Intrafibrillar silicification of collagen scaffolds for sustained release of stem cell homing chemokine in hard tissue regeneration

Abstract

Traditional bone regeneration strategies relied on supplementation of biomaterials constructs with stem or progenitor cells or growth factors. By contrast, cell homing strategies employ chemokines to mobilize stem or progenitor cells from host bone marrow and tissue niches to injured sites. Although silica-based biomaterials exhibit osteogenic and angiogenic potentials, they lack cell homing capability. Stromal cell-derived factor-1 (SDF-1) plays a pivotal role in mobilization and homing of stem cells to injured tissues. In this work, we demonstrated that 3-dimensional collagen scaffolds infiltrated with intrafibrillar silica are biodegradable and highly biocompatible. They exhibit improved compressive stress-strain responses and toughness over nonsilicified collagen scaffolds. They are osteoconductive and up-regulate expressions of osteogenesis- and angiogenesis-related genes more significantly than nonsilicified collagen scaffolds. In addition, these scaffolds reversibly bind SDF-1α for sustained release of this chemokine, which exhibits in vitro cell homing characteristics. When implanted subcutaneously in an in vivo mouse model, SDF-1α-loaded silicified collagen scaffolds stimulate the formation of ectopic bone and blood capillaries within the scaffold and abrogate the need for cell seeding or supplementation of osteogenic and angiogenic growth factors. Intrafibrillar-silicified collagen scaffolds with sustained SDF-1α release represent a less costly and complex alternative to contemporary cell seeding approaches and provide new therapeutic options for in situ hard tissue regeneration.

Figure 1.

A) Effect of pH value of the incubating medium on the release of silicic acid from the SCSs. B) Effect of collagen silicification on the degradation of CSs. Each CS was immersed in 5.2 ml of PBS containing 0.1 mg/ml of collagenase derived from Clostridium histolyticum. C) Unstained TEM image of an SCS with localized loss of intrafibrillar silica (arrows) after immersion in PBS at pH 7.4. D) Unstained TEM image showing degradation of a collagen fibril (open arrowheads) along the periphery of a SCS. Note that part of the same fibril (right side of image) remained silicified. Partial loss of intrafibrillar silica can also be observed (arrow) in some collagen fibrils within the scaffold. Scale bars = 100 nm.

Figure 2.

Evaluation of biomechanical properties. A–D) Compressive stress-strain curves obtained for the 4 groups of CSs: CS (A), SCS (B), SCS in PBS for 28 d (SCS-P; C), and SCS in PBS for 28 d and collegenase for 7 d (SCS-C; D). E) Tangent moduli of the 4 groups of CSs. Groups with the same uppercase letter designations are not statistically significant (P>0.05). F) Modulus of resilience of the 4 groups of CSs. Groups with the same lowercase letter designations are not statistically significant (P>0.05). Note that data from the CS group were not included in the statistical analyzes.

Figure 3.

In vitro bioactivity of an SCS after immersion in simulated body fluid, as demonstrated by STEM-EDX analysis. Top left panel: darkfield image of a collagen leaflet (consisting of multiple collagen fibrils) within the scaffold. Needle-shaped crystals were deposited along the periphery of the collagen leaflet. (High-resolution brightfield images can be found in Supplemental Fig. S2A). Top right, middle, and bottom left panels: elemental mappings of the distribution of oxygen (top right panel), silicon (middle left panel), calcium (middle right panel), and phosphorus (bottom left panel) within and around the collagen leaflet. Bottom right panel: bright magnification, merged distributions of calcium, phosphorus and silicon. Banded appearance of the collagen fibrils was reproduced by the hierarchical distribution of intrafibrillar silicon (arrow).

Figure 4.

A) Results of MTT assays. Mitochondrial succinic dehydrogenase activities were normalized against the Teflon negative control, which was taken to be 100%. B) Summary of the combined percentage of early and late apoptotic cells in flow cytometry test. For the MSCs, groups identified with the same uppercase letters are not statistically significant (P>0.05). For the EPCs, groups identified with the same lowercase letters are not statistically significant (P>0.05). PMMA, polymethyl methacrylate.

Figure 5.

A) Alizarin red S staining of extracellular bone nodules produced by differentiated MDCs after they were exposed to the Teflon control (top left panel), CSs (top right panel), and SCSs (bottom left panel) for 72 h. Bar chart (bottom right panel) summarizes the results of in vitro osteogenesis. Groups designated by the same uppercase letters are not statistically significant (P>0.05). B) Tube formation by EPCs represents a simple model of angiogenesis in which induction or inhibition of tube formation by exogenous signals may be monitored. Representative light microscopy images show EPCs after they were exposed to extracts derived from the Teflon control for 24 h (top left panel), CSs for 24 h (top right panel), and SCSs (bottom left panel) for 24 h. Scale bars = 100 μm. Bar chart (bottom right panel) summarizes the results of in vitro proangiogenesis. Extracts from SCSs were examined after the immersion of these scaffolds in phosphate-buffered saline (pH 7.4) for different time periods (1, 6, 12, and 24 h). Groups designated by the same numeric descriptors are not statistically significant (P>0.05).

Figure 6.

A) Release kinetics of rmSDF-1α after binding of different concentrations of the chemokine to silicified CSs. Two-phase exponential association models provided an excellent fit for the relationship between the cumulative rmSDF-1α release and rmSDF-1α concentration in each isotherm. Each model is characterized by an initial period of burst release (Y_max1, with rate constant K₁) that is followed by a period of slow, sustained release (Y_max2, with rate constant K₂). Values of these parameters and the corresponding half-lives are summarized in the bottom panel. B) Ability of rmSDF-1α-loaded SCSs to act as a chemoattractant for MSCs or EPCs. Representative crystal violet-stained images of MSCs (top left panel) and EPCs (top right panel) that migrated through the pores (arrowhead) of the transwell membrane to the bottom of the transwell. Scale bars = 100 μm. Bar chart (bottom panel) summarizes the in vitro cell homing results. NC, negative control (no SCS or rmSDF-1α); PC, positive control (no SCS, 100 ng/ml rmSDF-1α dissolved in culture medium); SCS-0 to SCS-1000, rmSDF-1α-loaded SCSs with relative concentrations of 0 to 1000 ng/ml. For MSCs, groups designated by the same uppercase letters are not statistically significant (P>0.05). For EPCs, groups designated by the same lowercase letters are not statistically significant (P>0.05).

Figure 7.

Light microscopy images of hematoxylin-eosin-stained sections showing ectopic bone and blood capillary vessel formation in a mouse subcutaneous implantation model. A) Cell seeding approach using an SCS loaded with murine MSCs. B) Cell homing approach using an rmSDF-1α-loaded SCS. Top panels: ×20 view; scale bars =100 μm. Bottom panels: ×40 view; scale bars = 50 μm. Solid arrowheads indicate remnant silicified collagen leaflets derived from the implanted scaffolds. Arrows indicate osteocyte-like cells within newly formed bone trabeculae. Pointers indicate capillaries containing red blood cells.