Biologic scaffolds composed of central nervous system extracellular matrix
Introduction
The extracellular matrix (ECM) represents the secreted product of the resident cells of each tissue and organ and thus logically defines the ideal substrate or scaffold for maintenance of tissue-specific cell phenotype. The ECM is a critical determinant of cell behavior and is known to affect intracellular signaling pathways, cell differentiation events, and cell proliferation among other important characteristics of tissue identity [1], [2], [3], [4], [5], [6], [7], [8]. These events are mediated through integrins and other cell surface receptors in response to ligands present within the ECM of every tissue [9], [10], [11]. Subtle changes in ECM structure and mechanical properties can affect cell transcriptional events and associated cell phenotype and function [12], [13].
Biologic scaffolds composed of ECM have been commonly used for the therapeutic reconstruction of many tissues including myocardium [14], [15], [16], kidney [17], lower urinary tract [18], [19], musculotendinous tissues [20], [21], [22], esophagus [23], and peripheral nerve [24], among others. There is clinical precedent for the application of ECM scaffolds in reconstruction of central nervous system (CNS) structures [25], [26], but the development of ECM scaffolds for CNS regenerative medicine strategies has received relatively scarce attention [27], [28], [29]. It has been suggested that ECM harvested from specific tissues is the preferred substrate for cells native to those respective tissues if maintenance of phenotypic characteristics is important [3], [4], [5], [6], [7], [8], [30]. The methods by which ECM scaffolds are prepared vary greatly and such methods can markedly affect the composition, architecture, and material properties of the resulting construct [31], [32], [33], [34] as well as the host response following implantation [35], [36], [37], [38]. Therefore, the methods of preparing ECM scaffolds intended for use in the repair and reconstruction of complex vital tissues such as heart, liver, kidney, and the CNS must be carefully considered as regenerative medicine strategies are developed for these tissues and organs.
The objectives of the present study were to (1) develop a method for decellularization of a variety of CNS tissues, (2) characterize the resulting CNS ECM scaffolds in terms of composition and in vitro cytocompatibility, and (3) investigate potential tissue-specific advantages of CNS ECM scaffolds compared to non-CNS ECM scaffolds by evaluating in vitro modulation of PC12 cell line mitogenesis, chemotaxis, and differentiation.
Section snippets
Preparation of CNS ECM
Porcine optic nerve, spinal cord, and brain tissues were obtained from animals (∼120 kg) at a local abattoir (Thoma's Meat Market, Saxonburg, PA). Tissues were frozen (>16 h at −80 °C), thawed completely, and separated from all non-CNS tissue. Dura mater was removed, and optic nerve and spinal cord tissues were longitudinally quartered and cut into lengths (<3 cm). The decellularization process consisted of a series of agitated baths: water (type I reagent water per ASTM D1193; 16 h at 4 °C;
Efficacy of decellularization method
No residual nuclei were visible in H&E and DAPI images of ECM derived from optic nerve, spinal cord, or brain (Fig. 2A–M). Maximum fragment size of residual DNA in CNS ECM scaffolds did not exceed 200 bp (Fig. 2N) [31]. Quantification of dsDNA using PicoGreen showed that CNS ECM scaffolds retained <50 ng dsDNA per mg dry ECM (Fig. 2P–R). Concentrations of dsDNA were 44.6 ± 7.9 ng/mg in optic nerve ECM, 37.9 ± 7.7 ng/mg in spinal cord ECM, and 40.2 ± 3.8 ng/mg in brain ECM.
CNS ECM constituents
Histologic staining
Discussion
This study describes a versatile decellularization method which can be applied to three different CNS tissues: optic nerve, spinal cord, and brain (Fig. 1). The full protocol from tissue to ECM requires <24 h, a duration which compares favorably to previously reported CNS tissue decellularization methods [27], [28], [29]. The resulting matrix is sufficiently acellular (Fig. 2) to obviate adverse host immune responses [35], [36], [37], [38] and contagion such as virus transmission [47], [48],
Conclusion
A variety of acellular biologic scaffolds can be derived from CNS tissues such as optic nerve, spinal cord, and brain by a combination of enzymatic and chemical processing (Fig. 1, Fig. 2). These CNS ECM scaffolds meet or exceed established decellularization criteria [31], are cytocompatible (Fig. 5), and retain neurosupportive proteins and growth factors present within the tissues of origin (Fig. 3, Fig. 4) which are known to modify neural cell behaviors. The resultant acellular biologic
Acknowledgments
This work was supported in part by the NIH (5R01AR053603), 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), and an NIBIB training grant (T32EB001026). The authors also thank Deanna Rhoads for histologic sectioning and Chris Carruthers for experimental assistance.
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These authors contributed equally to this work.