(W1230-03-19) Characteristic and Bio-Functionality of a Novel Multilayered Crosslinked Nanoscaffold for Tuberculosis Granuloma Modelling

Purpose: Tuberculosis (TB) has impacted globally since the 19^th century and became the leading cause of death after COVID-19 in 2021 with one person dying every 42 minutes (Global tuberculosis report, 2022). As the disease progress into the latent state, the complete TB eradication becomes more challenging since the bacteria may survive inside the host for an extended period of time without causing symptoms (Carranza et al., 2020). Host-directed therapy comes as a new strategy which targets host factor instead of the pathogen itself. This new approach is considered to be an effective strategy for infectious disease therapy, including TB (Kolloli and Subbian, 2017). Granuloma is the pathological hallmark of latent TB, which consist of orchestral immune cells. The structure first performs host-protective activities, but eventually turns pathogenic when it becomes the bacilli's shell to multiply (Gautam et al., 2018; Pagán and Ramakrishnan, 2018). There are currently few effective in vitro models for Granuloma with their drawbacks to the lack of extracellular matrix (ECM) which is necessary for cell regulation and the development of three-dimensional (3D) organisation (Woo et al., 2003). The combined use of natural and synthetic biomaterials in nanoscaffolds generation offers several advantages such as improved biomimicry of the ECM (Gautam et al., 2014). Multi-layered electrospun nanoscaffolds have been shown to exhibit improved qualities that are highly correlated with their bio-functionality in tissue engineering applications (Wu et al., 2017). The aim of this study was to develop a novel design of a multi-layered crosslinked nanoscaffolds by leveraging the use of Polyvinyl Alcohol (PVA), Poloxamer (P407) and Polycaprolactone (PCL) as synthethic polymer, Pectin as natural polymer, and Calcium Chloride (CaCl₂) as crosslinker to improve the bio-functionality and biorelevance for TB granuloma modelling.

Methods: Electrospinning method was used to create nanoscaffold with multi-layered design. Three types of layering design were developed with the following composition: Single-layered nanoscaffold consist of PVA/Pectin/P407, Bi-layered nanoscaffold consist of PVA/Pectin/P407-PCL, and Tri-layered nanoscaffold consist of PVA/Pectin/P407-PCL-PVA/Pectin/P407, with the CaCl₂ added as crosslinker. The characteristic of nanoscaffolds were evaluated based on: moisture content, porosity, morphology and fibre diameter using Scanning Electron Microscopy (SEM), and the spectrum of absorption using Attenuated Total Reflection Fourier-Transform Infrared (ATR-FTIR) spectroscopy. As the macrophage plays a crucial role in the development of TB granuloma, THP-1 cell line was used to assess nanoscaffold’s biofunctionality through Alamar Blue cell viability study.

Results: All polymer combinations successfully generated nanoscaffold with a smooth and continuous surface without visible defects or irregularities like cracks and beads. The fibre diameter produced are ranged between ~166-262 nm. Meanwhile, the thickness of nanofibre matt was found within the range of ~79-1242 µm, with the greatest value found in bi-layered non-crosslinked nanoscaffold. Compared to the non-crosslinked formula, crosslinked formula produced a lower thickness of nanofibre mat for the same design. The moisture content of all samples was found to be low ( < 3%) and the porosity is high ( >70%). It was also observed that the addition of crosslinking agent and formation of multi-layered contributed to the increasing moisture content and nanofibrous matt porosity. The ATR-FTIR spectra of the multi-layered nanoscaffold showed the characteristic of PCL by the occurrence of peak at 2945 cm−1 and 2868 cm−1. The single-layered nanoscaffold did not show the prescence of those PCL spectra, but clearly depicted peaks around 3550–3200 cm-1 which belongs to the O–H from the intermolecular and intramolecular hydrogen bonds and 1750-1735 cm-1 from C=O bond of PVA. The ATR-FTIR spectra of all nanoscaffold confirmed the prescence of principal peak of each component used to fabricate the nanoscaffold which also proved a successful compounding of the material within nanoscaffold structure. Cell viability study with Alamar Blue indicated the increasing proliferation of THP-1 cells affected by the prescence of nanoscaffold with the non-crosslinked produced a higher value than the crosslinked sample. Compared to the single-layered PCL, the single-layered non-crosslinked PVA/Pectin/P407 produced a better proliferation. Moreover, an improved biocompatibility demonstrated by the multi-layered non-crosslinked compared to single layer and crosslinked sample, with the highest value obtained by the tri-layered non-crosslinked PVA/Pectin/P407-PCL-PVA/Pectin/P407 nanoscaffold.

Conclusion: This study effectively constructed a novel multi-layered crosslinked nanoscaffold using the combination of PVA, P407, PCL, and Pectin, with CaCl₂ as crosslinking agent. The multi-layered design demonstrated an improved physical characteristic than single-layered, in terms of porosity, moisture content, and fibre morphology. The combination of natural and synthethic polymer improved the ability of nanoscaffold to support the THP-1 cell growth and proliferation, thus providing a better biofunctionality as synthethic extracellular matrix for TB granuloma modelling. The study highlights the potential of multilayered nanoscaffold design as a suitable substrate for tissue engineering.

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Acknowledgements:

Funding: Author Amalia Miranda is funded by Centre of Doctoral Studies King’s College London, for her PhD scholarship. Conflict of Interest Statements: The authors declare no conflict of interest. The funding sources had no input into the study's planning, data collecting, analysis, or interpretation, manuscript writing, or the choice to publish the findings.

SEM images of top surface of nanoscaffolds: (a) single-layered PVA/Pectin/P407; (b) single-layered PCL; (c) bi-layered PVA/Pectin/P407-PCL; (c) tri-layered PVA/Pectin/P407-PCL-PVA/Pectin/P407; (d) single-layered crosslinked PVA/Pectin/P407

Fibre diameter and matt thickness measurement of nanoscaffold

The mean fluorescence indicating the THP-1 cell viability when cultured on the samples