Evaluation of hernia surgical meshes sterilised with ethylene oxide for adoption under UK regulations.

BACKGROUND: Low-cost meshes (LCM) have been successfully used in low-income countries (LIC) over the past decades, demonstrating comparable surgical outcomes to commercial meshes at a fraction of the cost. However, LIC sterilisation standards (autoclave sterilisation at 121 °C) do not meet UK regulations for medical devices, which require either ethylene oxide (EO) sterilisation or steam sterilisation at 134 °C. Therefore, the aim of this study was to sterilise UK LCM and characterise their mechanical properties and in vitro biocompatibility to verify whether EO sterilisation causes changes in the mechanical properties and biocompatibility of LCM. METHODS: EO sterilised LCM were used. Uniaxial tensile tests were performed to measure mechanical properties. Biocompatibility was measured through viability and morphology of Human Dermal Fibroblasts (HDFs) cultured in mesh-conditioned media, and by calculating the metabolic activity and proliferation of HDFs attached on the meshes, with alamarBlue assay. RESULTS: Break stress of LCM1 was significantly higher than LCM2 (p < 0.0001), while Young's modulus of LCM1 was significantly lower than LCM2 (p < 0.05) and there was no significant difference in break strain. Viability and morphology showed no significant difference between LCM and control. Attachment and proliferation of HDFs on LCM showed a better proliferation on LCM2 than LCM1, with values similar to the control at the final time point. CONCLUSIONS: We demonstrated that EO sterilisation affects LCM mechanical properties, but they still have values closer to the native tissues than the commercially available ones. We also showed that in vitro biocompatibility of LCM2 is not affected by EO sterilisation, as HDFs attached and proliferated on the mesh, while EO affected attachment on LCM1. A more detailed cost analysis of the potential savings for healthcare systems around the world needs to be performed to strengthen the cost-effectiveness of this frugal innovation.

A Sustainable, Green-Processed, Ag-Nanoparticle-Incorporated Eggshell-Derived Biomaterial for Wound-Healing Applications.

The eggshell membrane (ESM) is a natural biomaterial with unique physical and mechanical properties that make it a promising candidate for wound-healing applications. However, the ESM's inherent properties can be enhanced through incorporation of silver nanoparticles (AgNPs), which have been shown to have antimicrobial properties. In this study, commercially produced AgNPs and green-processed AgNPs were incorporated into ESM and evaluated for their physical, biological, and antimicrobial properties for potential dermal application. The ESM was extracted using various techniques, and then treated with either commercially produced AgNPs (Sigma-Aldrich, Poole, UK) or green-synthesized AgNPs (Metalchemy, London, UK) to produce AgNPs-ESM samples. The physical characteristics of the samples were evaluated using scanning electron microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy, and the biological properties were assessed through in vitro studies using human dermal fibroblasts (HDFs) and BJ cells. The SEM analysis of the AgNPs-ESM samples showed localization of AgNPs on the ESM surface, and that the ESM maintained its structural integrity following AgNP incorporation. The FTIR confirmed loading of AgNPs to ESM samples. The biological studies showed that the 5 µg/mL AgNPs-ESM samples were highly biocompatible with both HDFs and BJ cells, and had good viability and proliferation rates. Additionally, the AgNPs-ESM samples demonstrated pro-angiogenic properties in the CAM assay, indicating their potential for promoting new blood vessel growth. Assessment of the antimicrobial activity of the enhanced AgNPs/ESMs was validated using the International Standard ISO 16869:2008 methodology and exploited Cladosporium, which is one of the most commonly identified fungi in wounds, as the test microorganism (≥5 × 106 cells/mL). The AgNPs-ESM samples displayed promising antimicrobial efficacy as evidenced by the measured zone of inhibition. Notably, the green-synthesized AgNPs demonstrated greater zones of inhibition (~17 times larger) compared to commercially available AgNPs (Sigma-Aldrich). Although both types of AgNP exhibited long-term stability, the Metalchemy-modified samples demonstrated a slightly stronger inhibitory effect. Overall, the AgNPs-ESM samples developed in this study exhibited desirable physical, biological, and antimicrobial properties for potential dermal wound-dressing applications. The use of green-processed AgNPs in the fabrication of the AgNPs-ESM samples highlights the potential for sustainable and environmentally friendly wound-healing therapies. Further research is required to assess the long-term biocompatibility and effectiveness of these biomaterials in vivo.

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.

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.

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.

Controlling human corneal stromal stem cell contraction to mediate rapid cell and matrix organization of real architecture for 3-dimensional tissue equivalents.

The architecture of the human corneal stroma consists of a highly organized extracellular matrix (ECM) interspersed with keratocytes. Their progenitor cells; corneal stromal stem cells (CSSC) are located at the periphery, in the limbal stroma. A highly organized corneal ECM is critical for effective transmission of light but this structure may be compromised during injury or disease, resulting in loss of vision. Re-creating normal organization in engineered tissue equivalents for transplantation often involves lengthy culture times that are inappropriate for clinical use or utilisation of synthetic substrates that bring complications such as corneal melting. CSSC have great therapeutic potential owing to their ability to reorganize a disorganized matrix, restoring transparency in scarred corneas. We examined CSSC contractile behavior to assess whether this property could be exploited to rapidly generate cell and ECM organization in Real Architecture For 3D Tissues (RAFT) tissue equivalents (TE) for transplantation. Free-floating collagen gels were characterized to assess contractile behavior of CSSC and establish optimum cell density and culture times. To mediate cell and collagen organization, tethered collagen gels seeded with CSSC were cultured and subsequently stabilized with the RAFT process. We demonstrated rapid creation of biomimetic RAFT TE with tunable structural properties. These displayed three distinct regions of varying degrees of cellular and collagen organization. Interestingly, increased organization coincided with a dramatic loss of PAX6 expression in CSSC, indicating rapid differentiation into keratocytes. The organized RAFT TE system could be a useful bioengineering tool to rapidly create an organized ECM while simultaneously controlling cell phenotype. STATEMENT OF SIGNIFICANCE: For the first time, we have demonstrated that human CSSC exhibit the phenomenon of cellular self-alignment in tethered collagen gels. We found this mediated rapid co-alignment of collagen fibrils and thus subsequently exploited this property in vitro to improve the architecture of engineered RAFT tissue equivalents of the corneal stroma. Existing techniques are extremely lengthy and carry significant risk and cost for GMP manufacture. This rapid and tunable technique takes just 8 h of culture and is therefore ideal for clinical manufacture, creating biomimetic tissue equivalents with both cellular and ECM organization. Thus, cellular self-alignment can be a useful bioengineering tool for the development of organized tissue equivalents in a variety of applications.

Human corneal stromal stem cells support limbal epithelial cells cultured on RAFT tissue equivalents.

Human limbal epithelial cells (HLE) and corneal stromal stem cells (CSSC) reside in close proximity in vivo in the corneal limbal stem cell niche. However, HLE are typically cultured in vitro without supporting niche cells. Here, we re-create the cell-cell juxtaposition of the native environment in vitro, to provide a tool for investigation of epithelial-stromal cell interactions and to optimize HLE culture conditions for potential therapeutic application. RAFT (Real Architecture For 3D Tissue) tissue equivalents (TEs) were used as a 3-dimensional substrate for co-culturing HLE and CSSC. Our results demonstrate that a monolayer of HLE that maintained expression of p63α, ABCB5, CK8 and CK15 (HLE markers), formed on the surface of RAFT TEs within 13 days of culture. CSSC remained in close proximity to HLE and maintained expression of mesenchymal stem cell markers. This simple technique has a short preparation time of only 15 days with the onset of HLE layering and differentiation observed. Furthermore, co-cultivation of HLE with another niche cell type (CSSC) directly on RAFT TEs, eliminates the requirement for animal-derived feeder cells. RAFT TEs may be useful for future therapeutic delivery of multiple cell types to restore the limbal niche following ocular surface injury or disease.

Functional limbal epithelial cells can be successfully isolated from organ culture rims following long-term storage.

PURPOSE: Because of a shortage of fresh corneal tissue for research, it was of interest to investigate the potential of successfully isolating human limbal epithelial cells (hLECs) from organ culture corneal-scleral (OCCS) rims. METHODS: Superficial segments of corneal limbus were dissected and digested using collagenase (0.5 mg/mL, 16 hours at 37 °C). Cell suspensions were separated into four different growth conditions: corneal epithelial cell medium (CM); CM + 3T3-Swiss albino cells; stromal stem cell medium (SM); and SM + 3T3 cells. Colony number, hLEC count, cell density, and colony forming efficiency (CFE) were quantified to assess different growth conditions. The expression profile associated with basal hLECs was assessed by immunofluorescence, and epithelial integrity was measured using our real architecture for 3D tissue (RAFT) corneal tissue equivalent. RESULTS: Human limbal epithelial cells can be successfully isolated from OCCS rims following 4 weeks in storage with an 80.55% success rate with 36 corneal rims. Stromal stem cell medium + 3T3s provided optimal growth conditions. Colony number, total cell number, and cell density were significantly higher at day 7 in cultures with SM than in CM. There were no significant differences between SM and CM when assessing CFE and the expression profile associated with basal hLECs. Cells maintained in SM were found to produce a higher quality epithelium than that cultured in CM. CONCLUSIONS: Organ culture corneal-scleral rims can be a valuable source for hLEC. Using a combination of collagenase-based isolation and medium designed for stromal stem cell isolation, a high number of good quality hLECs can be cultured from tissue that would have otherwise been ignored.

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 0.6ml of 3mg/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.

Tissue Engineering the Cornea: The Evolution of RAFT.

Corneal blindness affects over 10 million people worldwide and current treatment strategies often involve replacement of the defective layer with healthy tissue. Due to a worldwide donor cornea shortage and the absence of suitable biological scaffolds, recent research has focused on the development of tissue engineering techniques to create alternative therapies. This review will detail how we have refined the simple engineering technique of plastic compression of collagen to a process we now call Real Architecture for 3D Tissues (RAFT). The RAFT production process has been standardised, and steps have been taken to consider Good Manufacturing Practice compliance. The evolution of this process has allowed us to create biomimetic epithelial and endothelial tissue equivalents suitable for transplantation and ideal for studying cell-cell interactions in vitro.

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.

Human corneal stromal stem cells exhibit survival capacity following isolation from stored organ-culture corneas.

PURPOSE: To assess the suitability of human donor corneas maintained in long-term organ culture for the isolation and expansion of viable and functional corneal stromal stem cells (CSSCs). These cells display properties similar to mesenchymal stem cells and demonstrate the ability to reproduce an organized matrix in vitro. Therefore, CSSCs have great potential for the development of cell-based therapies for corneal blindness or stromal tissue bioengineering. METHODS: Human donor corneas that had been stored either in organ-culture medium (OC) up to 4 weeks (n = 3) or in Optisol medium (OS) up to 6 days (n = 3) were used for isolation of CSSCs and maintained in culture until passage 4. Cell phenotype of isolated CSSCs was assessed with light microscopy and immunocytochemistry (PAX6, CD73, and CD90). PAX6 protein expression was further confirmed with immunoblot analysis. RESULTS: A comparison of CSSCs isolated from corneas stored under OC and OS conditions revealed no obvious differences in their morphology. Immunocytochemistry revealed CSSCs from both OC and OS corneas maintained positive staining for PAX6 and mesenchymal stem cell markers CD73 and CD90. Immunoblotting confirmed protein expression of PAX6 in cells from both tissue types. CONCLUSIONS: Human CSSCs exhibit survival capacity by retaining their phenotype following isolation from long storage, OC corneas. This advantageous property enables the retrieval of CSSCs from OC corneas that are more abundantly available for research than OS-stored corneas. Organ-culture corneas are also often discarded for retrieval of other cell types, such as corneal epithelial and endothelial cells, which require high tissue quality for their preservation.

Challenges in the development of a reference standard and potency assay for the clinical production of RAFT tissue equivalents for the cornea.

AIM: To develop a reference standard and potency assay for Real Architecture For 3D Tissues (RAFT) tissue equivalents intended for use in limbal epithelial stem cell (LESC) therapy for the cornea. METHODS: RAFT, a cell-seeded plastic compressed collagen construct with LESCs cultured on the surface, was manufactured with the goal of achieving GMP compliance. RAFTs were tested for reproducibility of manufacture (reference standard) and subsequently wounded and monitored for re-epithelialization (potency assay). RESULTS: RAFT tissue equivalents produced with cells from different biological donors were capable of supporting multilayered epithelium in culture. The potency assay demonstrated re-epithelialization following wounding, indicating the potential efficacy of RAFT constructs. CONCLUSION: We have presented our attempts at creating a reference standard and potency assay for the clinical manufacture of RAFT for the treatment of LESC deficiency. However, it remains challenging for adult stem cell therapies (including LESC therapy) to fully meet regulatory requirements when dealing with a limited source of autologous cells with inherent biological variation between donors.

Alignment hierarchies: engineering architecture from the nanometre to the micrometre scale.

Natural tissues are built of metabolites, soluble proteins and solid extracellular matrix components (largely fibrils) together with cells. These are configured in highly organized hierarchies of structure across length scales from nanometre to millimetre, with alignments that are dominated by anisotropies in their fibrillar matrix. If we are to successfully engineer tissues, these hierarchies need to be mimicked with an understanding of the interaction between them. In particular, the movement of different elements of the tissue (e.g. molecules, cells and bulk fluids) is controlled by matrix structures at distinct scales. We present three novel systems to introduce alignment of collagen fibrils, cells and growth factor gradients within a three-dimensional collagen scaffold using fluid flow, embossing and layering of construct. Importantly, these can be seen as different parts of the same hierarchy of three-dimensional structure, as they are all formed into dense collagen gels. Fluid flow aligns collagen fibrils at the nanoscale, embossed topographical features provide alignment cues at the microscale and introducing layered configuration to three-dimensional collagen scaffolds provides microscale- and mesoscale-aligned pathways for protein factor delivery as well as barriers to confine protein diffusion to specific spatial directions. These seemingly separate methods can be employed to increase complexity of simple extracellular matrix scaffolds, providing insight into new approaches to directly fabricate complex physical and chemical cues at different hierarchical scales, similar to those in natural tissues.

Matrix mechanical properties of transversalis fascia in inguinal herniation as a model for tissue expansion.

Inguinal herniation represents a common condition requiring surgical intervention. Despite being regarded as a connective tissue disorder of uncertain cause, research has focused predominantly on biochemical changes in the key tissue layer, the transversalis fascia (TF) with little direct analysis of functional tissue mechanics. Connective tissue tensile properties are dominated by collagen fibril density and architecture. This study has correlated mechanical properties of herniated TF (HTF) and non-herniated TF (NHTF) with fibrillar properties at the ultrastructural level by quasi-static tensile mechanical analysis and image analysis of collagen electron micrographs. No significant difference was found between any of the key mechanical properties (break stress, strain or modulus) for HTF and NHTF. In addition, no significant differences were found in average collagen fibril diameter, density or fibre bundle spacing. However, both groups displayed anisotropy with greater break stress (p=0.001) on average in the transverse anatomical plane compared to the longitudinal plane in a mean ratio of 2:1 (anisotropy ratio), though there was no evidence of a difference in this ratio for HTF and NHTF for both break stress and modulus. It was noted that this anisotropy ratio corresponds closely with the expected force distribution on a model cylindrical structure loaded axially. The absence of other functional differences does not support the idea of a failing (injured) tissue but is consistent with it being a tissue undergoing chronic growth/expansion under multi-vectored mechanical loading. These findings provide new clues to collagen tissue herniation for mathematical modelling and model tissue engineering.