Manufacturing of 3D-Printed Hybrid Scaffolds with Polyelectrolyte Multilayer Coating in Static and Dynamic Culture Conditions

Abstract

Three-dimensional printing (3DP) has emerged as a promising method for creating intricate scaffold designs. This study assessed three 3DP scaffold designs fabricated using biodegradable poly(lactic) acid (PLA) through fused deposition modelling (FDM): mesh, two channels (2C), and four channels (4C). To address the limitations of PLA, such as hydrophobic properties and poor cell attachment, a post-fabrication modification technique employing Polyelectrolyte Multilayers (PEMs) coating was implemented. The scaffolds underwent aminolysis followed by coating with SiCHA nanopowders dispersed in hyaluronic acid and collagen type I, and finally crosslinked the outermost coated layers with EDC/NHS solution to complete the hybrid scaffold production. The study employed rotating wall vessels (RWVs) to investigate how simulating microgravity affects cell proliferation and differentiation. Human mesenchymal stem cells (hMSCs) cultured on these scaffolds using proliferation medium (PM) and osteogenic media (OM), subjected to static (TCP) and dynamic (RWVs) conditions for 21 days, revealed superior performance of 4C hybrid scaffolds, particularly in OM. Compared to commercial hydroxyapatite scaffolds, these hybrid scaffolds demonstrated enhanced cell activity and survival. The pre-vascularisation concept on 4C hybrid scaffolds showed the proliferation of both HUVECs and hMSCs throughout the scaffolds, with a positive expression of osteogenic and angiogenic markers at the early stages.

Keywords: 3DP hybrid scaffolds; co-culture; fused deposition modelling; polyelectrolyte multilayers; pre-vascularisation; rotating wall vessels; stem cells.

Figure 1

Viability of hMSCs seeded on all four scaffold designs cultured under static condition OM and PM after 1 day (for 3DP hybrid scaffolds) and 3 days (for HA scaffolds). Yellow scale bar = 500 μm.

Figure 2

Cell viability for different scaffold designs after 21 days cultured under static and dynamic conditions in OM and PM. Yellow scale bar = 500 μm.

Figure 3

(a) Comparison of the amount of DNA associated with hMSCs and (b) the percentages of LDH activity on HA, 2C, 4C and mesh scaffolds in different culture conditions after 21 days (ns ≥ 0.05; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Figure 4

The comparison of the total protein produced of hMSCs after 21 days cultured on HA, 2C, 4C, and mesh scaffolds in different culture conditions. (ns ≥ 0.05; *** p ≤ 0.001, **** p ≤ 0.0001).

Figure 5

(a) ALP activity of hMSCs after 21 days cultured on HA, 2C, 4C, and mesh scaffolds in different culture conditions (ns ≥ 0.05; * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001). (b) ALP staining for different scaffold designs after 21 days cultured under static and dynamic conditions in OM and PM. Red scale bar = 1 mm.

Figure 6

(a): X-ray Micro-CT of different structural designs of dry scaffolds (before seeding). (b): Density maps of HA, 2C, 4C, and mesh scaffolds after 21 days in different culture conditions. This figure demonstrated the comparisons of the formation of the mineralised matrix (designated by red area) by hMSCs on different scaffold designs under different culture conditions.

Figure 7

Cell morphology of the co-culture system in the channel of the 4C scaffolds after (a) 3 and (b) 10 days post-hMSCs addition. HUVECs (labelled in red) were distributed in the entire channels after 10 days post hMSCs (labelled in blue) addition. Scale bar = 250 μm.

Figure 8

The level of (a) PDGF-BB and (b) VEGF release by hMSCs alone, HUVECs alone, and co-culture model after day 3 HUVECs seeding only and day 6 and 10 post-hMSCs additions. (ns ≥ 0.05; **** p ≤ 0.0001).