3D Collagen Scaffolds As Supports For Neural Cell Expansion

Tissue Engineering Part A, 2017

Recent studies in the field of neuro-tissue engineering have demonstrated the promising effects of aligned contact guidance cue scaffolds of enhancement and direction of neuronal growth. In-vivo, neurons grow and develop neurites in a complex 3-Dimensional (3D) extra cellular matrix (ECM) surrounding. Studies have utilized hydrogel scaffolds derived from ECM molecules to better simulate natural growth. While many efforts have been made to control neuronal growth on 2D surfaces, the development of 3D scaffolds with an elaborate oriented topography to direct neuronal growth still remains a challenge. In this study we designed a method for growing neurons in an aligned and oriented 3D collagen hydrogel. We aligned collagen fibers by inducing controlled uniaxial strain on gels. To examine the collagen hydrogel as a suitable scaffold for neuronal growth we evaluated the physical properties of the hydrogel and measured collagen fiber properties. By combining the neuronal culture in 3D collagen hydrogels with strain induced alignment, we were able to direct neuronal growth in the direction of the aligned collagen matrix. Quantitative evaluation of neurite extension and directionality within aligned gels was performed. The analysis showed neurite growth aligned with collagen matrix orientation while maintaining the advantageous 3D growth.

Journal of Molecular Neuroscience, 2013

The aim of our work is to utilize the crosstalk between the vascular and the neuronal system to enhance directed neuritogenesis in uniaxial guidance scaffolds for the repair of spinal cord injury. In this study, we describe a method for angioneural regenerative engineering, i.e., for generating biodegradable scaffolds, produced by a combination of controlled freezing (freeze-casting) and lyophilization, which contain longitudinally oriented channels, and provide uniaxial directionality to support and guide neuritogenesis from neuronal cells in the presence of endothelial cells. The optimized scaffolds, composed of 2.5 % gelatin and 1 % genipin crosslinked, were characterized by an elastic modulus of~51 kPa and longitudinal channels of~50 μm diameter. The scaffolds support the growth of endothelial cells, undifferentiated or NGF-differentiated PC12 cells, and primary cultures of fetal chick forebrain neurons. The angioneural crosstalk, as generated by first forming endothelial cell monolayers in the scaffolds followed by injection of neuronal cells, leads to the outgrowth of long aligned neurites in the PC12/endothelial cell co-cultures also in the absence of exogenously added nerve growth factor. Neuritogenesis was not observed in the scaffolds in the absence of the endothelial cells. This methodology is a promising approach for neural tissue engineering and may be applicable for regenerative spinal cord injury repair.

2010

Background: 3D-scaffolds have been shown to direct cell growth and differentiation in many different cell types, with the formation and functionalisation of the 3Dmicroenvironment being important in determining the fate of the embedded cells.

Biochemical and Biophysical Research Communications, 2012

Collagen is one component of the extracellular matrix that has been widely used for constructive remodeling to facilitate cell growth and differentiation. The 3-D distribution and growth of cells within the porous scaffold suggest a clinical significance for nerve tissue engineering. In the current study, we investigated proliferation and differentiation of neuron cancer stem cells (NCSCs) on a 3-D porous collagen scaffold that mimics the natural extracellular matrix. We first generated green fluorescence protein (GFP) expressing NCSCs using a lentiviral system to instantly monitor the transitions of morphological changes during growth on the 3-D scaffold. We found that proliferation of GFP-NCSCs increased, and a single cell mass rapidly grew with unrestricted expansion between days 3 and 9 in culture. Moreover, immunostaining with neuronal nuclei (NeuN) revealed that NCSCs grown on the 3-D collagen scaffold significantly enhanced neurite outgrowth. Our findings confirmed that the 80 lm porous collagen scaffold could enhance attachment, viability and differentiation of the cancer neural stem cells. This result could provide a new application for nerve tissue engineering and nerve regeneration.

Annals of Biomedical Engineering, 2007

In vitro models of brain injury that use thick 3-D cultures and control extracellular matrix constituents allow evaluation of cell-matrix interactions in a more physiologically relevant configuration than traditional 2-D cultures. We have developed a 3-D cell culture system consisting of primary rat cortical neurons distributed throughout thick (>500 lm) gels consisting of type IV collagen (Col) conjugated to agarose. Neuronal viability and neurite outgrowth were examined for a range of agarose (AG) percentages (1.0-3.0%) and initial collagen concentrations ([Col] i ; 0-600 lg/ mL). In unmodified AG, 1.5% gels supported viable cultures with significant neurite outgrowth, which was not found at lower (£1.0%) concentrations. Varying [Col] i in 1.25% AG revealed the formation of dense, 3-D neurite networks at [Col] i of 300 lg/mL, while neurons in unmodified AG and at higher [Col] i (600 lg/mL) exhibited significantly less neurite outgrowth; although, neuronal survival did not vary with [Col] i. The effect of [Col] i on acute neuronal response following high magnitude, high rate shear deformation (0.50 strain, 30 s)1 strain rate) was evaluated in 1.5% AG for [Col] i of 30, 150, and 300 lg/mL, which supported cultures with similar baseline viability and neurite outgrowth. Conjugation of Col to AG also increased the complex modulus of the hydrogel. Following high rate deformation, neuronal viability significantly decreased with increasing [Col] i , implicating cell-matrix adhesions in acute mechanotransduction events associated with traumatic loading. These results suggest interrelated roles for matrix mechanical properties and receptor-mediated cell-matrix interactions in neuronal viability, neurite outgrowth, and transduction of high rate deformation. This model system may be further exploited for the elucidation of mechanotransduction mechanisms and cellular pathology following mechanical insult.

Experimental Neurology, 2004

The mammalian central nervous system (CNS) has little capacity for self-repair after injury, and neurons are not capable of proliferating. Therefore, neural tissue engineering that combines neural stem and progenitor cells and biologically derived polymer scaffolds may revolutionize the medical approach to the treatment of damaged CNS tissues. Neural stem and progenitor cells isolated from embryonic rat cortical or subcortical neuroepithelium were dispersed within type I collagen, and the cell -collagen constructs were cultured in serum-free medium containing basic fibroblast growth factor. The collagen-entrapped stem and progenitors actively expanded and efficiently generated neurons, which developed neuronal polarity, neurotransmitters, ion channels/receptors, and excitability. Ca 2+ imaging showed that differentiation from BrdU + /TuJ1 À to BrdU À /TuJ1 + cells was accompanied by a shift in expression of functional receptors for neurotransmitters from cholinergic and purinergic to predominantly GABAergic and glutamatergic. Spontaneous postsynaptic currents were recorded by patch-clamping from precursor cell-derived neurons and these currents were partially blocked by 10-AM bicuculline, and completely blocked by additional 10 AM of the kainate receptor antagonist CNQX, indicating an appearance of both GABAergic and glutamatergic synaptic activities. Staining with endocytotic marker FM1-43 demonstrated active synaptic vesicle recycling occurring among collagen-entrapped neurons. These results show that neural stem and progenitor cells cultured in 3D collagen gels recapitulate CNS stem cell development; this is the first demonstration of CNS stem and progenitor cell-derived functional synapse and neuronal network formation in a 3D matrix. The proliferative capacity and neuronal differentiating potential of neural progenitors in 3D collagen gels suggest their potential use in attempts to promote neuronal regeneration in vivo.

ACS Applied Bio Materials, 2019

This work describes for first time the fabrication and characterization of multicomponent interpenetrating networks composed of collagen I, hyaluronic acid, and poly(ethylene glycol) diacrylate for the 3D culture of human neural stem cells, astrocytes, and microglia. The chemical composition of the scaffolds can be modulated while maintaining values of complex moduli within the range of the mechanical performance of brain tissue (≈ 6.9kPa) and having cell viability exceeding 84%. The developed scaffolds are a promising new family of biomaterials that can potentially serve as 3D in vitro models for studying the physiology and physiopathology of the central nervous system.

Folia Neuropathologica, 2017

The biomimetic, standardized conditions for in vitro cultures of human neural progenitors derived from induced pluripotent stem cells (hiPSC-NPs) should meet the requirements to serve as the template and protective environment for therapeutically competent cell population. In this study, two different collagen scaffolds: bi-component consisting of collagen and chondroitin sulphate (Col-CS), and collagen modified by crosslinking agent 2,3-dialdehyde cellulose (Col-DAC) have been used for the first time to encapsulate hiPSC-NPs and compared for the ability to create permissive microenvironment enabling cell survival, growth and differentiation. In our previous report, physicochemical comparison of the scaffolds revealed different elasticity, and diverse size and distribution of the pores within the 3D structure. Binary systems of Col-CS and Col-DAC tested in the current study have the correct balance of properties to serve as a biomimetic niche: they accommodate hiPSC-NPs sustaining their ability to proliferate and differentiate into neural lineages. However, a dense, network structure and rounded in shape pores of the Col-DAC microenvironment resulted in differential cell distributions within the scaffolds, with a tendency for augmented formation of highly proliferating cell aggregates as compared to Col-CS scaffolds. In contrast, Col-CS, which exhibited formation of the network of ellipsoidal and inner interconnected parallel pore channels, promoted enhanced cell viability and neuronal differentiation.

Annals of Biomedical Engineering, 2014

Electrical and chemical stimulation have been studied as potent mechanisms of enhancing nerve regeneration and wound healing. However, it remains unclear how electrical stimuli affect nerve growth, particularly in the presence of neurotrophic factors. The objective of this study was to explore (1) the effect of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) supplementation to support neurite outgrowth in a 3D scaffold, and (2) the effect of brief, low voltage, electrical stimulation (ES) on neurite outgrowth prior to neurotrophin supplementation. Dissociated E11 chick dorsal root ganglia (DRG) were seeded within a 1.5 mg/mL type-I collagen scaffold. For neurotrophin treatments, scaffolds were incubated for 24 hrs in culture media containing nerve growth factor (NGF, 10 ng/mL) or BDNF (200 ng/mL), or both. For ES groups, scaffolds containing neurons were stimulated for 10 min at 8-10 V/m DC, then incubated for 24 hrs with neurotrophin. Fixed and labeled neurons were imaged to measure neurite growth and directionality. BDNF supplementation was not as effective as NGF at supporting DRG neurite outgrowth. ES prior to NGF supplementation improved DRG neurite outgrowth compared to NGF alone. This combination of brief ES with NGF treatment was the most effective treatment compared to NGF or BDNF alone. Brief ES had no impact on neurite directionality in the 3D scaffolds. These results demonstrate that ES improves neurite outgrowth in the presence of neurotrophins, and could provide a potential therapeutic approach to improve nerve regeneration when coupled with neurotrophin treatment.

Biotechnology and Bioengineering, 2009

We have designed and developed a microfluidic system to study the response of cells to controlled gradients of mechanical stiffness in 3D collagen gels. An ‘H’-shaped, source–sink network was filled with a type I collagen solution, which self-assembled into a fibrillar gel. A 1D gradient of genipin—a natural crosslinker that also causes collagen to fluoresce upon crosslinking—was generated in the cross-channel through the 3D collagen gel to create a gradient of crosslinks and stiffness. The gradient of stiffness was observed via fluorescence. A separate, underlying channel in the microfluidic construct allowed the introduction of cells into the gradient. Neurites from chick dorsal root ganglia explants grew significantly longer down the gradient of stiffness than up the gradient and than in control gels not treated with genipin. No changes in cell adhesion, collagen fiber size, or density were observed following crosslinking with genipin, indicating that the primary effect of genipin was on the mechanical properties of the gel. These results demonstrate that (1) the microfluidic system can be used to study durotactic behavior of cells and (2) neurite growth can be directed and enhanced by a gradient of mechanical properties, with the goal of incorporating mechanical gradients into nerve and spinal cord regenerative therapies. Biotechnol. Bioeng. 2009;102: 632–643. © 2008 Wiley Periodicals, Inc.