioned medium. Various components of peripheral nerve were examined separately as substrata for regenerating neuntes: (i) Schwann cells and their ECM; (ii) Schwann cells alone; (iii) Schwann cell ECM alone; (iv) Schwann cells, fibroblasts, and their assembled ECM; (v) Schwann cells, their ECM and neurites; and (vi) purified laminin. Regenerating peripheral neurites were from expiants of foetal rat dorsal root ganglia, which had been cultured for several weeks to rid them of accompanying non-neuronal cells, or from expiants of foetal rat superior cervical ganglia, which contained non-neuronal cells. CNS neurites from the somatosensory cortex of embryonic rats were also studied; these neurites may be either first growing or regenerating.Neurites from all types of expiants studied grew longer and were guided on a substratum of Schwann cells or Schwann cell ECM compared with a collagen substratum. The presence of fibroblasts during ECM assembly did not enhance the neurite growth-promoting activity. The design of the experiments suggested that the factors by which the Schwann cells or their ECM promoted and guided neurite outgrowth were surface-bound rather than medium-borne. Electron microscopic examination showed that neurites grew on either Schwann cell surfaces or basal lamina material. Attempts to define the chemical nature of the neurite growth-promoting effect of ECM by partial enzymatic digestion did not identify any single component as essential.Purified laminin was a more effective promoter of outgrowth of peripheral neurites than were Schwann cells or Schwann cell ECM. Cortical expiants also grew on laminin, but neurites were accompanied on this substratum by a massive migration of non-neuronal cells; the neurites appeared to extend primarily on the non-neuronal cells rather than by direct attachment to the laminin substratum. This characteristic outgrowth of cortical non-neuronal cells on laminin was not consistently seen on Schwann cell ECM.In conclusion, either the Schwann cell surface or the ECM produced and assembled by Schwann cells promotes neurite outgrowth and guides that outgrowth from the several types of peripheral and CNS neurons studied in this report.     

A Schwann cell matrix component of neuromuscular junctions and peripheral nerves

Molecules localized to the synapse are potential contributors to processes unique to this specialized region, such as synapse formation and maintenance and synaptic transmission. We used an immunohistochemical strategy to uncover such molecules by generating antibodies that selectively stain synaptic regions and then using the antibodies to analyse their antigens. In this study, we utilized a monoclonal antibody, mAb 6D7, to identify and characterize an antigen concentrated at frog neuromuscular junctions and in peripheral nerves. In adult muscle, immunoelectron microscopy indicates that the antigen is located in the extracellular matrix around perisynaptic Schwann cells at the neuromuscular junction and in association with myelinated and nonmyelinated axons in peripheral nerves. The maintenance of the mAb 6D7 epitope is innervation-dependent but is muscle-independent; it disappears from the synaptic region within 2 weeks after denervation, but persists after muscle damage when the nerve is left intact. mAb 6D7 immunolabelling is also detected at the neuromuscular junction in developing tadpoles. Biochemical analyses of nerve extracts indicate that mAb 6D7 recognizes a glycoprotein of 127 kDa with both N- and O-linked carbohydrate moieties. Taken together, the results suggest that the antigen recognized by mAb 6D7 may be a novel component of the synaptic extracellular matrix overlying the terminal Schwann cell. The innervation-sensitivity of the epitope at the neuromuscular junction suggests a function in the interactions between nerves and Schwann cells.     

An alginate hydrogel dura mater replacement for use with intracortical electrodes

The collagenous dura mater requires a secure closure following implantation of neural prosthetic devices to avoid complications due to cerebrospinal fluid leakage and infections. Alginate was previously suggested for use as a dural sealant. The liquid application and controllable gelling conditions enable alginate to conform to the unique geometries of a neural prosthetic device and the surrounding dura mater to create a barrier with the external environment. In this study, we evaluated the use of alginate as a method to securely reclose a dural defect and seal around an untethered microscale neural probe in the rabbit model. After 3 days and 3 weeks, the sealing strength of alginate remained eight times greater than normal rabbit intracranial pressure and similar in both the presence and absence of a penetrating neural probe. For time points up to 3 months, there was no significant difference in dura mater fibrosis or thickness between alginate and controls. Application of alginate to a dural defect results in a watertight seal that remains intact while the dura mater reforms. These findings indicate that alginate is an effective tool for sealing around microscale neural probes and suggests broader application as a sealant for larger neural prosthetic devices.     

A novel use of genipin-fixed gelatin as extracellular matrix for peripheral nerve regeneration

Application of combining herbal medicine and biomedical material science to nerve regeneration is a new approach. In this study, we describe a novel use of purified genipin, which can be extracted from Gardenia jasminoides Ellis, fixing the gelatin to be an extracellular matrix for peripheral nerve regeneration. A 10-mm gap of rat sciatic nerve was created between the proximal and distal nerve stumps, which were sutured into silicone rubber tubes filled with either the genipin-fixed gelatin or collagen gel. Silicone rubber tubes filled with saline were used as controls. Six weeks after implantation, regeneration across the nerve gaps occurred in 80 and 90% of the animals from the groups of genipin-fixed gelatin and collagen, respectively, whereas only 30% in the control group. Large numbers of myelinated axons were also seen in the genipin-fixed gelatin (5104 +/- 3278) and the collagen groups (8063 +/- 1807). These findings indicated that the genipin-fixed gelatin could be an acceptable extracellular matrix for nerve regeneration.     

Characterization of leukemic cell behaviors in a soft marrow mimetic alginate hydrogel

Alginate hydrogels possess tunable mechanical properties that can mimic soft marrow tissue and present three-dimensional (3D) cues. This study evaluates its utility for supporting leukemic cell growth in vitro and its impact on cell survival, growth, and differentiation. Our results showed that the standard viscosity alginates had compromised leukemia cell viability but lower viscosity alginates recovered cell viability and improved 3D cell proliferation (27 fold) compared to 2D cultures (18 fold). Conjugation with RGD peptides promoted further cell growth (43 folds). In general, 3D hydrogels supported high-density cultures better than 2D cultures. Leukemic cells formed densely packed cell clusters in alginate hydrogels and spontaneously differentiated into a more diverse myeloid population. The cell cycle data suggested that more cells go into active cycling with a G2/M arrest in alginate hydrogels and the presence of multiploidy confirmed maturation toward megakaryocytes. In summary, superior culture of leukemia cells in 3D hydrogels is demonstrated in this study accompanied by a potential role of physical cues influencing cell fate decision. Manipulation of biophysical and biochemical properties of alginate hydrogels permits the study of specific interactions and serves to provide a robust 3D platform for studying extrinsic contributions inside the bone marrow. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.     

Integration of layered chondrocyte-seeded alginate hydrogel scaffolds

Motivated by the necessity to engineer appropriately stratified cartilage, the shear mechanics of layered, bovine chondrocyte-seeded 20mg/mL alginate scaffolds were investigated and related to the structure and biochemical composition. Chondrocyte-seeded alginate scaffolds were exposed to a calcium-chelating solution, layered, crosslinked in CaCl(2), and cultured for 10 weeks. The shear mechanical properties of the layered gels were statistically similar to those of the non-layered controls. Shear modulus of layered gels increased by approximately six-fold while toughness and shear strength increased by more than two-fold during the culture period. Hydroxyproline content in both layered gels and controls had statistically significant increases after 6 weeks. Glycosaminoglycan (GAG) content of controls increased throughout culture while GAG content in layered gels leveled off after 4 weeks. Hematoxylin and eosin histological staining showed tissue growth at the interface over the first 4 weeks. Shear mechanical properties in the engineered tissues showed significant correlations to hydroxyproline content. Dependence of interfacial mechanical properties on hydroxyproline content was most evident for layered gels when compared to controls, especially for toughness and shear strength. Additionally, interfacial properties showed almost no dependence on GAG content. These findings demonstrate the feasibility of creating stratified engineered tissues through layering and that collagen deposition is necessary for interfacial integrity.     

Encapsulation of fibroblasts causes accelerated alginate hydrogel degradation

Calcium-alginate hydrogel has been widely studied as a material for cell encapsulation for tissue engineering. At present, the effect that cells have on the degradation of alginate hydrogel is largely unknown. We have shown that fibroblasts encapsulated at a density of 7.5 × 10⁵ cells ml^?1 in both 2% and 5% w/v alginate remain viable for at least 60 days. Rheological analysis was used to study how the mechanical properties exhibited by alginate hydrogel changed during 28 days in vitro culture. Alginate degradation was shown to occur throughout the study but was greatest within the first 7 days of culture for all samples, which correlated with a sharp release of calcium ions from the construct. Fibroblasts were shown to increase the rate of degradation during the first 7 days when compared with acellular samples in both 2% and 5% w/v gels, but after 28 days both acellular and cell-encapsulating samples retained disc-shaped morphologies and gel-like spectra. The results demonstrate that although at an early stage cells influence the mechanical properties of encapsulating alginate, over a longer period of culture, the hydrogels retain sufficient mechanical integrity to exhibit gel-like properties. This allows sustained immobilization of the cells at the desired location in vivo where they can produce extracellular matrix and growth factors to expedite the healing process.     

A hydrogel derived from decellularized dermal extracellular matrix

The ECM of mammalian tissues has been used as a scaffold to facilitate the repair and reconstruction of numerous tissues. Such scaffolds are prepared in many forms including sheets, powders, and hydrogels. ECM hydrogels provide advantages such as injectability, the ability to fill an irregularly shaped space, and the inherent bioactivity of native matrix. However, material properties of ECM hydrogels and the effect of these properties upon cell behavior are neither well understood nor controlled. The objective of this study was to prepare and determine the structure, mechanics, and the cell response in?vitro and in?vivo of ECM hydrogels prepared from decellularized porcine dermis and urinary bladder tissues. Dermal ECM hydrogels were characterized by a more dense fiber architecture and greater mechanical integrity than urinary bladder ECM hydrogels, and showed a dose dependent increase in mechanical properties with ECM concentration. In?vitro, dermal ECM hydrogels supported greater C2C12 myoblast fusion, and less fibroblast infiltration and less fibroblast mediated hydrogel contraction than urinary bladder ECM hydrogels. Both hydrogels were rapidly infiltrated by host cells, primarily macrophages, when implanted in a rat abdominal wall defect. Both ECM hydrogels degraded by 35 days in?vivo, but UBM hydrogels degraded more quickly, and with greater amounts of myogenesis than dermal ECM. These results show that ECM hydrogel properties can be varied and partially controlled by the scaffold tissue source, and that these properties can markedly affect cell behavior.     

Evaluation of alginate dialdehyde cross-linked gelatin hydrogel as a biodegradable sealant for polyester vascular graft

Vascular grafts are devices intended to replace compromised arteries in the body and grafts made of polyethylene terephthalate (PET) fabric have been used mainly for synthetic grafting procedures involving medium to large diameter vascular grafts. Though porosity of the graft permits tissue in-growth, it would lead to bleeding through the graft walls immediately after implantation. So it is essential to seal the pores either by preclotting with patient's own blood or by other sealing materials prior to implantation in order to prevent blood leakage through the graft wall. Biodegradable hydrogel materials are ideal candidates for this purpose. Apart from sealing the pores, they offer biocompatible and low-thrombogenic surfaces when coated on vascular graft. In the present study, a biodegradable hydrogel, derived from oxidized alginate and gelatin, has been deposited on PET grafts by dip coating and were characterized for its efficacy on sealing the pores of the graft. Water permeability in the static and pulsatile conditions, burst strength, in vitro cell culture cytotoxicity, hemocompatibility, and endothelial cell adhesion and proliferation of the coated grafts were investigated. Results showed that the alginate dialdehyde cross-linked gelatin hydrogel was nontoxic, hemocompatible, and was efficient in sealing the pores of the graft. Blood perfusion study showed that when hydrogel-coated grafts were exposed to blood for 30 min, they showed little affinity toward platelets or leukocytes. Hemolytic potential of PET was significantly reduced when it was coated with hydrogel. Improved adhesion and proliferation of endothelial cells were observed when PET grafts were coated with hydrogel. Results also showed that coating with hydrogel did not affect the burst strength of the PET graft.     

Improvement of the biocompatibility of alginate/poly-L-lysine/alginate microcapsules by the use of epimerized alginate as a coating

Alginate/poly-L-lysine(PLL)/alginate capsules are used widely for the microencapsulation of cells. Alginate consists of guluronic acid and mannuronic acid, the ratio and sequence of which affect the properties of the alginate. Using C5-epimerases, mannuronic acid can be converted to guluronic acid in the alginate polymer. Such an enzyme, AlgE4, was used to convert blocks of mannuronic acid (M-blocks) to blocks of alternating sequence (MG-blocks). The aims of this study were 1) to investigate whether the use of epimerized alginate as a coating could improve the biocompatibility of alginate/PLL/alginate capsules and 2) to study the biocompatibility of simple alginate beads prepared with epimerized alginate. Four different capsules, two of which contained epimerized alginate, were investigated after implantation in C57BL/6 mice for 1 week. The biocompatibility of alginate/PLL/alginate capsules, as measured by retrieval rates of the capsules and DNA contents and glucose oxidation rates of the cellular overgrowth, was improved when an epimerized coating alginate was used. There were, however, no statistically significant differences in the biocompatibility of simple alginate beads made from epimerized alginate when compared with non-epimerized alginate beads. In general, such beads produced without a PLL coating swelled to a higher extent than the conventional alginate/PLL/alginate capsules. In conclusion, the use of an epimerized coating on alginate-PLL-alginate can improve the biocompatibility of such capsules but still cannot completely eliminate the detrimental effects of PLL on the biocompatibility of the capsules.     

Fibrin/Schwann cell matrix in poly-epsilon-caprolactone conduits enhances guided nerve regeneration

The goal of this study was to investigate if a three dimensional matrix, loaded homogeneously with Schwann cells and the neurotrophic factor LIF (leukemia inhibitory factor), enhances regeneration in a biodegradable nerve guidance channel as compared to non-structured cell suspensions. Therefore a 10 mm nerve gap in the buccal branch of the rat's facial nerve was bridged with tubular PCL (poly-epsilon-caprolactone) conduits filled with no matrix, Schwann cells, the three dimensional fibrin/Schwann cell matrix or the fibrin/Schwann cell matrix added with LIF Four weeks after the nerve defects were bridged histological and morphometric analyses of the implants were performed. In conclusion, the three dimensional fibrin/Schwann cells matrix enhanced the quantity and the quality of peripheral nerve regeneration through PCL conduits. The application of LIF prevented hyperneurotization. Therefore, tissue engineered fibrin/Schwann cells matrices are new invented biocompatible and biodegradable devices for enhancing peripheral nerve regeneration as compared to non-structured cell suspensions without neurotrophic factors.     

一种简便的雪旺细胞培养方法

目的介绍一种简便的雪旺细胞培养方法。方法采用反复组织块种植法,进一步在2.5%胎牛血清条件下抑制成纤维细胞生长,进行雪旺细胞培养;以S-100抗体染色鉴定雪旺细胞。结果获得纯度在98%以上的大量高活性雪旺细胞。结论本方法可以简便的获得较高纯度的雪旺细胞,值得进一步推广。     

Cloning hybridomas in a reversible three-dimensional alginate matrix.

Alginate is a transparent polymer of guluronic and mannuronic acids that provide a favorable microenvironment for cell growth. Alginate gelation is calcium dependent and temperature independent. To facilitate the isolation of stable and productive antibody-producing hybridomas, we have developed a technique of cloning hybridomas in the three-dimensional alginate matrix. To provide cavities for hybridoma growth, we encorporated 10-15% (v/v) gelatin into the alginate prior to gelation. We have cloned more than 90 monoclonal antibody-producing hybridomas using the alginate matrix. The alginate matrix is readily reversible with the addition of a calcium chelator. The alginate matrix permits efficient cloning in limited incubator space, without the use of a feeder layer, and with minimal amount of medium. The transparent matrix also permits easy screening for clonality and growth.     

Blood-aggregating hydrogel particles for use as a hemostatic agent

The body is unable to control massive blood loss without treatment. Available hemostatic agents are often expensive, ineffective or raise safety concerns. Synthetic hydrogel particles are an inexpensive and promising alternative. In this study we synthesized and characterized N-(3-aminopropyl)methacrylamide (APM) hydrogel particles and investigated their use as a hemostatic material. The APM hydrogel particles were synthesized via inverse suspension polymerization with a narrow size distribution and rapid swelling behavior. In vitro coagulation studies showed hydrogel particle blood aggregate formation as well as bulk blood coagulation inhibition. In vivo studies using multiple rat injury and ovine liver laceration models demonstrated the particles’ ability to aid in rapid hemostasis. Subsequent hematoxylin and eosin and Carstairs’ method staining of the ovine liver incision sites showed significant hemostatic plug formation. This study suggests that these cationic hydrogel particles form a physical barrier to blood loss by forming aggregates, while causing a general decrease in coagulation activity in the bulk. The formation of a rapid sealant through aggregation and the promotion of local hemostasis through electrostatic interactions are coupled with a decrease in overall coagulation activity. These interactions require the interplay of a variety of mechanisms stemming from a simple synthetic platform.     

A thermo-sensitive poly(organophosphazene) hydrogel used as an extracellular matrix for artificial pancreas

A poly(organophosphazene) bearing alpha-amino-omega-methyl-poly(ethylene glycol) (AMPEG) and hydrophobic L-isoleucine ethyl ester (IleOEt) side groups has been synthesized. This material exhibited 4 phase transitions in an aqueous solution on gradually increasing the temperature, i.e., a transparent sol, a transparent gel, an opaque gel and a turbid sol. A 10 wt% buffered solution of the polymer was employed to entrap islets of Langerhans in an artificial pancreas. Rat islets entrapped in the gel showed prolonged insulin secretion in response to basal (5.5 mM) glucose concentration compared to free rat islets and islets entrapped in other types of polymer gels. Over a 28-day culture period, the rat islets in the poly(organophosphazene) hydrogel maintained higher cell viability and insulin production versus rat islets in different hydrogels and free islets. This thermo-sensitive injectable, biodegradable matrix can be used with several cell types, including nerve cells, to promote nerve regeneration.     

Embryonic Schwann cell development: the biology of Schwann cell precursors and early Schwann cells

The cellular events leading to the generation of Schwann cells from the neural crest have recently been clarified and it is now possible to outline a relatively simple model of the Schwann cell lineage in the rat and mouse. Neural crest cells have to undergo 3 main developmental transitions to become mature Schwann cells. These are the formation of Schwann cell precursors from crest cells, the formation of immature Schwann cells from precursors and, lastly, the postnatal and reversible generation of non-myelin- and myelin-forming Schwann cells. Axonal signals involving neuregulins are important regulators of these events, in particular of the survival, proliferation and differentiation of Schwann cell precursors.     

Self-crosslinked oxidized alginate/gelatin hydrogel as injectable,adhesive biomimetic scaffolds for cartilage regeneration

Biopolymeric hydrogels that mimic the properties of extracellular matrix have great potential in promoting cellular migration and proliferation for tissue regeneration. The authors reported earlier that rapidly gelling, biodegradable, injectable hydrogels can be prepared by self-crosslinking of periodate oxidized alginate and gelatin in the presence of borax, without using any toxic crosslinking agents. The present paper investigates the suitability of this hydrogel as a minimally invasive injectable, cell-attractive and adhesive scaffold for cartilage tissue engineering for the treatment of osteoarthritis. Time and frequency sweep rheology analysis confirmed gel formation within 20 s. The hydrogel integrated well with the cartilage tissue, with a burst pressure of 70 ± 3 mmHg, indicating its adhesive nature. Hydrogel induced negligible inflammatory and oxidative stress responses, a prerequisite for the management and treatment of osteoarthritis. Scanning electron microscopy images of primary murine chondrocytes encapsulated within the matrix revealed attachment of cells onto the hydrogel matrix. Chondrocytes demonstrated viability, proliferation and migration within the matrix, while maintaining their phenotype, as seen by expression of collagen type II and aggrecan, and functionality, as seen by enhanced glycosoaminoglycan (GAG) deposition with time. DNA content and GAG deposition of chondrocytes within the matrix can be tuned by incorporation of bioactive signaling molecules such as dexamethasone, chondroitin sulphate, platelet derived growth factor (PDGF-BB) and combination of these three agents. The results suggest that self-crosslinked oxidized alginate/gelatin hydrogel may be a promising injectable, cell-attracting adhesive matrix for neo-cartilage formation in the management and treatment of osteoarthritis.     

Sodium alginate as a novel therapeutic option in experimental colitis

The potential therapeutic effect of low-viscosity sodium alginate (LVA) was studied in a rat model of acute colitis induced by intracolonic administration of acetic acid. This experimental model produced a significant ulcerative colitis. Induction of colitis also significantly enhanced the serum and colonic mucosal cytokine (IL-6 and TNF-alpha) and eicosanoid (LTB4 and PGE2) levels, which paralleled with the severity of colitis. LVA solution was administered orally as drinking water at concentration of 0.5% (W/V) for 1 week. The tolerability and inhibitory effect of LVA on matrix metalloproteinase-2 (MMP-2) were tested using WEHI-164 cell line and zymography method. The results showed that LVA therapy is able to significantly reduce colonic damage score, histological lesion, serum and colonic mucosal IL-6, TNF-alpha, LTB4 and PGE2 levels in treated group compared with nontreated controls. Moreover, in vitro examinations revealed that treatment with LVA could diminish MMP-2 activity. It is concluded that LVA is able to suppress acetic acid-induced colitis in rats. Some of the action of LVA may be associated with its inhibitory effects on cytokine and eicosanoid production and MMP-2 activity. Our data suggest that LVA could potentially be a novel therapeutic option for inflammatory bowel disease.     

Nanotube-doped alginate gel as a novel carrier for BSA immobilization

In this paper different dopants, including carbon nanotubes (CNTs), silica nanotubes (SiNTs), graphite and silica gel, were incorporated into alginate (ALG) gel to form nanotube/alginate or nanoparticle/alginate composites used for bovine serum albumin (BSA) immobilization carrier. During encapsulation, first BSA was adsorbed on the dopants, and then BSA and dopants were suspended in alginate solution, followed by being added dropwise into the CaCl2 solution to form the biocomposites. The BSA leakage decreased significantly in these biocomposites as the following order: BSA-ALG-SiNT > BSA-ALG-CNT > BSA-ALG-SiO2 > BSA-ALG-graphite, which was mainly due to firstly the protein molecule adsorption on the dopants which could not be washed out easily and secondly, during the biocomposites formation, water loss in BSA-ALG-dopant biocomposites became less than that in BSA-ALG biocomposite. In addition, the mechanical properties of these biocomposites were remarkably reinforced in the same order as BSA leakage decrease.