Hypothesis / aims of study
Although Pelvic organ prolapse (POP) affects 1 in every 4 women in the world, the aetiology of POP is less understood. The histologic failure of commercial transvaginal mesh to integrate with native vaginal tissue has led to erosion and pain that has instigated the development of newer therapeutics for POP patients. It was recently shown that the foreign body immune response is associated with mesh erosion and pelvic pain. To overcome these hurdles in pelvic floor repair surgeries, it is pivotal to achieve significant tissue integration and an anti-inflammatory foreign body response. Electrospinning polymeric biomaterials have emerged as a promising approach to overcome some of the challenges of PP transvaginal mesh. They provide nano/micro-textured surfaces enabling the design of scalable materials that mimic the structural and mechanical cues of tissue extracellular matrix (ECM) and promote cell adhesion. Melt electrospinning (MES) based 3D printing is a newer approach that allows direct writing mode from a computer-aided design (CAD) to manufacture orderly more adaptable meshes and scaffolds with potential to specifically match the requirements of the tissue defect. It combines technologies from conventional solution electrospinning techniques with conventional FDM techniques commonly used in 3D printing to allow controlled fabrication of meshes with defined structures and porosities vital for biomedical and tissue engineering. In this study, we aimed to develop novel degradable surgical constructs using 3D printing of biocompatible polymers. We further engineered our constructs with endometrial derived Mesenchymal Stem cells (eMSCs) to drive immunomodulation by secreting paracrine factors around our 3D printed mesh with the aim of improving constructs-host integration. Given the immunomodulatory properties of eMSCs, we hypothesized that eMSC based tissue engineered 3D printed constructed will enable better host-construct integration.
Study design, materials and methods
We fabricated 3D printed poly ε-caprolactone (PCL) scaffolds as vaginal constructs using melt electrospinning (MES) at different temperatures using a GMP clinical grade GESIM Bioscaffolder. Electron microscopy and atomic force microscopy revealed that MES meshes at 3D printed at 1000C and speed (20 mm/sec) had the highest open pore diameter (47.2 ± 11.4 μm), strand thickness (118 ± 46 μm) and favourable eMSC attachment We also developed a plant based Aloe Vera-Sodium Alginate (AV-ALG) hydrogel using (1:1) (1%AV-1%ALG) for bioprinting controlled delivery of eMSCs. The final surgical constructs bioprinted with eMSCs were implanted into NSG mice to assess the foreign body response. Tissue integration and immune modulation of the implanted meshes were assessed through image analysis for multinucleated giant cells and the macrophage markers F4/80, CCR7 (M1 macrophage marker), CD206 (M2 macrophage marker).
This study identified the fabrication parameters for PCL MES mesh were 100°C with a speed of 20 mm/sec that generated uniformly distributed fibre sizes with cell-compatible pore sizes and a suitable topographic surface that promoted cell adhesion of bioprinted eMSC. A mixture of two plant-based materials, alginate (ALG) and aloe vera (AV), each at a concentration of 1%, formed immediate and spontaneous gelling, supported eMSC adhesion and proliferation, and provided a hydrogel for bioprinting the cells. In a pre-clinical small animal model, these MES meshes with the bioprinted eMSC-hydrogel provided an ideal substrate for eMSC retention in vivo, therefore, promoting better tissue integration with minimal foreign body reaction and fibrotic encapsulation. The retention of eMSCs promoted a significantly greater and rapid M2 macrophage anti-inflammatory response compared to the mesh alone and the mesh-hydrogel composite without cells.
Interpretation of results
Our results strongly demonstrate that eMSCs play an important immunomodulatory role in the foreign body response to our 3D bioprinted constructs. Importantly, our results highlight that the AV-ALG hydrogel has the capacity to reduce the acute macrophage-mediated inflammation associated with non-cellular constructs. Our method appears to be a suitable platform for future fabricating personalised meshes with minimal human interception. The AV-ALG based hydrogel was simple to formulate requiring no additional cross-linking and contained sheer thinning properties that effectively delivered and retained viable and proliferative eMSC at the implantation site. Considering the controversies of the current commercial meshes, our results validate a two-step fabrication process combining eMSCs, MES and 3D bioprinting to produce a clinical grade cellular product that can effectively enhance native tissue integration and regeneration.