[Show abstract][Hide abstract] ABSTRACT: Nerve regeneration in an injured spinal cord is often restricted, contributing to the devastating outcome of neurologic impairment below the site of injury. Although implantation of tissue-engineered scaffolds has evolved as a potential treatment method, the outcomes remain sub-optimal. One possible reason may be the lack of topographical signals from these constructs to provide contact guidance to invading cells or regrowing axons. Nanofibers mimic the natural extracellular matrix architecturally and may therefore promote physiologically relevant cellular phenotypes. In this study, the potential application of electrospun collagen nanofibers (diameter=208.2±90.4 nm) for spinal cord injury (SCI) treatment was evaluated in vitro and in vivo. Primary rat astrocytes and dorsal root ganglias (DRGs) were seeded on collagen-coated glass cover slips (two-dimensional [2D] substrate controls), and randomly oriented or aligned collagen fibers to evaluate scaffold topographical effects on astrocyte behavior and neurite outgrowth, respectively. When cultured on collagen nanofibers, astrocyte proliferation and expression of glial fibrillary acidic protein (GFAP) were suppressed as compared to cells on 2D controls at days 3 (p<0.05) and 7 (p<0.01). Aligned fibers resulted in elongated astrocytes (elongation factor >4, p<0.01) and directed the orientation of neurite outgrowth from DRGs along fiber axes. In the contrast, neurites emanated radially on randomly oriented collagen fibers. By forming collagen scaffolds into spiral tubular structures, we demonstrated the feasibility of using electrospun nanofibers for the treatment of acute SCI using a rat hemi-section model. At days 10 and 30 postimplantation, extensive cellular penetration into the constructs was observed regardless of fiber orientation. However, scaffolds with aligned fibers appeared more structurally intact at day 30. ED1 immunofluorescent staining revealed macrophage invasion by day 10, which decreased significantly by day 30. Neural fiber sprouting as evaluated by neurofilament staining was observed as early as day 10. In addition, GFAP immunostained astrocytes were found only at the boundary of the lesion site, and no astrocyte accumulation was observed in the implantation area at any time point. These findings indicate the feasibility of fabricating 3D spiral constructs using electrospun collagen fibers and demonstrated the potential of these scaffolds for SCI repair.
Tissue Engineering Part A 02/2012; 18(9-10):1057-66. DOI:10.1089/ten.TEA.2011.0430 · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nerve regeneration after spinal cord injuries (SCI) remains suboptimal despite recent advances in the field. One major hurdle is the rapid clearance of drugs from the injury site, which greatly limits therapeutic outcomes. Nanofiber scaffolds represent a potential class of materials for enhancing nerve regeneration because of its biomimicking architecture. In this study, we investigated the feasibility of incorporating neurotrophin-3 (NT-3) and chondroitinase ABC (ChABC) onto electrospun collagen nanofibers for SCI treatment. By using microbial transglutaminase (mTG) mediated crosslinking, proteins were loaded onto electrospun collagen nanofibers at an efficiency of ∼45-48%. By combining NT-3 with heparin during the protein incorporation process, a sustained release of NT-3 was obtained (∼96% by day 28). As indicated by dorsal root ganglion outgrowth assay, NT-3 incorporated collagen scaffolds supported neuronal culture and neurite outgrowth for a longer time period than bolus delivery of NT-3. The presence of heparin also protected ChABC from degradation. Specifically, as evaluated by dimethylmethylene blue assay, bioactive ChABC was detected from collagen scaffolds for at least 32 days in vitro in the presence of heparin (∼32% of bioactivity retained). In contrast, ChABC bioactivity was only ∼1.9% by day 22 in the absence of heparin. Taken together, these results clearly demonstrated the feasibility of incorporating NT-3 and ChABC via mTG immobilization to produce protein-incorporated collagen nanofibers. Such biofunctional nanofiber constructs may find useful applications in SCI treatment by providing topographical signals and multiple biochemical cues that can promote nerve regeneration while antagonizing axonal growth inhibition for CNS regeneration.
Journal of Biomedical Materials Research Part A 01/2012; 100(1):236-42. DOI:10.1002/jbm.a.33271 · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Currently available crosslinking methods for electrospun collagen nanofibers do not preserve the fibrous architecture over prolonged periods of time. In addition, electrospinning of collagen often involves solvents that lead to extensive protein denaturation. In this study, we demonstrate the advantage of acetic acid over 1,1,1,3,3,3 hexafluoroisopropanol (HFP) in preventing collagen denaturation. A novel photochemical crosslinking method using rose bengal as the photoinitiator is also introduced. Using circular dichorism analyses, we demonstrate the fraction of collagen helical structure to be significantly greater in acetic acid-spun fibers than HFP-spun fibers (28.9 +/- 5.9% vs. 12.5 +/- 2.0%, p < 0.05). By introducing 0.1% (w/v) rose bengal into collagen fibers and subjecting these scaffolds to laser irradiation at a wavelength of 514 nm for 100 sec, biodegradable crosslinked scaffolds were obtained. Scaffold degradation as evaluated by soaking crosslinked collagen scaffolds in PBS at 37 degrees C, indicated a mass loss of 47.7 +/- 7.4% and 68.9 +/- 24.7% at day 7 and day 15, respectively. However, these scaffolds retained fibrous architecture for at least 21 days under physiological conditions. Neural stem cell line, C17.2, cultured on crosslinked collagen scaffolds proliferated after 7 days by forming a confluent layer of cells with extensive cellular projections that were indicative of neurite outgrowth. Taken together, these findings support the potential of acetic acid-electrospun photochemical crosslinked collagen nanofibers for neural tissue engineering.
Journal of Biomedical Materials Research Part A 10/2010; 95(1):276-82. DOI:10.1002/jbm.a.32831 · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The repairing process in the nervous system is complicated and brings great challenges to researchers. Tissue engineering scaffolds provide an alternative approach for neural regeneration. Sub-micron and nano-scale fibrous scaffolds which mimic the topography of natural extracellular matrix (ECM) can be potential scaffold candidates for neural tissue engineering. Two fiber-fabrication methods have been explored in the field of nerve regeneration: electrospinning and self-assembly. Electrospinning produces fibers with diameters ranging from several micrometers to hundreds of nanometers. The fibrous nerve conduits can be introduced at lesion sites by implantation. Self-assembly fibers have diameters of tens of nanometers and can be injected for central nervous system (CNS) injury repair. Both fibrous scaffolds would enhance neurite extension and axon regrowth. These functional nanofibrous scaffolds can serve as powerful tools for neural tissue engineering.