Biologic scaffolds composed of central nervous system extracellular matrix

Abstract

Acellular biologic scaffolds are commonly used to facilitate the constructive remodeling of three of the four traditional tissue types: connective, epithelial, and muscle tissues. However, the application of extracellular matrix (ECM) scaffolds to neural tissue has been limited, particularly in the central nervous system (CNS) where intrinsic regenerative potential is low. The ability of decellularized liver, lung, muscle, and other tissues to support tissue-specific cell phenotype and function suggests that CNS-derived biologic scaffolds may help to overcome barriers to mammalian CNS repair. A method was developed to create CNS ECM scaffolds from porcine optic nerve, spinal cord, and brain, with decellularization verified against established criteria. CNS ECM scaffolds retained neurosupportive proteins and growth factors and, when tested with the PC12 cell line in vitro, were cytocompatible and stimulated proliferation, migration, and differentiation. Urinary bladder ECM (a non-CNS ECM scaffold) was also cytocompatible and stimulated PC12 proliferation but inhibited migration rather than acting as a chemoattractant over the same concentration range while inducing greater rates of PC12 differentiation compared to CNS ECM. These results suggest that CNS ECM may provide tissue-specific advantages in CNS regenerative medicine applications and that ECM scaffolds in general may aid functional recovery after CNS injury.

Fig. 1

Biologic scaffolds derived from porcine CNS tissues. (A) Native optic nerve tissue (left) and optic nerve ECM (right). (B) Native spinal cord tissue (left) and spinal cord ECM (right). (C) Native brain tissue (left) and brain ECM (right). All tissues and ECM are shown in their lyophilized state. Ruler divisions are 1.0 mm.

Fig. 2

Characterization of residual DNA in CNS ECM scaffolds. After H&E staining, cell nuclei were visible in (A) native optic nerve tissue but not in (B) optic nerve ECM. Cell nuclei were also visible in (C) native spinal cord tissue but not in (D) spinal cord ECM and in (E) native brain tissue but not in (F) brain ECM. DAPI showed similar results in (G) native optic nerve compared to (H) optic nerve ECM, (J) native spinal cord compared to (K) spinal cord ECM, and (L) native brain compared to (M) brain ECM. Magnification is 100×. (N) Gel electrophoresis showed that residual DNA fragments did not exceed 200 bp in optic nerve ECM, spinal cord ECM, or brain ECM. Arrows denote (left-right) 1500 bp, 1000 bp, 500 bp, and 200 bp (red arrows). (P–R) DNA quantification showed lower concentrations of DNA in CNS ECM compared to native tissues, with <50 ng DNA per mg ECM dry weight for decellularized optic nerve, spinal cord, and brain (note y axis is log scale; n = 3 for all groups; p < 0.005 for each native-ECM pair). Red lines denote established DNA criteria against which decellularization was verified [31]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Protein content of CNS ECM scaffolds. Myelin was present in (A) native optic nerve tissue, (B) optic nerve ECM, (C) native spinal cord tissue, (D) spinal cord ECM, (E) native brain tissue, and (F) brain ECM as shown by luxol fast blue staining. Laminin was also present in (G) native optic nerve, (H) optic nerve ECM, (J) native spinal cord, (K) spinal cord ECM, (L) native brain, and (M) brain ECM as shown by immunohistochemistry with hematoxylin counterstain. Magnification is 100×. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Growth factor content of CNS ECM scaffolds. Optic nerve ECM, spinal cord ECM, brain ECM, and urinary bladder ECM retained detectable concentrations of (A) VEGF and (B) bFGF, which were also present in native tissues (n = 3–5). (C) Optic nerve ECM was the only matrix that retained a detectable concentration of NGF (n = 3–4).

Fig. 5

Cytocompatibility of CNS ECM scaffolds. (A) Normal viability of undifferentiated PC12 cells was not different from viability of PC12 cells cultured with (B) optic nerve ECM, (C) spinal cord ECM, (D) brain ECM, or (E) urinary bladder ECM as determined by Live/Dead assay, including (F) quantification of live and dead cells in images. Cells were cultured in each ECM at a concentration of 100 μg protein per ml medium. Magnification is 400×.

Fig. 6

Mitogenic effects of CNS ECM scaffolds. Undifferentiated PC12 cell proliferation was modulated by (A) optic nerve ECM, (B) spinal cord ECM, (C) brain ECM, and (D) urinary bladder ECM as determined by BrdU incorporation during PC12 cell mitosis. Changes in mitogenesis ranged from increases of 53% (brain ECM, 25 μg protein per ml) to decreases of 18% (optic nerve ECM, 100 μg protein per ml). * Indicates p < 0.05 by one-way ANOVA with Tukey–Kramer post hoc analysis.

Fig. 7

Chemotactic effects of CNS ECM scaffolds. Undifferentiated PC12 cell migration was modulated by (A) optic nerve ECM, (B) spinal cord ECM, (C) brain ECM, and (D) urinary bladder ECM as determined by transmembrane PC12 cell migration. Changes in chemotaxis ranged from increases of 53% (brain ECM, 100 μg protein per ml) to decreases of 50% (urinary bladder ECM, 25 μg protein per ml). * Indicates p < 0.05 by one-way ANOVA with Tukey–Kramer post hoc analysis.

Fig. 8

Differentiation effects of CNS ECM scaffolds. (A) PC12 neuronal differentiation induced by CNS and non-CNS ECM as indicated by neurite extension. Differentiation was compared using the following medium supplements: (B) PBS as a negative control, (C) optic nerve ECM at 100 μg/ml, (D) spinal cord ECM at 100 μg/ml, (E) brain ECM at 100 μg/ml, urinary bladder ECM at 100 μg/ml, or (G) bFGF at 0.20 μg/ml as a positive control. * Urinary bladder ECM at 100 μg protein per ml induced greater differentiation than any other condition including the positive control (bFGF, 0.20 μg/ml). ** Spinal cord ECM or brain ECM at 100 μg/ml induced differentiation at rates comparable to the positive control which were greater than all other conditions except urinary bladder ECM at 100 μg protein per ml. *** Optic nerve ECM at 100 μg protein per ml or urinary bladder ECM at 10 μg protein per ml induced differentiation at greater rates than the negative control (no ECM: 0 μg/ml). # Differentiation rates increased with concentration for spinal cord ECM, brain ECM, and urinary bladder ECM (10 μg protein per ml vs. 100 μg protein per ml). Significant differences were determined between all groups shown by one-way ANOVA with Tukey–Kramer post hoc analysis (p < 0.05).