Micropore-induced capillarity enhances bone distribution in vivo in biphasic calcium phosphate scaffolds

Abstract

The increasing demand for bone repair solutions calls for the development of efficacious bone scaffolds. Biphasic calcium phosphate (BCP) scaffolds with both macropores and micropores (MP) have improved healing compared to those with macropores and no micropores (NMP), but the role of micropores is unclear. Here, we evaluate capillarity induced by micropores as a mechanism that can affect bone growth in vivo. Three groups of cylindrical scaffolds were implanted in pig mandibles for three weeks: MP were implanted either dry (MP-Dry), or after submersion in phosphate buffered saline, which fills pores with fluid and therefore suppresses micropore-induced capillarity (MP-Wet); NMP were implanted dry. The amount and distribution of bone in the scaffolds were quantified using micro-computed tomography. MP-Dry had a more homogeneous bone distribution than MP-Wet, although the average bone volume fraction, BVF‾, was not significantly different for these two groups (0.45±0.03 and 0.37±0.03, respectively). There was no significant difference in the radial bone distribution of NMP and MP-Wet, but the BVF‾, of NMP was significantly lower among the three groups (0.25±0.02). These results suggest that micropore-induced capillarity enhances bone regeneration by improving the homogeneity of bone distribution in BCP scaffolds. The explicit design and use of capillarity in bone scaffolds may lead to more effective treatments of large and complex bone defects.

Statement of significance: The increasing demand for bone repair calls for more efficacious bone scaffolds and calcium phosphate-based materials are considered suitable for this application. Macropores (>100μm) are necessary for bone ingrowth and vascularization. However, studies have shown that microporosity (<20μm) also enhances growth, but there is no consensus on the controlling mechanisms. In previous in vitro work, we suggested that micropore-induced capillarity had the potential to enhance bone growth in vivo. This work illustrates the positive effects of capillarity on bone regeneration in vivo; it demonstrates that micropore-induced capillarity significantly enhances the bone distribution in the scaffold. The results will impact the design of scaffolds to better exploit capillarity and improve treatments for large and load-bearing bone defects.

Keywords: Bone regeneration; Calcium phosphate scaffold; Capillary action; Micro-computed tomography; Microporosity.

Figure 1. Scaffold macro- and microstructure.

A. Photograph of a BCP scaffold used in this study. Scaffolds were 8 mm diameter and consisted of alternating layers of orthogonal rods. B. Schematic showing a lattice of scaffold rods. So-called macropores make the space between the rods. C. Scanning electron micrographs of rod microstructure. Rods were either microporous (MP) or solid and non-microporous (NMP).

Figure 2. Tissue and cells in the scaffold macro- and micropores.

Histology slices were taken at the center of the scaffolds. Samples were stained with Stevenel’s blue and counterstained with picro-fuchsin. Bone was pink/red; soft tissue, osteoid, cell cytoplasm were light blue; and cell nuclei were dark blue. Mineralized bone (b) was observed in macropores between scaffold rods (s) for all three groups. Fibrous soft tissue (st) was prevalent in the center of NMP scaffolds (E). Osteoblast-like cells (➙) lined mineralized bone in the macropores (B, D). Osteocytes (➞) were in lacunae (B, D, F). Osteoclast-like cells (⇾) were found on rod and bone surfaces in some areas (D). In the macropores of MP scaffolds, mineralized bone is anchored in rods (B, D, insets). In NMP, bone is not anchored (F, inset). Soft tissue in MP-Wet and NMP is contracted and frequently not in contact with the rods (C- E).

Figure 3. Blood vessel in macropore and scaffold microstructure after implantation.

(A-C) for MP-dry, (D) for MP-Wet and (E) for NMP. A blood vessel in a macropore (A1) confirms that vasculature develops in implanted scaffolds. Red blood cells inside the blood vessel appear aggregated and without nuclei. Blue-stained cells, which are not identified, are visible within the micropores of rods (A-D). In contrast, there are no cells in the rods of NMP which does not have micropores (E). In MP-Dry, some cells appear in aggregates and are uniformly dark blue (►) with no cell nucleus (B, C). They conform to the shape of surrounding cells and the pore walls (B1). Other cells in micropores are predominantly isolated, show an apparent nucleus, and stain lighter blue (▻). In some micropores, cells are surrounded by bone (B2). Osteoclast-like cells (⇾), characterized by their larger size and multiple nuclei, are on bone (A) and rod surfaces (B, D, E).

Figure 4. Representative image showing results from the automated segmentation algorithm used to quantify bone ingrowth in scaffold macropores.

A. Original 2D micro-CT image from a 3D z-stack of images corresponding to a MP-Dry scaffold. In this image, we see horizontal rods from one scaffold layer. Mineralized bone is in the macropore space between the scaffold rods. B. Label matrix resulting from the segmentation of A. Scaffold pixels are white, bone pixels are light gray and soft tissue or background pixels are dark gray.

Figure 5. Average bone volume fraction in the scaffolds, BVF¯.

Both types of MP scaffolds had a significantly higher $\overline{\mathit{\text{BVF}}}$ than NMP (p < 0.05). There was no difference in $\overline{\mathit{\text{BVF}}}$ between MP-Dry and MP-Wet (p = 0.10).

Figure 6. Quantitative evaluation of the bone distribution.

A. Average heatmaps of bone volume fraction. The BVF(x,y) values for MP-Dry are qualitatively more homogeneous throughout the heatmap compared to MP-Wet and NMP. Both MP-Wet and NMP have a distinct central region with less bone, while the central region is less distinct for MP-Dry. B. Bone growth front. The bone growth front for each individual sample is shown in gray and the average for each group is in red. The growth front in MP-Dry extends the furthest to the center and shows less variability compared to MP-Wet and NMP. The front is significantly closer to the center in MP-Dry than in MP-Wet and NMP (p < 0.01). There is no difference in the depth of the growth front between MP-Wet and NMP. C. Radial bone volume fraction, $\mathit{\text{BVF}}(\tilde{r}).$ For all groups, $\mathit{\text{BVF}}(\tilde{r})$ is higher at the scaffold-defect edge than in the center of the scaffold. The dashed lines correspond to the exponential fits given by Equation (1) and Table 1. MP-Wet and NMP have a larger discrepancy in $\mathit{\text{BVF}}(\tilde{r})$ between the periphery and the center as compared to MP-Dry. D. Average root-mean-square deviation, RMSD, of $\mathit{\text{BVF}}(\tilde{r})/\overline{\mathit{\text{BVF}}}$ from 1 across all $\tilde{r}$. RMSD is significantly less (p > 0.05) for MP-Dry than for MP-Wet and NMP. There is no difference between MP-Wet and NMP. Therefore, $\mathit{\text{BVF}}(\tilde{r})$ is closer to $\overline{\mathit{\text{BVF}}}$ for MP-Dry compared to MP-Wet and NMP.