Optimization of Optical and Mechanical Properties of Real Architecture for 3-Dimensional Tissue Equivalents: Towards Treatment of Limbal Epithelial Stem Cell Deficiency

Limbal epithelial stem cell (LESC) deficiency can cause blindness. Transplantation of cultured human limbal epithelial cells (hLE) on human amniotic membrane (HAM) can restore vision but clinical graft manufacture can be unreliable. We have developed a reliable and robust tissue equivalent (TE) alternative to HAM, Real Architecture for 3D Tissue (RAFT). Here, we aimed to optimize the optical and mechanical properties of RAFT TE for treatment of LESC deficiency in clinical application. The RAFT TE protocol is tunable; varying collagen concentration and volume produces differing RAFT TEs. These were compared with HAM samples taken from locations proximal and distal to the placental disc. Outcomes assessed were transparency, thickness, light transmission, tensile strength, ease of handling, degradation rates and suitability as substrate for hLE culture. Proximal HAM samples were thicker and stronger with poorer optical properties than distal HAM samples. RAFT TEs produced using higher amounts of collagen were thicker and stronger with poorer optical properties than those produced using lower amounts of collagen. The ‘optimal’ RAFT TE was thin, transparent but still handleable and was produced using 0.6 ml of 3 mg/ml collagen. Degradation rates of the ‘optimal’ RAFT TE and HAM were similar. hLE achieved confluency on ‘optimal’ RAFT TEs at comparable rates to HAM and cells expressed high levels of putative stem cell marker p63α. These findings support the use of RAFT TE for hLE transplantation towards treatment of LESC deficiency

In vitro characterisation of low-cost synthetic meshes intended for hernia repair in the UK

Purpose: Low-cost meshes (LCM) were repurposed for the repair of hernias in the developing world. In vivo studies have shown LCM to have comparable results to commercial meshes (CM) at a fraction of the cost. However, little has been done to characterise the mechanical and biocompatible properties of LCM, preventing its clinical use in the UK. The objectives of the research are to assess mechanical and ultrastructural properties of two UK-sourced low-cost meshes (LCM) and the characterisation of the LCMs in vitro biocompatibility. Methods: Mechanical properties of the two LCM were measured through uniaxial tensile test and ultrastructure was evaluated with Scanning Electron Microscopy. LIVE/DEAD Viability/Cytotoxicity Assay kit and alamarBlue were used to assess cellular viability and proliferation, respectively. Images were acquired with a fluorescence microscope and analysed using ImageJ (NIH, USA). Results: LCM1 and LCM2 were both multifilament meshes, with the first having smaller pores than the latter. LCM1 exhibited significantly higher tensile strength (p < 0.05) than LCM2 but significantly lower extensibility (p < 0.0001), while Young’s Modulus of the two samples was not significantly different. No significant difference was found in the cellular viability and morphology cultured in LCM1 and LCM2 conditioned media. Metabolic assay and fluorescence imaging showed cellular attachment and proliferation on both LCMs over 14 days. Conclusion: The characterisation of the two UK-sourced LCMs showed in vitro biocompatibility and mechanical and ultrastructural properties comparable to the equivalent CM. This in vitro data represents a step forward for the feasibility of adopting LCM for surgical repair of hernias in the UK.

The Isolation, Culture, and Cryopreservation of Human Rectus Sheath Fibroblasts

The vast majority of pelvic and intra-abdominal surgery is undertaken through at least one incision, through either the linea alba or the rectus sheath. These connective tissue layers are formed from the aponeuroses of the rectus muscles (anterior and posterior rectus sheath) and are vital for the structural integrity of the abdominal wall. Poor healing of these connective tissues after surgery can lead to significant morbidity for patients, who can develop unsightly and painful incisional hernias. Fibroblasts within the rectus sheath are responsible for laying down and remodeling collagen during the healing process after surgery. Despite their importance for this healing process, such cells have not been studied in vitro. In order to carry out such work, researchers must first be able to isolate these cells from human tissue and culture them successfully so they may be used for experimentation. This article provides an extensive and detailed protocol for the isolation, culture, cryopreservation, and thawing of human rectus sheath fibroblasts (RSFs). In our hands, this protocol develops confluent cultures of primary fibroblasts within 2 weeks, and sufficient cultures ready for freezing and storage after a further 2 to 4 weeks. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Collagenase digestion of human rectus sheath and isolation of RSFs Alternate Protocol: Collagenase digestion of human rectus sheath and isolation of RSFs, digestion in flask Support Protocol: Cryopreservation and thawing of human RSFs

Double standards in healthcare innovations: the case of mosquito net mesh for hernia repair

With over two decades of evidence available including from randomised clinical trials, we explore whether the use of low-cost mosquito net mesh for inguinal hernia repair, common practice only in low-income and middle-income countries, represents a double standard in surgical care. We explore the clinical evidence, biomechanical properties and sterilisation requirements for mosquito net mesh for hernia repair and discuss the rationale for its use routinely in all settings, including in high-income settings. Considering that mosquito net mesh is as effective and safe as commercial mesh, and also with features that more closely resemble normal abdominal wall tissue, there is a strong case for its use in all settings, not just low-income and middle-income countries. In the healthcare sector specifically, either innovations should be acceptable for all contexts, or none at all. If such a double standard exists and worse, persists, it raises serious questions about the ethics of promoting healthcare innovations in some but not all contexts in terms of risks to health outcomes, equitable access, and barriers to learning

Characterisation of human posterior rectus sheath reveals mechanical and structural anisotropy

Background: Our work aims to investigate the mechanical properties of the human posterior rectus sheath in terms of its ultimate tensile stress, stiffness, thickness and anisotropy. It also aims to assess the collagen fibre organisation of the posterior rectus sheath using Second-Harmonic Generation microscopy. Methods: For mechanical analysis, twenty-five fresh-frozen samples of posterior rectus sheath were taken from six different cadaveric donors. They underwent uniaxial tensile stress testing until rupture either in the transverse (n = 15) or longitudinal (n = 10) plane. The thickness of each sample was also recorded using digital callipers. On a separate occasion, ten posterior rectus sheath samples and three anterior rectus sheath samples underwent microscopy and photography to assess collagen fibre organisation. Findings: samples had a mean ultimate tensile stress of 7.7 MPa (SD 4.9) in the transverse plane and 1.2 MPa (SD 0.8) in the longitudinal plane (P < 0.01). The same samples had a mean Youngs modulus of 11.1 MPa (SD 5.0) in the transverse plane and 1.7 MPa (SD 1.3) in the longitudinal plane (P < 0.01). The mean thickness of the posterior rectus sheath was 0.51 mm (SD 0.13). Transversely aligned collagen fibres could be identified within the posterior sheath tissue using Second-Harmonic Generation microscopy. Interpretation: The posterior rectus sheath displays mechanical and structural anisotropy with greater tensile stress and stiffness in the transverse plane compared to the longitudinal plane. The mean thickness of this layer is around 0.51 mm – consistent with other studies. The tissue is constructed of transversely aligned collagen fibres that are visible using Second-Harmonic Generation microscopy

A Systematic Review Examining the Experimental Methodology Behind In Vivo Testing of Hiatus Hernia and Diaphragmatic Hernia Mesh

INTRODUCTION: Mesh implants are regularly used to help repair both hiatus hernias (HH) and diaphragmatic hernias (DH). In vivo studies are used to test not only mesh safety, but increasingly comparative efficacy. Our work examines the field of in vivo mesh testing for HH and DH models to establish current practices and standards. METHOD: This systematic review was registered with PROSPERO. Medline and Embase databases were searched for relevant in vivo studies. Forty-four articles were identified and underwent abstract review, where 22 were excluded. Four further studies were excluded after full-text review-leaving 18 to undergo data extraction. RESULTS: Of 18 studies identified, 9 used an in vivo HH model and 9 a DH model. Five studies undertook mechanical testing on tissue samples-all uniaxial in nature. Testing strip widths ranged from 1-20 mm (median 3 mm). Testing speeds varied from 1.5-60 mm/minute. Upon histology, the most commonly assessed structural and cellular factors were neovascularisation and macrophages respectively (n = 9 each). Structural analysis was mostly qualitative, where cellular analysis was equally likely to be quantitative. Eleven studies assessed adhesion formation, of which 8 used one of four scoring systems. Eight studies measured mesh shrinkage. DISCUSSION: In vivo studies assessing mesh for HH and DH repair are uncommon. Within this relatively young field, we encourage surgical and materials testing institutions to discuss its standardisation

3D culture model of fibroblast-mediated collagen creep to identify abnormal cell behaviour

Native collagen gels are important biomimetic cell support scaffolds, and a plastic compression process can now be used to rapidly remove fluid to any required collagen density, producing strong 3D tissue-like models. This study aimed to measure the mechanical creep properties of such scaffolds and to quantify any enhanced creep occurring in the presence of cells (cell-mediated creep). The test rig developed applies constant creep tension during culture and measures real-time extension due to cell action. This was used to model extracellular matrix creep, implicated in the transversalis fascia (TF) in inguinal hernia. Experiments showed that at an applied tension equivalent to 15% break strength, cell-mediated creep over 24-h culture periods was identified at creep rates of 0.46 and 0.38%/h for normal TF and human dermal fibroblasts, respectively. However, hernia TF fibroblasts produced negligible cell-mediated creep levels under the same conditions. Raising the cell culture temperature from 4 to 37 ◦C was used to demonstrate live cell dependence of this creep. This represents the first in vitro demonstration of TF cell-mediated collagen creep and to our knowledge the first demonstration of a functional, herniarelated cell abnormality

Advanced imaging and tissue engineering of the human limbal epithelial stem cell niche

The limbal epithelial stem cell niche provides a unique, physically protective environment in which limbal epithelial stem cells reside in close proximity with accessory cell types and their secreted factors. The use of advanced imaging techniques is described to visualize the niche in three dimensions in native human corneal tissue. In addition, a protocol is provided for the isolation and culture of three different cell types, including human limbal epithelial stem cells from the limbal niche of human donor tissue. Finally, the process of incorporating these cells within plastic compressed collagen constructs to form a tissue-engineered corneal limbus is described and how immunohistochemical techniques may be applied to characterize cell phenotype therein

Green Synthesis of Gold Nanoparticles Mediated by Garcinia Fruits andTheir Biological Applications

Background: Green synthesis of gold nanoparticles (AuNPs) using medicinal plant extract is an emerging area of research due to their applicability in nanomedicines. Methods: In this study, aqueous extracts prepared from fruit-pericarps of two Garcinia species, G. indica (GI) and G. cambogia (GC) fruits which are important medicinally and commercially have been utilized for the synthesis of AuNPs. Various analytical techniques were utilized to characterize the synthesized AuNPs. The synthesized AuNPs were investigated for their biological properties such as antioxidant activity using the (2,2-diphenyl-1-picrylhydrazyl) DPPH model, cytotoxicity against MCF-7 (breast) cancer cell line, and antibacterial activity against two bacterial strains viz. B. subtilis and E. coli. Results: The absorption peak of the AuNPs is observed at 541 nm using UV–Visible spectroscopy. The high resolution – scanning electron microscopy images showed spherical with a triangular shape AuNPs and their average sizes were ranging from 2 – 10 nm and it was found to be in good agreement with the particle size of 8 – 11 nm determined using X-ray diffraction analysis. Fourier-transform infrared spectroscopy revealed that water-soluble biomolecules from the aqueous extracts of the Garcinia species played a crucial role in the formation of AuNPs. The synthesized AuNPs exhibited considerable cytotoxicity with IC50 values 34.55 µg/ml (GI) and 35.69 µg/ml (GC) against the MCF-7 cancer cell line. Furthermore, synthesized AuNPs also demonstrated significant antioxidant and antibacterial properties comparable to the standards used. Conclusion: AuNPs have been synthesized using a simple green approach. The synthesized AuNPs demonstrated promising cytotoxicity, antioxidant, and antibacterial properties