Porous 3D Scaffolds Enhance MSC Vitality and Reduce Osteoclast Activity

Abstract

In the context of an aging population, unhealthy Western lifestyle, and the lack of an optimal surgical treatment, deep osteochondral defects pose a great challenge for the public health system. Biodegradable, biomimetic scaffolds seem to be a promising solution. In this study we investigated the biocompatibility of porous poly-((D,L)-lactide-ε-caprolactone)dimethacrylate (LCM) scaffolds in contrast to compact LCM scaffolds and blank cell culture plastic. Thus, morphology, cytotoxicity and metabolic activity of human mesenchymal stromal cells (MSC) seeded directly on the materials were analyzed after three and six days of culturing. Further, osteoclastogenesis and osteoclastic activity were assessed using reverse-transcriptase real-time PCR of osteoclast-specific genes, EIA and morphologic aspects after four, eight, and twelve days. LCM scaffolds did not display cytotoxic effects on MSC. After three days, metabolic activity of MSC was enhanced on 3D porous scaffolds (PS) compared to 2D compact scaffolds (CS). Osteoclast activity seemed to be reduced at PS compared to cell culture plastic at all time points, while no differences in osteoclastogenesis were detectable between the materials. These results indicate a good cytocompatibility of LCM scaffolds. Interestingly, porous 3D structure induced higher metabolic activity of MSC as well as reduced osteoclast activity.

Keywords: 3D-printing; bone; cartilage; implant; mRNA expression; mesenchymal stromal cells; osteoclasts; poly-lactide-caprolactone; polymer; tartrate-resistant acid phosphatase.

Figure 1

MTT assay (A) after 3 (d) and (B) 6 days of incubation. CS—compact scaffolds, PS—porous scaffolds, C—control on cell culture plastic. * indicates a statistically significant likelihood of p ≤ 0.05. Circles and small asterisks denote outliers.

Figure 2

LDH assay (A) after 3 (d) and (B) 6 days of incubation. PS—porous scaffolds, CS—compact scaffolds, C—control on cell culture plastic.

Figure 3

MSC growing at the surface of porous scaffolds (A,D) and compact scaffolds (B,E) for 3 (A–C) and 6 days (D–F). Scale bar of F is representative for (A–E).

Figure 4

Relative expression of Nfatc1 after 4 (A), 8 (B), and 12 days (C). Small asterisks denote outliers.

Figure 5

Relative expression of DC-Stamp after 4 (A), 8 (B), and 12 days (C). Circles and small asterisks denote outliers.

Figure 6

TRAP5b assay after 4 (A), 8 (B), and 12 days (C). * indicates a statistically significant likelihood of p ≤ 0.05, ** indicates a statistically significant likelihood of p ≤ 0.01. Circles and small asterisks denote outliers.

Figure 7

Relative expression of CtsK after 4 (A), 8 (B), and 12 days (C). * indicates a statistically significant likelihood of p ≤ 0.05. Circles denote outliers.

Figure 8

Relative expression of CalcR after 4 (A), 8 (B), and 12 days (C). * indicates a statistically significant likelihood of p ≤ 0.05. Small asterisks denote outliers.

Figure 9

Relative expression of CA2 after 4 (A), 8 (B), and 12 days (C). Circles and small asterisks denote outliers.

Figure 10

Light microscopic observation of PBMC and osteoclasts after 4 (A–C), 8 (D–F), and 12 days (G–I) at the interface of PS (A,D,G), CS (B,E,H), and pure cell culture plastic (control, C,F,I). Scale bar of I is representative for (A–H).

Figure 11

Immunofluorescence microscopy of osteoclasts after eight days of incubation on porous scaffold (A,D), compact scaffold (B,E), and cell culture plastic (C,F). Green: actin labelling, blue: nuclei. Scale bar of (C) is representative for (A,B) and of (F) for (D,E).

Figure 12

Images of porous (PS) and compact LCM scaffolds (CS). (A) Overview of PS compared to CS that is nearly transparent. (B) Higher magnification of CS. (C) Scanning electron microscopic (SEM) picture of the surface of CS. (D) SEM overview of PS. (E) Detail SEM of PS.