Hydrogels derived from central nervous system extracellular matrix
Introduction
Biologic scaffolds composed of extracellular matrix (ECM) can facilitate the constructive remodeling of numerous tissues including esophagus [1], [2], lower urinary tract [3], [4], muscle and tendon [5], [6], and myocardium [7], [8], among others. Although the mechanisms by which ECM scaffolds promote a constructive and functional remodeling response are only partially understood, recruitment of endogenous multipotent progenitor cells [9], [10], modulation of the innate immune response [11], [12], scaffold degradation with the generation of bioactive molecular cues [13], [14], [15], and innervation [16] have all been shown to be important events in this process. The contribution of the innate three-dimensional ultrastructure, unique surface ligand distribution, or molecular composition to constructive, functional remodeling is largely unknown. However, hydrogel formulations of matrix scaffolds lack the native three-dimensional ultrastructure of the source tissue but still possess in vitro and in vivo biologic activity [17], [18], [19], [20], [21], [22], suggesting that the molecular composition of these materials is an active factor in remodeling events. There have also been reports that suggest tissue-specific biologic scaffold materials have properties that enhance greater site-appropriate phenotypic cell differentiation compared to ECM scaffolds derived from non-homologous tissue sources [23], [24], [25], [26].
The use of biologic scaffold materials within either the central or peripheral nervous system has not been extensively investigated [27]. However, it has been shown that innervation of remodeled scaffold materials is an early event when such materials are placed in several different anatomic locations and represents a predictor of constructive and functional outcomes [16], [28], [29]. It has also been shown that innervation is a critical event in robust regenerative responses that occur in species such as the newt and axolotl [30], [31], [32]. Methods for the isolation of central nervous system (CNS) ECM have recently been described. The objectives of the present study were to develop a method to create hydrogel forms of brain and spinal cord ECM, examine the biomolecular composition and mechanical properties of the resulting hydrogels, and evaluate the in vitro neural cytocompatibility and neurotrophic potential of CNS-ECM hydrogels versus a hydrogel prepared from a non-CNS-ECM; specifically, porcine urinary bladder matrix.
Section snippets
Overview of experimental design
Following decellularization of porcine brain and spinal cord, the resulting brain and spinal cord ECM (B-ECM and SC-ECM, respectively) were solubilized. The ECM materials were analyzed for collagen and sulfated glycosaminoglycan content, ultrastructure, and hydrogel mechanical properties. A commonly used neural cell line for examining neurite extension, N1E-115 [33], [34], was used to identify the neurotrophic potential of ECM hydrogels in two- and three-dimensional culture. The results were
Collagen and sGAG quantification
Collagen concentration of B-ECM was 537.5 ± 26.9 μg collagen/mg dry weight, which was less than SC-ECM and UBM-ECM at 703.2 ± 47.3 and 702.5 ± 113.5 μg collagen/mg dry weight, respectively (p < 0.01) (Fig. 1A). B-ECM and UBM-ECM had a higher sGAG concentration, 5.1 ± 1.4 (p < 0.009) and 4.4 ± 0.4 (p < 0.02) μg sGAG/mg dry weight, respectively, compared to SC-ECM, which was 1.3 ± 0.9 μg sGAG/mg dry weight (Fig. 1B).
Qualitative assessment
B-ECM, SC-ECM, and UBM-ECM pre-gel solutions polymerized to form a hydrogel at
Discussion
The present study shows that biologic scaffolds derived from porcine brain and spinal cord can be processed to form hydrogels that retain selected ECM-specific constituents. At comparable ECM concentrations, these hydrogel forms of CNS-ECM have distinctive composition and biomechanical properties. Furthermore, CNS-ECM hydrogels are cytocompatible, promote N1E-115 cell differentiation, and support three-dimensional neurite extension.
The mechanical properties of SC-ECM hydrogels are similar to
Conclusions
B-ECM and SC-ECM hydrogels, while derived by similar decellularization methods from their source tissue, each have a unique biochemical composition, mechanical properties, and neurotrophic potential. The increase in neurite length for N1E-115 cells in response to B-ECM suggests a tissue-specific effect of B-ECM hydrogels on a brain derived cell line. Each ECM elicited unique cell responses as demonstrated by neurotrophic potential in their solubilized form and support of considerable
Acknowledgments
Christopher Medberry was partially supported by the NIH-NHLBI training grant (T32-EB001026) entitled “Cellular Approaches to Tissue Engineering and Regeneration.” Peter Crapo was partially supported by an Ocular Tissue Engineering and Regenerative Ophthalmology (OTERO) Fellowship from the Louis J. Fox Center for Vision Restoration (a joint program of UPMC and the University of Pittsburgh). Matthew Wolf was partially supported by the NIH-NHLBI training grant (T32-HL76124-6) entitled
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These authors contributed equally to this work.