Biomimetic oyster shell-replicated topography alters the behaviour of human skeletal stem cells

Abstract

The regenerative potential of skeletal stem cells provides an attractive prospect to generate bone tissue needed for musculoskeletal reparation. A central issue remains efficacious, controlled cell differentiation strategies to aid progression of cell therapies to the clinic. The nacre surface from Pinctada maxima shells is known to enhance bone formation. However, to date, there is a paucity of information on the role of the topography of P. maxima surfaces, nacre and prism. To investigate this, nacre and prism topographical features were replicated onto polycaprolactone and skeletal stem cell behaviour on the surfaces studied. Skeletal stem cells on nacre surfaces exhibited an increase in cell area, increase in expression of osteogenic markers ALP (p < 0.05) and OCN (p < 0.01) and increased metabolite intensity (p < 0.05), indicating a role of nacre surface to induce osteogenic differentiation, while on prism surfaces, skeletal stem cells did not show alterations in cell area or osteogenic marker expression and a decrease in metabolite intensity (p < 0.05), demonstrating a distinct role for the prism surface, with the potential to maintain the skeletal stem cell phenotype.

Keywords: Nacre; bone regeneration; osteogenic differentiation; skeletal stem cell; topography.

Figure 1.

Schematic representation of biofabrication of PCL topographical surfaces. (1) Polydimethylsiloxane (PDMS) was poured on shell and heat cured at 60°C for 60 min. The mould was then removed from the shell. (2) Polycaprolactone (PCL) beads were washed in methanol and left to air dry. PCL beads were then melted at 80°C and PDMS mould transferred to molten PCL and features replicated using hot embossing. At this point, flat control PCL surfaces were generated using glass slides. When the PCL had cooled, the PDMS stamp was removed. (3) PCL topographical surfaces were prepared for cell culture with plasma treatment. (4) SEM images of PCL-replicated nacre and prism topographies. To prepare for SEM, surfaces were sputter coated with 7 nm platinum.

Figure 2.

Morphology of SSCs cultured on nacre, prism and flat topographical surfaces. Cells were stained with CellTracker Green and imaged at (a–c) day 4 and (d–f) day 7. Mean cell area (μm²) was calculated at day 4 and day 7 (g), (n = 3 patients – females aged 51, 52 and 56), *p < 0.05; **p < 0.01.

Figure 3.

Changes in osteogenic mRNA and protein on shell topographies. (a–e) Expression of RUNX2, ALP, COL1A1, OCN and OPN mRNA in SSC cultured on nacre, prism and flat osteogenic media (flat OM) topographies over 28 days. n = 3 patient samples, *p < 0.05; **p < 0.01; ***p < 0.001; ⁺p < 0.0001. (f) Immunofluorescence of OPN on days 14 and 21 on flat, nacre and prism topographies. Negative control is SSCs stained in the absence of anti-OPN antibody, cultured on standard commercial tissue culture plastic. White arrows indicate area of OPN protein. Scale bar = 100 μm.

Figure 4.

Metabolomic analysis of SSC culture on nacre and prism shell topographies. Intensity ratio, relative to flat control, of (a and d) total metabolites, (b and e) carbohydrate-associated metabolites, and (c and f) lipid-associated metabolites for (a–c) day 10 and (d–f) day 21. Flat OM represents flat surface cultured in osteogenic media. Peak intensity ratio normalised to flat control, n = 3 replicates. PCA plots for (g) day 10 and (h) day 21.

Figure 5.

Nucleotide metabolism–associated canonical pathways for SSCs cultured on nacre, prism and control OM surfaces on days 10 and 21. IPA-generated canonical pathways associated with nucleotide metabolism for SSC on day 10 on (a) nacre topographies, (b) prism topographies and (c) flat osteogenic media control. Canonical pathways on day 21 on (d) nacre topographies, (e) prism topographies and (f) flat osteogenic media control. Shown in either red or green and on the numbers to the right of each bar was the number of metabolites identified in the sample out of all possible known associated metabolites. Threshold considered for the analysis was – log(p value) > 1.5. Legend on bottom left.