Fabrication and characterization of tunable polysaccharide hydrogel blends for neural repair

Hydrogels are an important class of biomaterials that have the potential to be used as three-dimensional tissue engineering scaffolds for regenerative medicine. This is especially true in the central nervous system, where neurons do not have the ability to regenerate due to the prohibitory local environment following injury. Hydrogels can fill an injury site, replacing the growth-prohibiting environment with a more growth-permissive one. In this study, dextran and chitosan were incorporated into a methylcellulose and agarose hydrogel blend. This created several thermally sensitive polysaccharide hydrogel blends that had tunable mechanical and surface charge properties. Cortical neurons were cultured on the hydrogels to determine the blend that had the greatest neuron compatibility. Our results show that softer, more positively charged polysaccharide hydrogel blends allow for greater neuron attachment and neurite extension, showing their promise as CNS regeneration scaffolds.     

Neurite outgrowth in fibrin gels is regulated by substrate stiffness

Fibrin is a promising matrix for use in promoting nerve repair given its natural occurrence in peripheral nerve injuries, and the biophysical properties of this matrix can be regulated to modulate tissue regeneration. In this study, we examined the effect of physical and mechanical properties of fibrin gels on dorsal root ganglia (DRG) neurite extension. Increases in fibrinogen concentration increased the number of fibrin strands, resulting in decreased pore size and increased stiffness. Neurite extension was reduced when DRG explants were cultured within fibrin gels of increasing fibrinogen concentrations (from 9.5 to 141 mg/mL). The addition of NaCl also increased the number of fibrin strands, reducing fiber diameter and porosity, while increasing mechanical strength, and reductions in neurite extension correlated with increases in NaCl content. We determined that neurite extension within fibrin gels is dependent on fibrinolysis and is mediated by the secretion of serine proteases and matrix metalloproteinases by entrapped DRGs, as confirmed by culturing cells in the presence of inhibitors against these enzymes and real-time-polymerase chain reaction. Taken together, the results of this study provide new insight into the effect of fibrin gel biophysical properties on neurite extension and suggest new opportunities to improve the efficacy of these materials when used as nerve guidance conduits.     

The influence of physical structure and charge on neurite extension in a 3D hydrogel scaffold

Understanding neural cell differentiation and neurite extension in three-dimensional scaffolds is critical for neural tissue engineering. This study explores the structure-function relationship between a 3D hydrogel scaffold and neural cell process extension and examines the role of ambient charge on neurite extension in 3D scaffolds. A range of agarose hydrogel concentrations was used to generate varied gel physical structures and the corresponding neurite extension was examined. Agarose gel concentration and the corresponding pore radius are important physical properties that influence neural cell function. The average pore radii of the gels were determined while the gel was in the hydrated state and in two different dehydrated states. As the gel concentration was increased, the average pore radius decreased exponentially. Similarly, the length of neurites extended by E9 chick DRGs cultured in agarose gels depends on gel concentration. The polycationic polysaccharide chitosan and the polyanionic polysaccharide alginate were used to incorporate charge into the 3D hydrogel scaffold, and neural cell response to charge was studied. Chitosan and alginate were covalently bound to the agarose hydrogel backbone using the bi-functional coupling agent 1,1'carbonyldiimldazole. DRGs cultured in chitosan-coupled agarose gel exhibited a significant increase in neurite length compared to the unmodified agarose control. Conversely, the alginate-coupled agarose gels significantly inhibited neurite extension. This study demonstrates a strong, correlation between the ability of sensory ganglia to extend neurites in 3D gels and the hydrogel pore radius. In addition, our results demonstrate that charged biopolymers influence neurite extension in a polarity dependent manner.     

Agarose gel stiffness determines rate of DRG neurite extension in 3D cultures

The optimization of scaffold mechanical properties for neurite extension is critical for neural tissue engineering. Agarose hydrogels can be used to stimulate and maintain three-dimensional neurite extension from primary sensory ganglia in vitro. The present study explores the structure-function relationship between dorsal root ganglion (DRG) neurite extension and agarose gel mechanical properties. A range of agarose gels of differing concentrations were generated and the corresponding rate of E9 DRG neurite extension was measured. Rate of neurite extension was inversely correlated to the mechanical stiffness of agarose gels in the range of 0.75-2.00% (wt/vol) gel concentrations. In addition, we postulate a physical model that predicts the rate of neurite extension in agarose gels, if gel stiffness is a known parameter. This model is based on Heidemann and Buxbaum's model of neurite extension. These results, if extended to scaffolds of other morphological and chemical features, would contribute significantly to the design criteria of three-dimensional scaffolds for neural tissue engineering.     

Survival and neurite outgrowth of rat cortical neurons in three-dimensional agarose and collagen gel matrices

To better understand interactions between neurons and extracellular matrix equivalents, embryonic day-18 rat cortical neurons were immobilized and maintained in culture for up to 24 days in agarose and type I collagen gels. Using live/dead staining, neuronal cultures in low density collagen gel lasted at least 3 weeks. At 14 days, over 50% of immobilized cells in collagen gel were found viable while in low density agarose gel no cells survived. In situ cell death detection showed that most, if not all, dead cells in either of the gels underwent apoptosis. The collagen-trapped neurons exhibited normal neuronal polarity and developed long neurites, estimated at over 500 microm. The results suggest that collagen, because it is a major extracellular matrix constituent, suppresses apoptosis and provides a suitable substrate for neuronal survival and differentiation.     

Three-dimensional culture of human disc cells within agarose or a collagen sponge: assessment of proteoglycan production

The objective of the present study was to assess proteoglycan production by human intervertebral disc cells cultured in vitro in selected cell carriers. Based on previous studies which evaluated disc cells seeded into collagen sponge, collagen gel, agarose, alginate or fibrin gel three-dimensional (3D) cell carriers, collagen sponge and agarose were found to provide superior microenvironments for formation of extracellular matrix (ECM). A standardized test design was used to evaluate ECM formed after 14 days of culture using the 1,9-dimethylmethylene blue (DMB) assay to assess sulfated glycosaminoglycan (S-GAG) production. Although agarose culture showed higher S-GAG levels compared to collagen sponge (2.94+/-2.20 (19) microg/ml S-GAG (mean+/-S.D. (n)) vs. 0.94+/-0.77 (22), respectively, p=0.0003), this is off-set by the significantly lower proliferation rate associated with culture of disc cells in agarose.     

Neurite bridging across micropatterned grooves

After injury, regenerating axons must navigate complex, three-dimensional (3D) microenvironments. Topographic guidance of neurite outgrowth has been demonstrated in vitro with culture substrates that contain micropatterned features on the nanometer-micron scale. In this study we report the ability of microfabricated biomaterials to support neurite extension across micropatterned grooves with feature sizes on the order of tens of microns, sizes relevant to the design of biomaterials and tissue engineering scaffolds. Neonatal rat dorsal root ganglion (DRG) neurons were cultured on grooved substrates of poly(dimethyl siloxane) coated with poly-L-lysine and laminin. Here we describe an unusual capability of a subpopulation of DRG neurons to extend neurites that spanned across the grooves, with no underlying solid support. Multiple parameters influenced the formation of bridging neurites, with the highest numbers of bridges observed under the following experimental conditions: cell density of 125,000 cells per sample, groove depth of 50 microm, groove width of 30 microm, and plateau width of 200 microm. Bridges were formed as neurites extended from a neuron in a groove, contacted adjacent plateaus, pulled the neuron up to become suspended over the groove, and the soma translocated to the plateau. These studies are of interest to understanding cytoskeletal dynamics and designing biomaterials for 3D axon guidance.     

不同结合状态硫酸软骨素多糖链对体外二维三维培养背根节神经突起生长影响研究

目的构建鸡胚背根节细胞3D生长模型,观察CSPG对不同浓度的琼脂糖水溶胶培养基对鸡胚背根节神经突起生长的影响。方法①利用多肽缩合剂1’1羰基二咪唑介导CS-B长链与琼脂糖凝胶共价结合。②分别配制0.5%、0.75%、1%、1.25%、1.5%的SeaPrep琼脂糖凝胶溶液和共价结合了CS-B多糖长链的琼脂糖凝胶溶液。取孵育9~10天后鸡胚背根节加入上述凝胶溶液加培养液培养。③培养24小时之后在24 h、48 h7、2 h、96 h进行观察拍照。观察各时间点神经突起生长情况。结果①鸡胚背根节神经突起在1.25%的琼脂糖凝胶溶液中开始呈3D生长,在小于1%的琼脂糖溶液中只能2D生长。②在同一时间点,呈2D生长的背根节突起和呈3D生长的背根节突起在突起长度方面没有统计学差异(P>0.05)。③3D生长的DRG神经突起在共价结合了CS-B的琼脂糖凝胶中生长长度明显短于普通琼脂糖水凝胶(P<0.05),并且可观察到部分突起长出短而直的成束突起。但是对于呈2D生长的DRG突起长度在两种培养基中却未见差异(P>0.05)。结论鸡胚背根节神经元周围突起生长长度在3D培养中与在2D培养中有没有明显的差异。硫酸软骨素能够有效抑制呈3D生长的神经节突起生长,并且影响到神经突起的生长方式。但是在2D培养基中,未能看到上述的抑制作用。     

The polarity and magnitude of ambient charge influences three-dimensional neurite extension from DRGs

Sulfated proteoglycans have inhibitory effects on neurite extension, and the negative charge of the glycosaminoglycan side chains may be involved in the inhibitory process. The main goal of this study is to investigate the effects of charge on three-dimensional neurite extension. Various concentrations of dermatan sulfate (DS), a chondroitin sulfate glycosaminoglycan, and consequently, various degrees of negative charge were presented on three-dimensional agarose hydrogels and the effect of charge on neurite extension from primary neurons was investigated. Dose-response experiments were also performed with the polycationic (positively charged) polysaccharide chitosan covalently coupled to agarose. The amount of DS or chitosan coupled to the agarose gel was quantified via metachromatic dye or Fourier transform infrared spectroscopy methods, respectively. The length of embryonic day 9 (E9) chick dorsal root ganglia neurites extended through charged agarose gels is dependent on the polarity and quantity of ambient charge. The inhibitory effects of the sulfated DS and the enhancing effects of the polycationic chitosan on neurite extension decrease as the amount of DS or chitosan coupled to agarose is decreased. These findings indicate that primary neural process extension is influenced by the polarity of ambient charge in a dose-responsive manner.     

Patterned PLG substrates for localized DNA delivery and directed neurite extension

Tissue engineering strategies that enable nerve regeneration will require methods that can promote and direct neurite extension across the lesion. In this report, we investigate an in vitro combinatorial approach to directed neurite outgrowth using gene delivery from topographically patterned substrates, which can induce expression of neurotrophic factors to promote neurite extension and direct the extending neurites. Poly(lactide-co-glycolide) (PLG), which has been used to fabricate conduits or bridges for regeneration, was compression molded to create channels with 100, 150, and 250 microm widths. DNA complexes were immobilized to the PLG, and cells cultured on the substrate were transfected with efficiencies dependent on channel width and DNA amount. A co-culture model consisting of primary neurons and accessory cells was employed to investigate neurite outgrowth within the channels. Localized secretion of nerve growth factor (NGF) by the accessory cells promoted neuron survival and neurite extension. Neurons cultured in channels with NGF expression exhibited longer primary neurites than in the absence of channels. Neurons cultured in smaller width PLG microchannels exhibited a greater degree of directionality and less secondary sprouting than larger channels. Finally, surface immobilization allowed for the delivery of distinct plasmids from each channel, which may enable channels to be tailored for specific nerve tracts. This approach demonstrates the ability to combine gene delivery with physical guidance, and can be tailored to target specific axonal populations with varying neurotrophic factor requirements.     

Anisotropic scaffolds facilitate enhanced neurite extension in vitro

Tissue engineering (TE) techniques to enhance nerve regeneration following nerve damage have had limited success in matching the performance of autografts across short nerve gaps (< 10 mm). For regeneration over longer nerve gaps, TE techniques have been less successful than autografts. Most engineered scaffolds do not present directional cues to the regenerating nerves. In our efforts to design a TE scaffold to replace the autograft, we hypothesize that anisotropic hydrogel scaffolds with gradients of a growth-promoting glycoprotein, laminin-1 (LN-1), may promote directional neurite extension and enhance regeneration. In this study we report the engineering of three-dimensional (3D) agarose scaffolds with photoimmobilized gradients of LN-1 of differing slopes. Dorsal root ganglia (DRG) from chicken embryos were cultured in the agarose scaffolds and their neurite extension rate was determined. DRG neurite extension rates were significantly higher in the anisotropic scaffolds, with a maximal growth rate in an anisotropic scaffold twice that of the maximal growth rate in isotropic scaffolds of LN-1. We suggest that these anisotropic scaffolds, presenting an optimal gradient of LN-1, may significantly impact nerve regeneration. Such anisotropic scaffolds may represent a new generation of tissue engineered materials with built-in directional cues for guided tissue or nerve regeneration.     

Effects of collagen 1, fibronectin, laminin and hyaluronic acid concentration in multi-component gels on neurite extension

Recovery after peripheral nerve injury remains a significant challenge. Extracellular matrix proteins and hydrogels of extracellular matrix components have been shown to improve regeneration in peripheral nerve entubulation models, especially over long distances. The chemical properties, ligand identity and density, and mechanical properties of the hydrogel can affect neurite extension. However, the importance of combinatorial effects between different components in co-gels of several extracellular matrix components is unclear. In this study, we investigated neurite extension from explanted dorsal root ganglia cultured within co-gels made from laminin, fibronectin, collagen 1 and hyaluronic acid. Laminin had a strong, dose-dependent effect on both neurite length and outgrowth. Fibronectin was slightly, but generally not significantly, inhibitory to neurite extension. The concentration of collagen 1 and hyaluronic acid did not have significant effects on neurite extension. The combinatorial effects among the four components were additive rather than synergistic. A co-gel made with 1.5 mg/ml collagen 1 and 1.5 mg/ml laminin was optimum in this study, resulting in an average neurite length of 1532 +/- 91 microm versus 976 +/- 32 microm for controls, and an increase in overall volume outgrowth (reflecting neurite length and branching) of 85.9+/-9.3% over controls. This co-gel provides a mechanically stable scaffold with high ligand density and biochemical affinity. The results of this study support the use of co-gels of laminin and collagen 1 for promoting regeneration in peripheral nerve injuries and suggest that interactions among hydrogel components are not significant.     

Effects of collagen 1, fibronectin,laminin and hyaluronic acid concentration in multi-component gels on neurite extension

Recovery after peripheral nerve injury remains a significant challenge. Extracellular matrix proteins and hydrogels of extracellular matrix components have been shown to improve regeneration in peripheral nerve entubulation models, especially over long distances. The chemical properties, ligand identity and density, and mechanical properties of the hydrogel can affect neurite extension. However, the importance of combinatorial effects between different components in co-gels of several extracellular matrix components is unclear. In this study, we investigated neurite extension from explanted dorsal root ganglia cultured within co-gels made from laminin, fibronectin, collagen 1 and hyaluronic acid. Laminin had a strong, dose-dependent effect on both neurite length and outgrowth. Fibronectin was slightly, but generally not significantly, inhibitory to neurite extension. The concentration of collagen 1 and hyaluronic acid did not have significant effects on neurite extension. The combinatorial effects among the four components were additive rather than synergistic. A co-gel made with 1.5 mg/ml collagen 1 and 1.5 mg/ml laminin was optimum in this study, resulting in an average neurite length of 1532 ± 91 μm versus 976 ± 32 μm for controls, and an increase in overall volume outgrowth (reflecting neurite length and branching) of 85.9±9.3% over controls. This co-gel provides a mechanically stable scaffold with high ligand density and biochemical affinity. The results of this study support the use of co-gels of laminin and collagen 1 for promoting regeneration in peripheral nerve injuries and suggest that interactions among hydrogel components are not significant.     

A laminin and nerve growth factor-laden three-dimensional scaffold for enhanced neurite extension.

Agarose hydrogel scaffolds were engineered to stimulate and guide neuronal process extension in three dimensions in vitro. The extracellular matrix (ECM) protein laminin (LN) was covalently coupled to agarose hydrogel using the bifunctional cross-linking reagent 1,19- carbonyldiimidazole (CDI). Compared to unmodified agarose gels, LN-modified agarose gels significantly enhanced neurite extension from three-dimensionally (3D) cultured embryonic day 9 (E9) chick dorsal root ganglia (DRGs), and PC 12 cells. After incubation of DRGs or PC 12 cells with YIGSR peptide or integrin beta1 antibody respectively, the neurite outgrowth promoting effects in LN-modified agarose gels were significantly decreased or abolished. These results indicate that DRG/PC 12 cell neurite outgrowth promoting effect of LN-modified agarose gels involves receptors for YIGSR/integrin beta1 subunits respectively. 1,2-bis(10, 12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC)-based lipid microcylinders were loaded with nerve growth factor (NGF), and embedded into agarose hydrogels. The resulting trophic factor gradients stimulated directional neurite extension from DRGs in agarose hydrogels. A PC 12 cell-based bioassay demonstrated that NGF-loaded lipid microcylinders can release physiologically relevant amounts of NGF for at least 7 days in vitro. Agarose hydrogel scaffolds may find application as biosynthetic 3D bridges that promote regeneration across severed nerve gaps.     

Fibrin glue mixed with gelatin/hyaluronic acid/chondroitin-6-sulfate tri-copolymer for articular cartilage tissue engineering: the results of real-time polymerase chain reaction

Autologous fibrin glue has been demonstrated as a potential scaffold with very good biocompatibility for neocartilage formation. However, fibrin glue has been reported not to provide enough mechanical strength, but with many growth factors to interfere the tissue growth. Gelatin/hyaluronic acid/chondroitin-6-sulfate (GHC6S) tri-copolymer sponge has been prepared as scaffold for cartilage tissue engineering and showed very good results, but problems of cell seeding and cell distribution troubled the researchers. In this study, GHC6S particles would be added into the fibrin glue to provide better mechanical strength, better cell distribution, and easier cell seeding, which would be expected to improve cartilage regeneration in vitro. Porcine cryo-precipitated fibrinogen and thrombin prepared from prothrombin activated by 10% CaCl(2) solution were used in two groups. One is the fibrin glue group in which porcine chondrocytes were mixed with thrombin-fibrinogen solution, which was then converted into fibrin glue. The other is GHC6S-fibrin glue in which GHC6S particles were added into the thrombin-fibrinogen solution with porcine chondrocytes. After culturing for 1-2 weeks, the chondrocytes cultured in GHC6S-fibrin glue showed a round shape with distinct lacuna structure and showed positive in S-100 protein immunohistochemical stain. The related gene expressions of tissue inhibitor of metalloproteinases-1, matrix metalloproteinase-2, MT1-MMP, aggrecan, decorin, type I, II, X collagen, interleukin-1 beta, transforming growth factor-beta 1 (TGF-beta1), and Fas-associating death domain were checked by real-time PCR. The results indicated that the chondrocytes cultured in GHC6S-fibrin glue would effectively promote extracellular matrix (ECM) secretion and inhibit ECM degradation. The evidence could support that GHC6S-fibrin glue would be a promising scaffold for articular cartilage tissue engineering.     

Matrix inclusion within synthetic hydrogel guidance channels improves specific supraspinal and local axonal regeneration after complete spinal cord transection

We have previously shown that a novel synthetic hydrogel channel composed of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) (pHEMA-MMA) is biocompatible and supports axonal regeneration after spinal cord injury. Our goal was to improve the number and type of regenerated axons within the spinal cord through the addition of different matrices and growth factors incorporated within the lumen of the channel. After complete spinal cord transection at T8, pHEMA-MMA channels, having an elastic modulus of 263+/-13 kPa were implanted into adult Sprague Dawley rats. The channels were then filled with one of the following matrices: collagen, fibrin, Matrigel, methylcellulose, or smaller pHEMA-MMA tubes placed within a larger pHEMA-MMA channel (called tubes within channels, TWC). We also supplemented selected matrices (collagen and fibrin) with neurotrophic factors, fibroblast growth factor-1 (FGF-1) and neurotrophin-3 (NT-3). After channel implantation, fibrin glue was applied to the cord-channel interface, and a duraplasty was performed with an expanded polytetrafluoroethylene (ePTFE) membrane. Controls included animals that had either complete spinal cord transection and implantation of unfilled pHEMA-MMA channels or complete spinal cord transection. Regeneration was assessed by retrograde axonal tracing with Fluoro-Gold, and immunohistochemistry with NF-200 (for total axon counts) and calcitonin gene related peptide (CGRP, for sensory axon counts) after 8 weeks survival. Fibrin, Matrigel, methylcellulose, collagen with FGF-1, collagen with NT-3, fibrin with FGF-1, and fibrin with NT-3 increased the total axon density within the channel (ANOVA, p<0.05) compared to unfilled channel controls. Only fibrin with FGF-1 decreased the sensory axon density compared to unfilled channel controls (ANOVA, p<0.05). Fibrin promoted the greatest axonal regeneration from reticular neurons, and methylcellulose promoted the greatest regeneration from vestibular and red nucleus neurons. With Matrigel, there was no axonal regeneration from brainstem motor neurons. The addition of FGF-1 increased the axonal regeneration of vestibular neurons, and the addition of NT-3 decreased the total number of axons regenerating from brainstem neurons. The fibrin and TWC showed a consistent improvement in locomotor function at both 7 and 8 weeks. Thus, the present study shows that the presence and type of matrix contained within synthetic hydrogel guidance channels affects the quantity and origin of axons that regenerate after complete spinal cord transection, and can improve functional recovery. Determining the optimum matrices and growth factors for insertion into these guidance channels will improve regeneration of the injured spinal cord.     

Blood-derived biomaterials: fibrin sealant,platelet gel and platelet fibrin glue

Blood-derived biomaterials include fibrin sealant (FS) (also called fibrin glue), platelet gel (PG), and platelet fibrin glue. They are used in many surgical fields because of their functional properties and unique physical advantages compared to synthetic products. FS can be made industrially by the fractionation of large plasma pools, or from single plasma donations. Thanks to a high content in fibrinogen, FS exhibits, after activation by thrombin and formation of a strong fibrin clot, tissue sealing and haemostatic properties. PG and platelet fibrin glue are made from single blood donations (platelet concentrates combined or not with cryoprecipitate). Owing to their richness in platelet, PG and PFG can release, upon thrombin activation, a myriad of growth factors that can stimulate cell growth and differentiation, generating much interest for hard and soft tissues regeneration and healing, as well as, increasingly, cell therapy protocols to replace fetal bovine serum. Blood-derived biomaterials have the advantages, over synthetic glues and other biomaterials, of being physiologically compatible with human tissues, and of not inducing tissue necrosis or other tissue reactions. They can be readily colonized by cells and are totally biodegradable in a matter of days to weeks. These blood-derived biomaterials are used increasingly as tissue engineering tools, allowing surgeons to influence and improve the in vitro or in vivo cellular environment to enhance the success of tissue grafting. We review here the three main types of biomaterials that can be made from human blood and describe their biochemical and physiological properties as well as their clinical applications.     

Novel soft alginate hydrogel strongly supports neurite growth and protects neurons against oxidative stress

Neural tissue engineering focuses on development of biomaterials that could support regeneration of neurons after trauma as well as injury caused by degenerative diseases. In this work we describe novel soft alginate hydrogels, which provide an adhesive matrix for rat and human neurons and facilitate neurite outgrowth. Only soft hydrogels, prepared with sub-stoichiometric concentrations of Ca2?, Ba2?, and Sr2? cations by cross-linking with no >10% of all potentially available gelation sites in alginate, facilitated rapid and abundant neurite outgrowth in primary neuronal monolayer cultures, neural spheroids, and neurons derived from rat and human neural stem cells. To support neurite growth, hydrogels did not require modification by any extracellular matrix components and were prepared from high as well as low viscous alginates of different origin. In addition, neurons cultured on soft hydrogels were resistant to oxidative stress injury induced by hydrogen peroxide. These findings, which apply both to rat and human neurons, go beyond the well-described role of alginates as inert materials for cell encapsulation. Such soft alginate hydrogels may be useful for the preparation of pharmaceutical compositions for prophylaxis and treatment of neurodegenerative disorders, for promoting neuronal regeneration in the peripheral and central nervous system and for neural tissue engineering applications.     

Resurfacing potential of heterologous chondrocytes suspended in fibrin glue in large full-thickness defects of femoral articular cartilage: an experimental study in the goat.

A large full-thickness articular-cartilage defect was created in the medial femoral condyle of 32 adult goats. The defects were xenografted with isolated rabbit chondrocytes suspended in fibrin glue. Sham operated goats, where only a standardized defect was created, were used as controls. Results of cartilage repair were assessed after 3, 8, 13, 26 and 52 weeks. The repair tissue was evaluated macroscopically, histologically and biochemically. Results indicated that xenografted rabbit chondrocytes survived the transplantation and maintained their potential to produce matrix in fibrin glue, particularly if they were located in a non-weight-bearing area. In terms of an immunological reaction to xenografted chondrocytes, only mild signs of synovitis were observed in both groups and rejection of transplanted cells did not occur. From 3 weeks gradually progressive resolvement of the fibrin glue was observed with subsequent replacement by fibrous tissue. Initially xenografted defects histologically showed better tendency for cartilage regeneration, however, 52 weeks after surgery no significant differences could be detected in the repair tissue of both groups macroscopically, histologically and on biochemical scoring. The amount of collagen type II in the newly synthesized matrix was 75% 1 year after surgery. This study shows that isolated heterologous chondrocytes can be used for transplantation in articular cartilage defects, however, fibrin glue does not offer enough biomechanical support to the cells to maintain its function as a three-dimensional scaffold.     

Effects of Reg‐2 on Survival of Spinal Cord Neurons In Vitro

Regeneration gene protein 2 (Reg‐2) is a small secreted protein expressed in motor and sensory neurons of spinal cord during developmental stages and following injury of peripheral nerves. Reg‐2 appears to act as a neurotrophic factor and protects injured neurons from death during regeneration. To illustrate these potential protective effects in vitro, we investigated the blocking effects of Reg‐2 antibodies on the survival of primary cultured spinal cord neurons and astrocytes, as well as on neurite outgrowth. In addition, the effects of Reg‐2 in neuron injury models induced by peroxide and mitochondrial poisoning were assessed. Our results showed that Reg‐2 antibody markedly reduced survival and neurite outgrowth from neurons, whereas astrocyte survival was unaffected. Addition of Reg‐2 into the culture medium had no effect on neuron survival or neurite outgrowth. However, the addition of the Reg‐2 into culture media after peroxide treatment or cellular hypoxia insult induced by mitochondrial poisoning can reduce lactate dehydrogenase release levels and cell death. Thus, the data suggests that Reg‐2 is essential for the survival and neurite outgrowth of developing spinal cord neurons but not the survival of glial cells, and that Reg‐2 plays protective effects on spinal cord neurons against injury in vitro. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.