Slipper Limpet (Crepidula fornicata) Shells Support In Vitro Osteogenesis of Human Adipose-Derived Stem Cells

Abstract

This study aimed to investigate a cost-effective alternative to man-made calcium phosphate ceramics for treating bone defects. The slipper limpet is an invasive species in European coastal waters, and its shells composed of calcium carbonate could potentially be a cost-effective source of bone graft substitutes. This research analyzed the mantle of the slipper limpet (Crepidula fornicata) shells to enhance in vitro bone formation. Discs machined from the mantle of C. fornicata were analyzed using scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS), X-ray crystallography (XRD), Fourier-transform infrared spectroscopy (FT-IR) and profilometry. Calcium release and bioactivity were also studied. Cell attachment, proliferation, and osteoblastic differentiation (RT-qPCR and alkaline phosphatase activity) were measured in human adipose-derived stem cells grown on the mantle surface. The mantle material was mainly composed of aragonite and showed a sustained Ca²⁺ release at physiological pH. In addition, apatite formation was observed in simulated body fluid after three weeks, and the materials supported osteoblastic differentiation. Overall, our findings suggest the mantle of C. fornicata shows potential as a material for fabricating bone graft substitutes and structural biomaterials for bone regeneration.

Keywords: Crepidula fornicata; calcium carbonate; mantle; mesenchymal stem cells; osteogenesis; slipper limpet shells.

Figure 1

(a) SEM image of C. fornicata outside surface (scale bar 2 μm), showing interlocking plates. (b) EDS compositional analysis of limpet shells, showing the elemental composition of C. fornicata shells.

Figure 2

X-ray diffraction patterns of Crepidula fornicata’s powder, showing the presence of aragonite.

Figure 3

FT-IR spectrum of C. fornicata powder, containing aragonite-CaCO₃.

Figure 4

Cumulative calcium release profile from C. fornicata’s discs in PBS, at 37 °C, over a period of 7 days. Each data point represents the mean ± SD (n = 3).

Figure 5

(a) Representative profilometry analysis of the mantle surface of C. fornicata discs, showing a micro-meter scale roughness. (b) Contact angle quantitative analysis of C. fornicata discs with and without coating with FBS (n = 3). Comparison between groups was assessed by t-test (*** p = 0.0001).

Figure 6

SEM images of C. fornicata surface after immersion in either deionized water (a) or SBF (b) for 21 days. The scale bar is 10 µm. (c) Percentage surface co-deposition of calcium and phosphorus as calculated by Imagej from SEM images, showing a significant increase in apatite formation. Results are reported as mean ± SD (n = 3). A t-test revealed the statistical difference between samples in SBF and controls (** p = 0.0026). (d). Representative EDS spectrum of the area highlighted by a square in (b).

Figure 7

Cell proliferation of hAdMSC to Crepidula fornicata shells. Cell proliferation was determined by a resazurin assay. Data are reported as a MEAN ± SD (n = 4). Two-way ANOVA returned p < 0.05 (* p < 0.05 and ** p < 0.01).

Figure 8

SEM images of hAdMSC grown on coverslips in either DMEM for 7 (a) and 21 days (b) or osteogenic medium for 7 (c) and 21 days (d) or on shells with DMEM for 7 (e) and 21 days (f) or with Osteogenic medium for 7 (g) and 21 days (h). Images were acquired at 20.00 kV. Arrows indicate mineral deposition on the biomaterial and controls. The scale bar is 50 µm.

Figure 9

ALP expression of hAdMSC grown on coverslips and shells at days 7 and 21. Data are reported as a MEAN ± SD (n = 4). Two-way ANOVA returned p < 0.05 (* p < 0.05, ** p < 0.01 and **** p < 0.0001).

Figure 10

Relative gene expression of RUNX2, COL1, OPN, OCN and Col1A1 of hAdMSC on shells on days 7 and 21. Data are reported as a MEAN ± SD (n = 4). Two-way ANOVA returned p < 0.05 (* p < 0.05, ** p < 0.01, ***p < 0.001 and **** p < 0.0001).

Figure 11

Fabrication procedure of C. fornicata discs. (a) Cylinders were shaped by coring the mantle of rinsed slipper limpet shells using a diamond core. (b) Cylinders had an average diameter of 5 mm and an average height of 2–3 mm. (c) Representative C. fornicata disc.