Periodic Reporting for period 1 - MEMS (Melt Electrowriting of Multi-layered Scaffolds for osteochondral defect repair (MEMS))
Okres sprawozdawczy: 2023-10-01 do 2025-03-31
Osteochondral (OC) defects are focal joint injuries that affect both the articular cartilage and the underlying subchondral bone. These lesions may result from acute trauma or underlying conditions such as osteochondritis dissecans or osteonecrosis. The melt electrowriting (MEW) technique has recently emerged as a novel additive manufacturing platform capable of producing polymeric scaffolds with fiber diameters in the submicron range enabling the production of scaffolds with features more mimetic of native tissues. The Melt-Electrowriting of Multi-layered Scaffolds for osteochondral defect repair project (MEMS) aimed to demonstrate the innovation potential of MEW scaffolds for synovial joints regeneration and further enhance their efficacy using biomimetic osteogenic and chondrogenic coatings. Building on previous foundational work, this Proof-of-Concept grant enabled us to assess the feasibility, reproducibility, and functional enhancement of MEW scaffolds through:
• Advanced architectural control at the microscale.
• Tailored MEW scaffold pore sizes and fibre orientations.
• Selective surface biofunctionalisation with osteogenic and chondrogenic cues.
The overarching objective was to develop a single-step additive manufacturing platform capable of producing clinically relevant 3D scaffolds (~5 mm thick) with zonally distinct features that reflect the transition from cartilage to bone. Specifically, the project focused on: (1) manufacturability, (2) surface functionalisation and (3) scaffold structure optimisation.
The MEMS project ultimately sought to bridge the gap between high-resolution 3D printing and translational tissue engineering by delivering a scalable, reproducible, and bioactive scaffold solution. These insights lay the groundwork for potential commercialisation routes in regenerative orthopaedics and position MEW as a next-generation platform for advanced medical implants.
• Advanced architectural control at the microscale.
• Tailored MEW scaffold pore sizes and fibre orientations.
• Selective surface biofunctionalisation with osteogenic and chondrogenic cues.
The overarching objective was to develop a single-step additive manufacturing platform capable of producing clinically relevant 3D scaffolds (~5 mm thick) with zonally distinct features that reflect the transition from cartilage to bone. Specifically, the project focused on: (1) manufacturability, (2) surface functionalisation and (3) scaffold structure optimisation.
The MEMS project ultimately sought to bridge the gap between high-resolution 3D printing and translational tissue engineering by delivering a scalable, reproducible, and bioactive scaffold solution. These insights lay the groundwork for potential commercialisation routes in regenerative orthopaedics and position MEW as a next-generation platform for advanced medical implants.
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.
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.
The changes made to the programming of the MEW and the MEW printer itself made it capable of producing clinically relevant MEW scaffold structures with multiple zonal architectures. In the future this will facilitate the development of a range of different scaffold structures targeting diverse applications. This project also developed a biomimetic, pro-chondrogenic coating containing intact native type II collagen fibrils with associated sGAGs. We will continue to explore the utility of these coatings for different tissue engineering and regenerative medicine applications.
An initial market analysis confirmed that a significant market exists for new scaffold-based therapies for cartilage and osteochondral defect repair. However, following an internal assessment of the technology developed as part of this project, it was concluded that the scientific developments emanating from the project did not justify the filing of a patent and further commercialisation at this time.
An initial market analysis confirmed that a significant market exists for new scaffold-based therapies for cartilage and osteochondral defect repair. However, following an internal assessment of the technology developed as part of this project, it was concluded that the scientific developments emanating from the project did not justify the filing of a patent and further commercialisation at this time.