sepharose and Spinal-Cord-Injuries

This review presents a summary of various types of scaffold biomaterials used alone or together with therapeutic drugs and cells to regenerate spinal cord injury (SCI). The inhibitory environment and loss of axonal connections after SCI give rise to critical obstacles to regeneration of lost tissues and neuronal functions. Biomaterial scaffolds can provide a bridge to connect lost tissues, an adhesion site for implanted or host cells, and sustained release of therapeutic drugs in the injured spinal cord. In addition, they not only provide a structural platform, but can play active roles by inhibiting apoptosis of cells, inflammation and scar formation, and inducing neurogenesis, axonal growth and angiogenesis. Many synthetic and natural biomaterial scaffolds have been extensively investigated and tested in vitro and in animal SCI models for these purposes. We summarized the literature on the biomaterials commonly used for spinal cord regeneration in terms of historical backgrounds and current approaches.

Topics: Alginates; Animals; Apoptosis; Axons; Biocompatible Materials; Chitosan; Collagen; Disease Models, Animal; Drug Delivery Systems; Fibrin; Humans; Hyaluronic Acid; Inflammation; Lactic Acid; Materials Testing; Peptides; Polyesters; Polymers; Sepharose; Spinal Cord; Spinal Cord Injuries; Spinal Cord Regeneration; Stem Cells; Tissue Engineering; Tissue Scaffolds

An agarose scaffold can be useful for supporting and guiding injured axons after spinal cord injury (SCI), but the electrophysiological signal of regenerated axon in scaffolds has not yet been determined. The current study investigated whether a Matrigel-loaded agarose scaffold would enhance the regeneration of axons after SCI. Moreover, the functional connectivity of regenerated axons within the channels of the scaffold was evaluated by directly recording motor evoked potentials. Our data showed that the agarose scaffold containing Matrigel can support and enhance linearly organized axon regeneration after SCI. Additionally, motor evoked potentials were successfully recorded from regenerated axons. These results demonstrate that an agarose scaffold loaded with Matrigel could promote the regeneration of axons and guide the reconnection of functional axons after SCI.

Topics: Animals; Axons; Biomimetic Materials; Collagen; Drug Combinations; Equipment Design; Equipment Failure Analysis; Guided Tissue Regeneration; Laminin; Male; Nerve Regeneration; Neuronal Outgrowth; Prostheses and Implants; Proteoglycans; Rats; Rats, Sprague-Dawley; Recovery of Function; Sepharose; Spinal Cord Injuries; Tissue Scaffolds; Treatment Outcome

The past 30 years of research in spinal cord injury (SCI) have revealed that, under certain conditions, some types of axons are able to regenerate. To aid these axons in bridging the lesion site, many experimenters place cellular grafts at the lesion. However, to increase the potential for functional recovery, it is likely advantageous to maximize the number of axons that reach the intact spinal cord on the other side of the lesion. Implanting linear-channeled scaffolds at the lesion site provides growing axons with linear growth paths, which minimizes the distance they must travel to reach healthy tissue. Moreover, the linear channels help the regenerating axons maintain the correct mediolateral and dorsoventral position in the spinal cord, which may also improve functional recovery by keeping the axons nearer to their correct targets. Here, we provide a protocol to perform a full spinal cord transection in rats that accommodates an implanted scaffold.

Topics: Anesthesia; Animals; Axons; Female; Guided Tissue Regeneration; Nerve Regeneration; Rats; Rats, Inbred F344; Sepharose; Spinal Cord; Spinal Cord Injuries; Tissue Scaffolds

Bioengineered scaffolds have the potential to support and guide injured axons after spinal cord injury, contributing to neural repair. In previous studies we have reported that templated agarose scaffolds can be fabricated into precise linear arrays and implanted into the partially injured spinal cord, organizing growth and enhancing the distance over which local spinal cord axons and ascending sensory axons extend into a lesion site. However, most human injuries are severe, sparing only thin rims of spinal cord tissue in the margins of a lesion site. Accordingly, in the present study we examined whether template agarose scaffolds seeded with bone marrow stromal cells secreting Brain-Derived Neurotrophic Factor (BDNF) would support regeneration into severe, complete spinal cord transection sites. Moreover, we tested responses of motor axon populations originating from the brainstem. We find that templated agarose scaffolds support motor axon regeneration into a severe spinal cord injury model and organize axons into fascicles of highly linear configuration. BDNF significantly enhances axonal growth. Collectively, these findings support the feasibility of scaffold implantation for enhancing central regeneration after even severe central nervous system injury.

Topics: Animals; Axons; Biocompatible Materials; Cell Enlargement; Equipment Design; Equipment Failure Analysis; Female; Guided Tissue Regeneration; Molecular Imprinting; Motor Neurons; Nerve Regeneration; Rats; Rats, Inbred F344; Sepharose; Spinal Cord Injuries; Tissue Scaffolds; Treatment Outcome

Previously we reported that templated agarose scaffolds can orient and guide local spinal cord axons after injury. In the present study we examined whether growth of long-projecting spinal cord axons could also be promoted into, and then beyond, templated agarose scaffolds placed into a spinal cord lesion site. Ascending spinal cord dorsal column sensory axons were transected at the C4 level. Animals were then subjected to combinatorial therapies consisting of: 1) templated agarose scaffolds implanted into the lesion site, seeded with autologous bone marrow stromal cells expressing a growth factor, neurotrophin-3 (NT-3), 2) lentiviral vectors expressing NT-3 beyond the lesion site (to promote axonal emergence from the scaffold along chemotropic gradients of growth factors), and 3) priming lesions ("conditioning lesions") of the sensory neuronal cell body to stimulate the endogenous growth state of the injured neuron. Control groups received either non-organized, NT-3-expressing cell suspension grafts in the lesion site, or templated scaffolds plus one of the two components of the combination therapy. Among groups that received templated agarose scaffolds, long-tract sensory axonal regeneration occurred into the spinal cord lesion site, and the growth of these axons was remarkably organized and linear compared to non-organized cell suspension grafts. Axonal penetration was maximal in subjects that received combination therapies; further, 83 + 13% of axons entering the scaffolds in combination-treated subjects continued to grow the full length of the lesion cavity to reach the distal aspect of the scaffold, over a 2 mm distance. In contrast, axons regenerating into cell suspension grafts lacking guidance scaffolds exhibited a parabolic decay of growth as a function of distance, and only 22 + 6% of axons extended the length of the lesion cavity. Moreover, axonal regeneration beyond the lesion site occurred only among subjects that received full combinatorial treatments (p < 0.05). However, axon growth beyond the scaffold was constrained to a reactive cell layer that formed between the distal aspect of the scaffold and host tissue, and did not continue further to re-penetrate the host spinal cord. Thus, templated agarose scaffolds substantially enhance the organization and distance over which long-tract axons extend through a spinal cord lesion site in the presence of combinatorial therapies, but host-scaffold reactive interfaces limit axon re-penetration of

Topics: Animals; Axons; Biomarkers; Female; Guided Tissue Regeneration; Implants, Experimental; Nerve Regeneration; Rats; Rats, Inbred F344; Sepharose; Spinal Cord; Spinal Cord Injuries; Tissue Scaffolds

Methylprednisolone (MP) has been shown to reduce acute inflammation resulting from a secondary damage cascade initiated by the primary physical injury to the spinal cord. The current clinical practice for delivering systemic MP is inefficient, and high doses are required, resulting in adverse, undesired, dose-related side effects in patients. Here, we report a novel, minimally invasive, localized drug delivery system for delivering MP to the contused adult rat spinal cord that potentially side-steps the deleterious consequences of systemic cortico-steroid therapy. MP was encapsulated in biodegradable PLGA based nanoparticles (NP), and these nanoparticles were embedded in an agarose hydrogel for localization to the site of contusion injury. To visualize and quantify its spatial distribution within the injured spinal cord, MP was conjugated to Texas-red cadaverine prior to encapsulation in nanoparticles. When delivered via the hydrogel-nanoparticle system, MP entered the injured spinal cord and diffused up to 1.5mm deep and up to 3mm laterally into the injured spinal cord within 2 days. Furthermore, topically delivered MP significantly decreased early inflammation inside the contusion injured spinal cord as evidenced by a significant reduction in the number of ED-1(+) macrophages/activated microglia. This decreased early inflammation was accompanied by a significantly diminished expression of pro-inflammatory proteins including Calpain and iNOS. Additionally, topically delivered MP significantly reduced lesion volume 7 days after contusion injury. The minimally invasive MP delivery system reported in this study has the potential to enhance the effectiveness of MP therapy after contusion injury to the spinal cord and avoid the side effects arising from high dose cortico-steroid therapy.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Delayed-Action Preparations; Drug Carriers; Male; Methylprednisolone; Rats; Rats, Sprague-Dawley; Sepharose; Spinal Cord Injuries; Tissue Distribution; Treatment Outcome

Although several approaches to stimulate axonal regeneration after spinal cord injury have succeeded in stimulating robust growth of axons into a lesion site, the growth is generally highly disorganized, losing the distinct arrangement of axonal tracts within the spinal cord. Previously described freeze-dried agarose scaffolds, composed of individual, uniaxial channels extending through their entire length, were prepared with and without recombinant Brain-Derived Neurotrophic Factor (BDNF) protein and tested in an adult rat model of spinal cord injury to determine whether regenerating axons could be guided across a site of injury in an organized fashion. After 1 month, both the cellular and axonal responses within and around scaffolds were evaluated. Scaffolds were found to be well integrated with host tissue, individual channels were penetrated by cells, and axons grew through scaffolds in a strikingly linear fashion. Furthermore, the regeneration was significantly augmented by the incorporation of BDNF protein into the walls and lumen of the scaffold. These findings clearly demonstrate that axonal regeneration can be organized and guided across a site of injury.

Topics: Animals; Astrocytes; Axons; Biocompatible Materials; Brain-Derived Neurotrophic Factor; Cell Enlargement; Female; Guided Tissue Regeneration; Implants, Experimental; Macrophages; Microglia; Neovascularization, Physiologic; Nerve Regeneration; Neurofilament Proteins; Neurons; Rats; Rats, Inbred F344; Schwann Cells; Sepharose; Spinal Cord; Spinal Cord Injuries

Permanent functional loss usually occurs after injury to the spinal cord. Currently, a clinical strategy to promote regeneration in the injured spinal cord does not exist. It has become evident that in order to promote regeneration, a growth permissive substrate at the injury site is critical. In this study, we report the utilization of an agarose scaffold that gels in situ, conformally filling an irregular, dorsal over-hemisection spinal cord defect in adult rats. Besides being growth permissive, the scaffolds also serve as carriers of trophic factors when embedded with BDNF releasing microtubules. We report that our thermo-reversible scaffolds are capable of supporting 3D neurite extension in vivo and are effective carriers of drug delivery vehicles for sustained local delivery of trophic factors. We demonstrate that BDNF encourages neurite growth into the scaffolds, and reduces further the minimal inflammatory response agarose gels generate in vivo as evidenced by quantitative analysis of the extent of NF-160 kDA positive neurons and axons, GFAP positive reactive astrocytes, and CS-56 positive chondroitin sulfate proteoglycan at the interface of the scaffold and host spinal cord. We suggest that these thermo-reversible scaffolds have great potential to serve as growth permissive 3D scaffolds, and to present neurotrophic factors and potentially anti-scar agents to the injury site and enhance regeneration after spinal cord injury.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Astrocytes; Axons; Brain-Derived Neurotrophic Factor; Chondroitin Sulfate Proteoglycans; Delayed-Action Preparations; Drug Carriers; Glial Fibrillary Acidic Protein; Guided Tissue Regeneration; Hydrogels; Implants, Experimental; Macrophages; Male; Nerve Regeneration; Neurofilament Proteins; Neurons; Phosphatidylcholines; Rats; Rats, Sprague-Dawley; Sepharose; Spinal Cord; Spinal Cord Injuries

Strategies to promote axonal extension through a site of injury, including the provision of nervous system growth factors and supportive substrates, produce growth of axons, that is highly random and does not extend past the lesion site and into the host tissue (Brain Res. Bull 57(6) (2002) 833). Physically guiding the linear growth of axons across a site of injury, in addition to providing neurotrophic and/or cellular support, would help to retain the native organization of regenerating axons across the lesion site and into distal host tissue, and would potentially increase the probability of achieving functional recovery. In the present study, a novel procedure was developed for using freeze-dry processing to create nerve guidance scaffolds made from agarose, with uniaxial linear pores. The hydrated scaffolds were soft and flexible, contained linear guidance pores extending through their full length, were stable under physiological conditions without chemical crosslinking, and could be readily loaded with diffusible growth stimulating proteins.

Topics: Absorption; Animals; Axons; Biocompatible Materials; Cell Adhesion; Cell Culture Techniques; Cell Line; Culture Media; Diffusion; Freezing; Humans; Hydrolysis; Nerve Growth Factor; Nerve Regeneration; Nerve Tissue; Neurons; Nitrogen; PC12 Cells; Polymers; Rats; Sepharose; Spinal Cord; Spinal Cord Injuries; Temperature; Time Factors; Water