WP 1: Surface functionalization of MEW scaffolds with cartilage-specific extracellular matrix (ECM) components.
The goal of this WP is to functionalise the surface of MEW scaffolds with solubilised cartilage extracellular matrix (ECM), and to assess how this impacts the capacity of such scaffolds to support chondrogenesis of human marrow stem/stromal cells (MSCs). For surface functionalisation of the MEW scaffolds, existing methods for polydopamine-facilitated collagen coatings were evaluated and compared with a novel method. The loading efficiency of each method was measured, which indicated the novel method led to a higher loading of collagen into the polydopamine coating. This novel method was then employed to investigate the chondrogenic potential of a range of different extracted collagens, with uncoated and polydopamine coated scaffolds serving as controls. Three collagen extractions were employed to coat the scaffolds:
• Pepsin solubilised collagen type II (PSC)
• Insoluble collagen type II (InC)
• Acid-solubilised collagen type I (TC-I)
The pepsin-solubilised collagen type II was significantly less chondrogenic in vitro than insoluble collagen type II and acid-solubilised collagen type I both in terms of collagen and sGAG deposition. The insoluble collagen type II coating led to significantly greater sGAG deposition in short-term culture, however there was no significant different between it and the TC-I after longer-term culture.
WP 2: Influence of MEW scaffold fiber architecture on neo-cartilage development.
MEW scaffolds were printed with square pores using a range of fiber spacings: 200 µm, 300 µm, 400 µm, 600 µm, and 800 µm. The scaffolds were seeded with mesenchymal stem/stromal cells, and cultured for 30 days in chondrogenic media. In all groups except the 200 µm pore size group, no significant differences were found between collagen coated samples and their non coated controls. For the 200 µm group, the presence of the coating lead to significantly higher deposition of sGAG compared to the non-coated. Therefore a 200 µm pore size was determined to be the preferred pore size for the cartilage phase of the MEMS device, with larger pore sizes as the scaffold transitions to bone.
WP 3: Influence of MEW scaffold fiber architecture on angiogenesis and osteogenesis in vivo AND WP 4: Manufacture and in vivo assessment of multi-layered MEW scaffolds designed for OC defect repair
Challenges arise when attempting to use MEW to print large, clinically relevant scaffolds for large bone or osteochondral defect repair, that is scaffolds that approach 5 mm in height (or over 300 layers). These challenges were identified as:
• Uneven deposition – caused by electrostatic attraction of the melt to elevated features in the print.
• Overshoot on corners – due to the inertia of the melt during rapid changes in print direction.
• Poor interlayer fusion – particularly prevalent in smaller pore sizes as a result of long layer print times leading to excess cooling of the previous layer.
A MATLAB-based print program generator was coded to reduce the amount of overlap in a MEW print to its minimum possible to prevent the appearance of structures that are taller than the surrounding print which would lead excess deposition and material starvation in surrounding print lines. Suitable acceleration, deceleration, and velocities were identified for each pore size investigated to minimise the risk of overshoot on corners. Corners were also staggered slightly to further separate them and reduce the risk of corner overshoot leading to deposition of the next line again resulting excess deposition and material starvation. A PID-controlled heater was installed in the MEW enclosure to combat excessive cooling. An Arduino was employed in the PID controller set-up with feedback from temperature humidity sensors inside the MEW enclosure. These allowed the printing of MEW structures over 5 mm high that maintained their defined pore structure. Due to the additional workload associated with the MEW of these larger scaffold structures, we were unable to complete the in vivo subcutaneous studies associated with these WPs.
