Osteogenic Differentiation of Mesenchymal Stem Cells Induced by Geometric Mechanotransductive 3D-Printed Poly-(L)-Lactic Acid Matrices

Abstract

Bone-related defects present a key challenge in orthopaedics. The current gold standard, autografts, poses significant limitations, such as donor site morbidity, limited supply, and poor morphological adaptability. This study investigates the potential of scaffold geometry to induce osteogenic differentiation of human adipose-derived stem cells (hADSCs) through mechanotransduction, without the use of chemical inducers. Four distinct poly-(L)-lactic acid (PLA) scaffold architectures-Traditional Cross (Tc), Triangle (T), Diamond (D), and Gyroid (G)-were fabricated using fused filament fabrication (FFF) 3D printing. hADSCs were cultured on these scaffolds, and their response was evaluated utilising an alkaline phosphatase (ALP) assay, immunofluorescence, and extensive proteomic analyses. The results showed the D scaffold to have the highest ALP activity, followed by Tc. Proteomics results showed that more than 1200 proteins were identified in each scaffold with unique proteins expressed in each scaffold, respectively Tc-204, T-194, D-244, and G-216. Bioinformatics analysis revealed structures with complex curvature to have an increased expression of proteins involved in mid- to late-stage osteogenesis signalling and differentiation pathways, while the Tc scaffold induced an increased expression of signalling and differentiation pathways pertaining to angiogenesis and early osteogenesis.

Keywords: adipose-derived stem cells (ADSCs); bone tissue engineering (BTE); mechanotransduction.

Figure 1

The diagrams show the 3D rendered computer-aided designs for each 3D-printed scaffold with the top view on the left and the orthogonal view on the right. (A1,A2) Traditional Cross (Tc) structure, (B1,B2) Triangle (T) structure, (C1,C2) Diamond (D) structure, and (D1,D2) Gyroid (G) structure. These four structures were printed in PLA and used for all subsequent tissue culture experiments and assays. Scale bar = 1 mm.

Figure 2

Scanning electron microscopy of each printed scaffold captured on a Zeiss EVO. This shows PLA layering and complex internal structures. (A) Traditional Cross (Tc) structure, (B) Triangle (T) structure, (C) Diamond (D) structure, and (D) Gyroid (G) structure. Magnification (20×), scale (1 mm), KeV (10.00).

Figure 3

Fluorescence imaging for the cells grown on each structure. Images were captured at 4×, 10×, and 20× magnification. The nuclei are stained with DAPI and appear in BLUE. Actin filaments are stained with phallodin-TRITC and seen in RED. (A1–A3) Traditional Cross (Tc) structure, (B1–B3) Triangle (T) structure, (C1–C3) Diamond (D) structure, and (D1–D3) Gyroid (G) structure. The white asterisks indicate the region of interest (ROI).

Figure 4

ALP activity across scaffold geometries. Error bars indicate the coefficient of variation. Time point significance: Scaffolds—T Day 3–14 (p = 0.0253), Tc Day 3–14 (p = 0.0141), and Tc Day 10–14 (p = 0.0216). Total group timepoint comparisons—Day 3 vs. Day 10 (p = 0.0423), Day 7 vs. Day 14 (0.0002), Day 3 vs. Day 14 (p = 0.0002). * p < 0.05.

Figure 5

Venn diagram displaying the breakdown of unique and shared proteins across the four structures.

Figure 6

(A) Gene ontology biological process analysis of proteins expressed across captured proteomes shows a large contingent of proteins linked to bone development, osteogenesis, and cytoskeletal and extracellular matrix organisation. (B) Key protein heatmap of the log10 expression changes across each sample type. Red: expression above the median; blue: expression below the median; white: median expression across samples.

Figure 6

(A) Gene ontology biological process analysis of proteins expressed across captured proteomes shows a large contingent of proteins linked to bone development, osteogenesis, and cytoskeletal and extracellular matrix organisation. (B) Key protein heatmap of the log10 expression changes across each sample type. Red: expression above the median; blue: expression below the median; white: median expression across samples.

Figure 7

Mechanotransduction pathways’ schematic and bar graphs showing the number of proteins expressed within each critical pathway hub. The x-axis indicates the number of identified proteins per pathway.

Figure 8

Number of proteins expressed by each scaffold associated with each BP on Day 14 of culture. (A) Early-stage osteogenic differentiation (ESOD). (B,C) Mid-stage osteogenic differentiation (MSOD), separated into two panels (part 1 and part 2) for visual clarity. (D) Late-stage osteogenic differentiation (LSOD).

Figure 9

(A,B) Number of proteins expressed, per scaffold, for BP and pathways related to angiogenic activity. The x-axis indicates the number of identified proteins per pathway.