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Background: Low back pain (LBP) is predominantly caused by degeneration of the intervertebral disc (IVD) and central nucleus pulposus (NP) region. Conservative treatments fail to restore disc function, motivating the exploration of nucleic acid therapies, such as the use of microRNAs (miRNAs). miRNAs have the potential to modulate expression of discogenic factors, while silencing the catabolic cascade associated with degeneration. To deliver these miRNAs, nonviral cell penetrating peptides (CPPs) are gaining favor given their low immunogenicity and strong targeting ability. Single miRNA therapies have been investigated for IVD repair, however dual miRNA delivery strategies have not been commonly examined and may augment regeneration., Materials and Methods: Transfection of four pro-discogenic miRNAs (miRNA mimics:140-5p; 149-5p and inhibitors: 141-3p; 221-3p) and dual delivery of six miRNA pairings was performed using two CPPs, RALA and GET peptide (FLR), in primary rat NP monolayer culture, and in an ex vivo organ culture model of rat caudal discs. Protein expression of discogenic (aggrecan, collagen type II, and SOX9) and catabolic markers (ADAMTS5 and MMP13) were assessed., Results: Monolayer investigations signified enhanced discogenic marker expression following dual miRNA delivery, signifying a synergistic effect when compared to single miRNA transfection. Utilization of an appropriate model was emphasized in our ex vivo organ culture experiment, revealing the establishment of a regenerative microenvironment characterized by reduced catabolic enzyme activity and enhanced matrix deposition, particularly following concurrent delivery of FLR-miRNA-149-5p mimic and miRNA-221-3p inhibitor. Bioinformatics analysis of miRNA-149-5p mimic and miRNA-221-3p inhibitor identified distinct targets, pathways, and interactions, suggesting a mode of action for this amplified response., Conclusion: Our findings suggest the potential of FLR-miRNA-149-5p + miRNA-221-3p inhibitor to create an anti-catabolic niche within the disc to foster regeneration in moderate cases of disc degeneration, which could be utilized in further studies with the overarching aim of developing treatments for LBP., Competing Interests: Conor T. Buckley is an Editorial Board member of JOR Spine and co‐author of this article. They were excluded from editorial decision‐making related to the acceptance of this article for publication in the journal. The remaining authors declare no conflicts of interest., (© 2024 The Author(s). JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Purpose: Buccal mucosal grafts have a well-established role in urology regarding the management of ureteric stricture disease. Despite its established use as a graft material there is a lack of data on the mechanical properties of buccal mucosa. We aim to compare the passive mechanical properties of porcine buccal mucosa with the ureter., Materials and Methods: Buccal mucosa (n = 20) and ureteric specimens (n = 21) were harvested from 19 domestic pigs at the time of euthanasia. Mechanical testing was performed using a uniaxial tensometer to generate stress-strain curves. From these curves, ultimate tensile strength and elastic modulus were calculated. Data was analysed, and one-way ANOVA testing was used to assess for significance variation in mechanical response due to direction of testing, and between tissue types., Results and Conclusions: Porcine ureteric specimens displayed mechanical anisotropy, with a significant difference in the elastic modulus depending on the direction of testing (p = 0.005), and a higher mean ultimate tensile strength in the longitudinal (498.09 kPa) compared to the transverse (263.99 kPa) direction (p = 0.0005). Buccal mucosa was isotropic with no significant difference in elastic modulus or ultimate tensile strength according to direction of testing. Comparison of tissue types showed a significantly greater mean ultimate tensile strength in the longitudinal direction (p = 0.002) in ureter compared to buccal mucosa. We present the first comparison of the mechanical properties of buccal mucosa with ureter, and demonstrate significant variation in elastic modulus and ultimate tensile strength between the tissue types. These findings have important clinical implications for physiological function and in the design of alternative approaches for reconstruction in buccal mucosa grafted ureters., Competing Interests: Declarations. Ethical approval: This study did not require research and ethics committee approval as no experiments were performed on human participants. All animals were treated according to national health product regulatory guidelines. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Articular cartilage has limited capacity for repair (or for regeneration) under pathological conditions, given its non-vascularized connective tissue structure and low cellular density. Our group has successfully developed an injectable hydrogel for cartilage repair, composed of collagen type I (Col I), collagen type II (Col II), and methacrylated-hyaluronic acid (MeHA), capable of supporting chondrogenic differentiation of mesenchymal stem cells (MSCs) towards articular cartilage-like phenotypes. Recent studies have demonstrated that silencing miR-221 may be an effective approach in promoting improved MSC chondrogenesis. Thus, this study aimed to develop a miR -activated hydrogel capable of offering a more effective and less invasive therapeutic approach to articular cartilage repair by delivering a pro-chondrogenic miR-221 inhibitor to MSCs using our MeHA-Col I/Col II hydrogel. The MeHA-Col I/Col II hydrogel was cast as previously shown and incorporated with cells transfected with miR-221 inhibitor (using a non-viral peptide delivery vector) to produce the miR -activated hydrogel. Down-regulation of miR-221 did not affect cell viability and enhanced MSCs-mediated chondrogenesis, as evidenced by significantly upregulated expression of key pro-chondrogenic articular cartilage genes ( COL2A1 and ACAN ) without promoting hypertrophic events ( RUNX2 and COL10A1 ). Furthermore, miR-221 down-regulation improved cartilage-like matrix formation in the MeHA-Col I/Col II hydrogel, with significantly higher levels of sulfated glycosaminoglycans (sGAG) and Col II produced by MSCs in the hydrogel. These results provide evidence of the potential of the miR- activated hydrogel as a minimally invasive therapeutic strategy for articular cartilage repair., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Cartilage defect repair with optimal efficiency remains a significant challenge due to the limited self-repair capability of native tissues. The development of bioactive scaffolds with biomimicking mechanical properties and degradation rates matched with cartilage regeneration while simultaneously driving chondrogenesis, plays a crucial role in enhancing cartilage defect repair. To this end, a novel composite scaffold with hierarchical porosity was manufactured by incorporating a pro-chondrogenic collagen type I/II-hyaluronic acid (CI/II-HyA) matrix to a 3D-printed poly(glycerol sebacate) (PGS) framework. Based on the mechanical enforcement of PGS framework, the composite scaffold exhibited a compressive modulus of 167.0 kPa, similar to that of native cartilage, as well as excellent fatigue resistance, similar to that of native joint tissue. In vitro degradation tests demonstrated that the composite scaffold maintained structural, mass, and mechanical stability during the initial cartilage regeneration period of 4 weeks, while degraded linearly over time. In vitro biological tests with rat-derived mesenchymal stem cell (MSC) revealed that, the composite scaffold displayed increased cell loading efficiency and improved overall cell viability due to the incorporation of CI/II-HyA matrix. Additionally, it also sustained an effective and high-quality MSC chondrogenesis and abundant de-novo cartilage-like matrix deposition up to day 28. Overall, the biomimetic composite scaffold with sufficient mechanical support, matched degradation rate with cartilage regeneration, and effective chondrogenesis stimulation shows great potential to be an ideal candidate for enhancing cartilage defect repair., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: To assess human in vivo intrarenal pressure (IRP) and peristaltic activity at baseline and after ureteric stent placement, using a narrow calibre pressure guidewire placed retrogradely in the renal pelvis., Patients and Methods: A prospective, multi-institutional study recruiting consenting patients undergoing ureteroscopy was designed with ethical approval. Prior to ureteroscopy, the urinary bladder was emptied and the COMET™ II pressure guidewire (Boston Scientific) was advanced retrogradely via the ureteric orifice to the renal pelvis. Baseline IRPs were recorded for 1-2 min. At procedure completion, following ureteric stent insertion, IRPs were recorded for another 1-2 min. Statistical analysis of mean baseline IRP, peristaltic waveforms and frequency of peristaltic contractions was performed, thereby analysing the influence of patient variables and ureteric stenting., Results: A total of 100 patients were included. Baseline mean (±SD) IRP was 16.76 (6.4) mmHg in the renal pelvis, with maximum peristaltic IRP peaks reaching a mean (SD) of 25.75 (17.9) mmHg. Peristaltic activity generally occurred in a rhythmic, coordinated fashion, with a mean (SD) interval of 5.63 (3.08) s between peaks. On univariate analysis, higher baseline IRP was observed with male sex, preoperative hydronephrosis, and preoperative ureteric stenting. On linear regression, male sex was no longer statistically significant, whilst the latter two variables remained significant (P = 0.004; P < 0.001). The mean (SD) baseline IRP in the non-hydronephrotic, unstented cohort was 14.19 (4.39) mmHg. Age, α-blockers and calcium channel blockers did not significantly influence IRP, and no measured variables influenced peristaltic activity. Immediately after ureteric stent insertion, IRP decreased (mean [SD] 15.18 [5.28] vs 16.76 [6.4] mmHg, P = 0.004), whilst peristaltic activity was maintained., Conclusions: Human in vivo mean (SD) baseline IRP is 14.19 (4.39) mmHg in normal kidneys and increases with both hydronephrosis and preoperative ureteric stenting. Mean (SD) peristaltic peak IRP values of 25.75 (17.9) mmHg are reached in the renal pelvis every 3-7 s and maintained in the early post-stent period., (© 2024 BJU International.)

The extracellular matrix plays a critical role in modulating cell behaviour in the developing and adult central nervous system influencing neural cell morphology, function and growth. Neurons and astrocytes, play vital roles in neural signalling and support respectively and respond to cues from the surrounding matrix environment. However, a better understanding of the impact of specific individual extracellular matrix proteins on both neurons and astrocytes is critical for advancing the development of matrix-based scaffolds for neural repair applications. This study aimed to provide an in-depth analysis of how different commonly used extracellular matrix proteins- laminin-1, Fn, collagen IV, and collagen I-affect the morphology and growth of trophic induced pluripotent stem cell (iPSC)-derived astrocyte progenitors and mouse motor neuron-like cells. Following a 7-day culture period, morphological assessments revealed that laminin-1, fibronectin, and collagen-IV, but not collagen I, promoted increased process extension and a stellate morphology in astrocytes, with collagen-IV yielding the greatest increases. Subsequent analysis of neurons grown on the different extracellular matrix proteins revealed a similar pattern with laminin-1, fibronectin, and collagen-IV supporting robust neurite outgrowth. fibronectin promoted the greatest increase in neurite extension, while collagen-I did not enhance neurite growth compared to poly-L-lysine controls. Super-resolution microscopy highlighted extracellular matrix-specific nanoscale changes in cytoskeletal organization, with distinct patterns of actin filament distribution where the three basement membrane-associated proteins (laminin-1, fibronectin, and collagen-IV) promoted the extension of fine cellular processes. Overall, this study demonstrates the potent effect of laminin-1, fibronectin and collagen-IV to promote both iPSC-derived astrocyte progenitor and neuronal growth, yielding detailed insights into the effect of extracellular matrix proteins on neural cell morphology at both the whole cell and nanoscale levels. The ability of laminin-1, collagen-IV and fibronectin to elicit strong growth-promoting effects highlight their suitability as optimal extracellular matrix proteins to incorporate into neurotrophic biomaterial scaffolds for the delivery of cell cargoes for neural repair., (© 2024 The Author(s). Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

Hydrogels with low toxicity, antimicrobial potency and shear-thinning behavior are promising materials to combat the modern challenges of increased infections. Here, we report on 8-arm star block copolypeptides based on poly(L-lysine), poly(L-tyrosine) and poly(S-benzyl-L-cysteine) blocks. Three star block copolypeptides were synthesized with poly(S-benzyl-L-cysteine) always forming the outer block. The inner block comprised either two individual blocks of poly(L-lysine) and poly(L-tyrosine) or a statistical block copolypeptide from both amino acids. The star block copolypeptides were synthesized by the Ring Opening Polymerization (ROP) of the protected amino acid N-carboxyanhydrides (NCAs), keeping the overall ratio of monomers constant. All star block copolypeptides formed hydrogels and Scanning Electron Microscopy (SEM) confirmed a porous morphology. The investigation of their viscoelastic characteristics, water uptake and syringe extrudability revealed superior properties of the star polypeptide with a statistical inner block of L-lysine and L-tyrosine. Further testing of this sample confirmed no cytotoxicity and demonstrated antimicrobial activity of 1.5-log and 2.6-log reduction in colony-forming units, CFU/mL, against colony-forming reference laboratory strains of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus , respectively. The results underline the importance of controlling structural arrangements in polypeptides to optimize their physical and biological properties.

Mobilizing endogenous progenitor cells to repair damaged tissue in situ has the potential to revolutionize the field of regenerative medicine, while the early establishment of a vascular network will ensure survival of newly generated tissue. In this study, a gene-activated scaffold containing a stromal derived factor 1α plasmid (pSDF1α), a pro-angiogenic gene that is also thought to be involved in the recruitment of mesenchymal stromal cells (MSCs) to sites of injury is described. It is shown that over-expression of SDF1α protein enhanced MSC recruitment and induced vessel-like structure formation by endothelial cells in vitro. When implanted subcutaneously, transcriptomic analysis reveals that endogenous MSCs are recruited and significant angiogenesis is stimulated. Just 1-week after implantation into a calvarial critical-sized bone defect, pSDF1α-activated scaffolds are recruited MSCs and rapidly activate angiogenic and osteogenic programs, upregulating Runx2, Dlx5, and Sp7. At the same time-point, pVEGF-activated scaffolds are recruited a variety of cell types, activating endochondral ossification. The early response induced by both scaffolds leads to complete bridging of the critical-sized bone defects within 4-weeks. The versatile cell-free gene-activated scaffold described in this study is capable of harnessing and enhancing the body's own regenerative capacity and has immense potential in a myriad of applications., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Introduction: There is currently limited guidance for researchers on Patient and Public Involvement (PPI) for preclinical spinal cord research, leading to uncertainty about design and implementation. This study aimed to develop evidence-informed principles to support preclinical spinal cord researchers to incorporate PPI into their research., Methods: This study used a modified Delphi method with the aim of establishing consensus on a set of principles for PPI in spinal cord research. Thirty-eight stakeholders including researchers, clinicians and people living with spinal cord injury took part in the expert panel. Participants were asked to rate their agreement with a series of statements relating to PPI in preclinical spinal cord research over two rounds. As part of Round 2, they were also asked to rate statements as essential or desirable., Results: Thirty-eight statements were included in Round 1, after which five statements were amended and two additional statements were added. After Round 2, consensus (> 75% agreement) was reached for a total of 27 principles, with 13 rated as essential and 14 rated as desirable. The principles with highest agreement related to diversity in representation among PPI contributors, clarity of the purpose of PPI and effective communication., Conclusion: This research developed a previously unavailable set of evidence-informed principles to inform PPI in preclinical spinal cord research. These principles provide guidance for researchers seeking to conduct PPI in preclinical spinal cord research and may also inform PPI in other preclinical disciplines., Patient and Public Involvement Statement: This study was conducted as part of a project aiming to develop PPI in preclinical spinal cord injury research associated with an ongoing research collaboration funded by the Irish Rugby Football Union Charitable Trust (IRFU CT) and the Science Foundation Ireland Centre for Advanced Materials and BioEngineering Research (SFI AMBER), with research conducted by the Tissue Engineering Research Group (TERG) at the RCSI University of Medicine and Health Sciences. The project aims to develop an advanced biomaterials platform for spinal cord repair and includes a PPI Advisory Panel comprising researchers, clinicians and seriously injured rugby players to oversee the work of the project. PPI is included in this study through the involvement of members of the PPI Advisory Panel in the conceptualisation of this research, review of findings, identification of key points for discussion and preparation of the study manuscript as co-authors., (© 2024 The Author(s). Health Expectations published by John Wiley & Sons Ltd.)

Background: The aim was to ascertain the impact of irrigation technique on human intrarenal pressure during retrograde intrarenal surgery., Methods: A parallel randomized trial recruited patients across three hospital sites. Patients undergoing retrograde intrarenal surgery for renal stone treatment with an 11/13-Fr ureteral access sheath were allocated randomly to 100 mmHg pressurized-bag (PB) or manual hand-pump (HP) irrigation. The primary outcome was mean procedural intrarenal pressure. Secondary outcomes included maximum intrarenal pressure, variance, visualization, HP force of usage, procedure duration, stone clearance, and clinical outcomes. Live intrarenal pressure monitoring was performed using a COMETTMII pressure guidewire, deployed cystoscopically to the renal pelvis. The operating team was blinded to the intrarenal pressure., Results: Thirty-eight patients were randomized between July and November 2023 (trial closure). The final analysis included 34 patients (PB 16; HP 18). Compared with PB irrigation, HP irrigation resulted in significantly higher mean intrarenal pressure (mean(s.d.) 62.29(27.45) versus 38.16(16.84) mmHg; 95% c.i. for difference in means (MD) 7.97 to 40.29 mmHg; P = 0.005) and maximum intrarenal pressure (192.71(106.23) versus 68.04(24.16) mmHg; 95% c.i. for MD 70.76 to 178.59 mmHg; P < 0.001), along with greater variance in intrarenal pressure (log transformed) (6.23(1.59) versus 4.60(1.30); 95% c.i. for MD 0.62 to 2.66; P = 0.001). Surgeon satisfaction with procedural vision reported on a scale of 10 was higher with PB compared with HP irrigation (mean(s.d.) 8.75(0.58) versus 6.28(1.27); 95% c.i. for MD 1.79 to 3.16; P < 0.001). Subjective HP usage force did not correlate significantly with transmitted intrarenal pressure (Pearson R = -0.15, P = 0.57). One patient (HP arm) developed urosepsis., Conclusion: Manual HP irrigation resulted in higher and more fluctuant intrarenal pressure trace (with inferior visual clarity) than 100-mmHg PB irrigation., Registration Number: osf.io/jmg2h (https://osf.io/)., (© The Author(s) 2024. Published by Oxford University Press on behalf of BJS Foundation Ltd.)

Background: Patient and public involvement in research (PPI) has many benefits including increasing relevance and impact. While using PPI in clinical research is now an established practice, the involvement of patients and the public in pre-clinical research, which takes place in a laboratory setting, has been less frequently described and presents specific challenges. This study aimed to explore the perspectives of seriously injured rugby players' who live with a spinal cord injury on PPI in pre-clinical research., Methods: Semi-structured interviews were conducted via telephone with 11 seriously injured rugby players living with spinal cord injury on the island of Ireland. A purposive sampling approach was used to identify participants. Selected individuals were invited to take part via gatekeeper in a charitable organisation that supports seriously injured rugby players. Interviews were transcribed verbatim and analysed thematically., Findings: Six themes were identified during analysis: 'appreciating potential benefits of PPI despite limited knowledge', 'the informed perspectives of people living with spinal cord injury can improve pre-clinical research relevance', 'making pre-clinical research more accessible reduces the potential for misunderstandings to occur', 'barriers to involvement include disinterest, accessibility issues, and fear of losing hope if results are negative', 'personal contact and dialogue helps people feel valued in pre-clinical research, and 'PPI can facilitate effective dissemination of pre-clinical research as desired by people living with spinal cord injury.', Conclusion: People affected by spinal cord injury in this study desire further involvement in pre-clinical spinal cord injury research through dialogue and contact with researchers. Sharing experiences of spinal cord injury can form the basis of PPI for pre-clinical spinal cord injury research., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Carroll et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The ureteric wall is a complex multi-layered structure. The ureter shows variation in passive mechanical properties, histological morphology and insertion forces along the anatomical length. Ureter mechanical properties also vary depending on the direction of tensile testing and the anatomical region tested. Compliance is greatest in the proximal ureter and lower in the distal ureter, which contributes to the role of the ureter as a high-resistance sphincter. Similar to other human tissues, the ureteric wall remodels with age, resulting in changes to the mechanical properties. The passive mechanical properties of the ureter vary between species, and variation in tissue storage and testing methods limits comparison across some studies. Knowledge of the morphological and mechanical properties of the ureteric wall can aid in understanding urine transport and safety thresholds in surgical techniques. Indeed, various factors alter the forces required to insert access sheaths or scopes into the ureter, including sheath diameter, safety wires and medications. Future studies on human ureteric tissue both in vivo and ex vivo are required to understand the mechanical properties of the ureter and how forces influence these properties. Testing of instrument insertion forces in humans with a focus on defining safe upper limits and techniques to reduce trauma are also needed. Last, evaluation of dilatation limits in the mid and proximal ureter and clarification of tensile strength anisotropy in human specimens are necessary., (© 2023. Springer Nature Limited.)

Messenger RNA (mRNA) represents a promising therapeutic tool in the field of tissue engineering for the fast and transient production of growth factors to support new tissue regeneration. However, one of the main challenges to optimizing its use is achieving efficient uptake and delivery to mesenchymal stem cells (MSCs), which have been long reported as difficult-to-transfect. The aim of this study was to systematically screen a range of nonviral vectors to identify optimal transfection conditions for mRNA delivery to MSCs. Furthermore, for the first time, we wanted to directly compare the protein expression profile from three different types of mRNA, namely, unmodified mRNA (uRNA), base-modified mRNA (modRNA), and self-amplifying mRNA (saRNA) in MSCs. A range of polymer- and lipid-based vectors were used to encapsulate mRNA and directly compared in terms of physicochemical properties as well as transfection efficiency and cytotoxicity in MSCs. We found that both lipid- and polymer-based materials were able to successfully condense and encapsulate mRNA into nanosized particles (<200 nm). The overall charge and encapsulation efficiency of the nanoparticles was dependent on the vector type as well as the vector:mRNA ratio. When screened in vitro , lipid-based vectors proved to be superior in terms of mRNA delivery to MSCs cultured in a 2D monolayer and from a 3D collagen-based scaffold with minimal effects on cell viability, thus opening the potential for scaffold-based mRNA delivery. Modified mRNA consistently showed the highest levels of protein expression in MSCs, demonstrating 1.2-fold and 5.6-fold increases versus uRNA and saRNA, respectively. In summary, we have fully optimized the nonviral delivery of mRNA to MSCs, determined the importance of careful selection of the mRNA type used, and highlighted the strong potential of mRNA for tissue engineering applications.

Tissue engineering approaches aim to provide biocompatible scaffold supports that allow healing to progress often in healthy tissue. In diabetic foot ulcers (DFUs), hyperglycemia impedes ulcer regeneration, due to complications involving accumulations of cellular methylglyoxal (MG), a key component of oxidated stress and premature cellular aging which further limits repair. In this study, we aim to reduce MG using a collagen-chondroitin sulfate gene-activated scaffold (GAS) containing the glyoxalase-1 gene (GLO-1) to scavenge MG and anti-fibrotic β-klotho to restore stem cell activity in diabetic adipose-derived stem cells (dADSCs). dADSCs were cultured on dual GAS constructs for 21 days in high-glucose media in vitro. Our results show that dADSCs cultured on dual GAS significantly reduced MG accumulation (-84%; p < 0.05) compared to the gene-free controls. Similar reductions in profibrotic proteins α-smooth muscle actin (-65%) and fibronectin (-76%; p < 0.05) were identified in dual GAS groups. Similar findings were observed in the expression of pro-scarring structural proteins collagen I (-62%), collagen IV (-70%) and collagen VII (-86%). A non-significant decrease in the expression of basement membrane protein E-cadherin (-59%) was noted; however, the dual GAS showed a significant increase in the expression of laminin (+300%). We conclude that dual GAS-containing Glo-1 and β-klotho had a synergistic MG detoxification and anti-fibrotic role in dADSC's. This may be beneficial to provide better wound healing in DFUs by controlling the diabetic environment and rejuvenating the diabetic stem cells towards improved wound healing.

Introduction: Patient and public involvement (PPI) in research is an embedded practice in clinical research, however, its role in preclinical or laboratory-based research is less well established and presents specific challenges. This study aimed to explore the perspectives of two key stakeholder groups, preclinical researchers and clinicians on PPI in preclinical research, using spinal cord research as a case study., Methods: Semi-structured interviews were conducted online with 11 clinicians and 11 preclinical researchers all working in the area of spinal cord injury (SCI). Interviews were transcribed verbatim and analysed thematically., Findings: Nine themes were developed through analysis. Participants' perspectives included that people living with SCI had a right to be involved, that PPI can improve the relevance of preclinical research, and that PPI can positively impact the experiences of researchers. They identified the distance between lab-based research and the daily experiences of living with SCI to be a barrier and proactive management of accessibility and the motivated and networked SCI community as key facilitators. To develop strong partnerships, participants suggested setting clear expectations, ensuring good communication, and demonstrating respect for the time of PPI contributors involved in the research., Conclusions: While traditionally PPI has been more commonly associated with clinical research, participants identified several potential benefits of PPI in preclinical spinal cord research that have applicability to preclinical researchers more broadly. Preclinical spinal researchers should explore how to include PPI in their work., Patient or Public Contribution: This study was conducted as part of a broader project aiming to develop an evidence base for preclinical PPI that draws on a 5-year preclinical research programme focused on the development of advanced biomaterials for spinal cord repair as a case study. A PPI Advisory Panel comprising seriously injured rugby players, clinicians, preclinical researchers, and PPI facilitators collaborated as co-authors on the conceptualisation, design of the interview protocol, data analysis and writing of this manuscript., (© 2024 The Authors. Health Expectations published by John Wiley & Sons Ltd.)

Treating bone infections and ensuring bone repair is one of the greatest global challenges of modern orthopedics, made complex by antimicrobial resistance (AMR) risks due to long-term antibiotic treatment and debilitating large bone defects following infected tissue removal. An ideal multi-faceted solution would will eradicate bacterial infection without long-term antibiotic use, simultaneously stimulating osteogenesis and angiogenesis. Here, a multifunctional collagen-based scaffold that addresses these needs by leveraging the potential of antibiotic-free antimicrobial nanoparticles (copper-doped bioactive glass, CuBG) to combat infection without contributing to AMR in conjunction with microRNA-based gene therapy (utilizing an inhibitor of microRNA-138) to stimulate both osteogenesis and angiogenesis, is developed. CuBG scaffolds reduce the attachment of gram-positive bacteria by over 80%, showcasing antimicrobial functionality. The antagomiR-138 nanoparticles induce osteogenesis of human mesenchymal stem cells in vitro and heal a large load-bearing defect in a rat femur when delivered on the scaffold. Combining both promising technologies results in a multifunctional antagomiR-138-activated CuBG scaffold inducing hMSC-mediated osteogenesis and stimulating vasculogenesis in an in vivo chick chorioallantoic membrane model. Overall, this multifunctional scaffold catalyzes killing mechanisms in bacteria while inducing bone repair through osteogenic and angiogenic coupling, making this platform a promising multi-functional strategy for treating and repairing complex bone infections., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

Fibroblasts are the most abundant cell type in dermal skin and keratinocytes are the most abundant cell type in the epidermis; both play a crucial role in wound remodeling and maturation. We aim to assess the functionality of a novel dual gene activated scaffold (GAS) on human adult dermal fibroblasts (hDFs) and see how the secretome produced could affect human dermal microvascular endothelial cells (HDMVECs) and human epidermal keratinocyte (hEKs) growth and epithelization. Our GAS is a collagen chondroitin sulfate scaffold loaded with pro-angiogenic stromal derived factor (SDF-1α) and/or an anti-aging β-Klotho plasmids. hDFs were grown on GAS for two weeks and compared to gene-free scaffolds. GAS produced a significantly better healing outcome in the fibroblasts than in the gene-free scaffold group. Among the GAS groups, the dual GAS induced the most potent pro-regenerative maturation in fibroblasts with a downregulation in proliferation (twofold, p < 0.05), fibrotic remodeling regulators TGF-β1 (1.43-fold, p < 0.01) and CTGF (1.4-fold, p < 0.05), fibrotic cellular protein α-SMA (twofold, p < 0.05), and fibronectin matrix deposition (twofold, p < 0.05). The dual GAS secretome also showed enhancements of paracrine keratinocyte pro-epithelializing ability (1.3-fold, p < 0.05); basement membrane regeneration through laminin (6.4-fold, p < 0.005) and collagen IV (8.7-fold, p < 0.005) deposition. Our findings demonstrate enhanced responses in dual GAS containing hDFs by proangiogenic SDF-1α and β-Klotho anti-fibrotic rejuvenating activities. This was demonstrated by activating hDFs on dual GAS to become anti-fibrotic in nature while eliciting wound repair basement membrane proteins; enhancing a proangiogenic HDMVECs paracrine signaling and greater epithelisation of hEKs., Competing Interests: The authors have no conflicts to disclose., (© 2024 Author(s).)

Objective: The aim of this study was to assess the in vivo efficacy of a novel regenerative collagen-based scaffold developed by the Royal College of Surgeons in Ireland in a chronic tympanic membrane perforation (TMP) using a chinchilla model., Methods: Bilateral TMPs were induced in 17 mixed gender chinchillas using tympanic membrane resection followed by a mixture of topical Mitomycin C and dexamethasone for 3 days. These were monitored with weekly otoscopy for 8 weeks. Animals were excluded if signs of infection developed in the follow up period (n = 8). At 8 weeks, intervention began and 18 TMPs were assigned to either treatment with the collagen-based scaffold (treated group) or spontaneous healing (control group). Animals were euthanized 6 weeks post-intervention. Otoscopic imaging and auditory brain response (ABR) were conducted at baseline, 8 weeks post-TMP induction and 6 weeks post-intervention. All TMPs were then evaluated at 6 weeks post-intervention and bullae underwent histologic evaluation., Results: At 6 weeks post-intervention, otoscopic imaging demonstrated various degrees of healing in the treated ears. The treated group was noted to have an increased rate of healing when compared to the control group. Histologic evaluation demonstrated a variation in the degree of perforation healing within groups, with some animals in the treated group showing high levels of perforation healing. At 8 weeks after the TMP procedure, most of the animals had worsened hearing response. At 6-week post the collagen-based scaffold treatment, about 50 % (4/8) of the treated ears had improved in hearing response as compared to those of non-treated ears., Conclusion: Given the initial histologic evidence of partial healing in scaffold-treated ears, the post-intervention period should be extended to monitor the potential for complete healing. Given the overall positive findings related to healing with the scaffold-treated ears, this material warrants further investigation., Competing Interests: Declaration of competing interest None., (Copyright © 2023. Published by Elsevier B.V.)

Very large bone defects significantly diminish the vascular, blood, and nutrient supply to the injured site, reducing the bone's ability to self-regenerate and complicating treatment. Delivering nanomedicines from biomaterial scaffolds that induce host cells to produce bone-healing proteins is emerging as an appealing solution for treating these challenging defects. In this context, microRNA-26a mimics (miR-26a) are particularly interesting as they target the two most relevant processes in bone regeneration-angiogenesis and osteogenesis. However, the main limitation of microRNAs is their poor stability and issues with cytosolic delivery. Thus, utilising a collagen-nanohydroxyapatite (coll-nHA) scaffold in combination with cell-penetrating peptide (RALA) nanoparticles, we aimed to develop an effective system to deliver miR-26a nanoparticles to regenerate bone defects in vivo. The microRNA-26a complexed RALA nanoparticles, which showed the highest transfection efficiency, were incorporated into collagen-nanohydroxyapatite scaffolds and in vitro assessment demonstrated the miR-26a-activated scaffolds effectively transfected human mesenchymal stem cells (hMSCs) resulting in enhanced production of vascular endothelial growth factor, increased alkaline phosphatase activity, and greater mineralisation. After implantation in critical-sized rat calvarial defects, micro CT and histomorphological analysis revealed that the miR-26a-activated scaffolds improved bone repair in vivo, producing new bone of superior quality, which was highly mineralised and vascularised compared to a miR-free scaffold. This innovative combination of osteogenic collagen-nanohydroxyapatite scaffolds with multifunctional microRNA-26a complexed nanoparticles provides an effective carrier delivering nanoparticles locally with high efficacy and minimal off-target effects and demonstrates the potential of targeting osteogenic-angiogenic coupling using scaffold-based nanomedicine delivery as a new "off-the-shelf" product capable of healing complex bone injuries., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Fergal O'Brien reports financial support was provided by Science Foundation Ireland. Fergal O'Brien reports financial support was provided by European Research Council. Joanna Sadowska reports financial support was provided by European Commission. Joanna Sadowska reports financial support was provided by ON Foundation. Seth Donahue reports financial support was provided by National Science Foundation. Helen O. McCarthy reports financial support was provided by Science Foundation Ireland. Helen O. McCarthy has patent #WO2014087023 issued to Helen O. McCarthy., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Angiogenesis is critical for successful bone repair, and interestingly, miR-210 and miR-16 possess counter-active targets involved in both angiogenesis and osteogenesis: miR-210 acts as an activator by silencing EFNA3 & AcvR1b, while miR-16 inhibits both pathways by silencing VEGF & Smad5. It was thus hypothesized that dual delivery of both a miR-210 mimic and a miR-16 inhibitor from a collagen-nanohydroxyapatite scaffold system may hold significant potential for bone repair. Therefore, this systems potential to rapidly accelerate bone repair by directing enhanced angiogenic-osteogenic coupling in host cells in a rat calvarial defect model at a very early 4 week timepoint was assessed. In vitro, the treatment significantly enhanced angiogenic-osteogenic coupling of human mesenchymal stem cells, with enhanced calcium deposition after just 10 days in 2D and 14 days on scaffolds. In vivo, these dual-miRNA loaded scaffolds showed more than double bone volume and vessel recruitment increased 2.3 fold over the miRNA-free scaffolds. Overall, this study demonstrates the successful development of a dual-miRNA mimic/inhibitor scaffold for enhanced in vivo bone repair for the first time, and the possibility of extending this 'off-the-shelf' platform system to applications beyond bone offers immense potential to impact a myriad of other tissue engineering areas. STATEMENT OF SIGNIFICANCE: miRNAs have potential as a new class of bone healing therapeutics as they can enhance the regenerative capacity of bone-forming cells. However, angiogenic-osteogenic coupling is critical for successful bone repair. Therefore, this study harnesses the delivery of miR-210, known to be an activator of both angiogenesis and osteogenesis, and miR-16 inhibitor, as miR-16 is known to inhibit both pathways, from a collagen-nanohydroxyapatite scaffold system to rapidly enhance osteogenesis in vitro and bone repair in vivo in a rat calvarial defect model. Overall, it describes the successful development of the first dual-miRNA mimic/inhibitor scaffold for enhanced in vivo bone repair. This 'off-the-shelf' platform system offers immense potential to extend beyond bone applications and impact a myriad of other tissue engineering areas., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: To evaluate the pressure range generated in the human renal collecting system during ureteroscopy (URS), in a large patient sample, and to investigate a relationship between intrarenal pressure (IRP) and outcome., Patients and Methods: A prospective multi-institutional study was conducted, with ethics board approval; February 2022-March 2023. Recruitment was of 120 consecutive consenting adult patients undergoing semi-rigid URS and/or flexible ureterorenoscopy (FURS) for urolithiasis or diagnostic purposes. Retrograde, fluoroscopy-guided insertion of a 0.036-cm (0.014″) pressure guidewire (COMET™ II, Boston Scientific, Marlborough, MA, USA) to the renal pelvis was performed. Baseline and continuous ureteroscopic IRP was recorded, alongside relevant operative variables. A 30-day follow-up was completed. Descriptive statistics were applied to IRP traces, with mean (sd) and maximum values and variance reported. Relationships between IRP and technical variables, and IRP and clinical outcome were interrogated using the chi-square test and independent samples t-test., Results: A total of 430 pressure traces were analysed from 120 patient episodes. The mean (sd) baseline IRP was 16.45 (5.99) mmHg and the intraoperative IRP varied by technique. The mean (sd) IRP during semi-rigid URS with gravity irrigation was 34.93 (11.66) mmHg. FURS resulted in variable IRP values: from a mean (sd) of 26.78 (5.84) mmHg (gravity irrigation; 12/14-F ureteric access sheath [UAS]) to 87.27 (66.85) mmHg (200 mmHg pressurised-bag irrigation; 11/13-F UAS). The highest single pressure peak was 334.2 mmHg, during retrograde pyelography. Six patients (5%) developed postoperative urosepsis; these patients had significantly higher IRPs during FURS (mean [sd] 81.7 [49.52] mmHg) than controls (38.53 [22.6] mmHg; P < 0.001)., Conclusions: A dynamic IRP profile is observed during human in vivo URS, with IRP frequently exceeding expected thresholds. A relationship appears to exist between elevated IRP and postoperative urosepsis., (© 2023 The Authors. BJU International published by John Wiley & Sons Ltd on behalf of BJU International.)

Objectives: To explore beliefs and practice patterns of urologists regarding intrarenal pressure (IRP) during ureteroscopy (URS). Methods: A customized questionnaire was designed in a 4-step iterative process incorporating a systematic review of the literature and critical analysis of topics/questions by six endourologists. The 19-item questionnaire interrogated perceptions, practice patterns, and key areas of uncertainty regarding ureteroscopic IRP, and was disseminated via urologic societies, networks, and social media to the international urologic community. Consultants/attendings and trainees currently practicing urology were eligible to respond. Quantitative responses were compiled and analyzed using descriptive statistics and chi-square test, with subgroup analysis by procedure volume. Results: Responses were received from 522 urologists, practicing in six continents. The individual question response rate was >97%. Most (83.9%, 437/515) respondents were practicing at a consultant/attending level. An endourology fellowship incorporating stone management had been completed by 59.2% (307/519). The vast majority of respondents (85.4%, 446/520) scored the perceived clinical significance of IRP during URS ≥7/10 on a Likert scale. Concern was uniformly reported, with no difference between respondents with and without a high annual case volume ( p  = 0.16). Potential adverse outcomes respondents associated with elevated ureteroscopic IRP were urosepsis (96.2%, 501/520), collecting system rupture (80.8%, 421/520), postoperative pain (67%, 349/520), bleeding (63.72%, 332/520), and long-term renal damage (26.1%, 136/520). Almost all participants (96.2%, 501/520) used measures aiming to reduce IRP during URS. Regarding the perceived maximum acceptable threshold for mean IRP during URS, 30 mm Hg (40 cm H 2 O) was most frequently selected [23.2% (119/463)], with most participants (78.2%, 341/463) choosing a value ≤40 mm Hg. Conclusions: This is the first large-scale analysis of urologists' perceptions of ureteroscopic IRP. It identifies high levels of concern among the global urologic community, with almost unanimous agreement that elevated IRP is associated with adverse clinical outcomes. Equipoise remains regarding appropriate IRP limits intraoperatively and the most appropriate technical strategies to ensure adherence to these.

Pressure ulcers (PUs) remain a chronic health problem with severe impacts on healthcare systems. Early detection is crucial to providing effective interventions. However, detecting PUs currently relies on subjective tissue evaluations, such as visual skin assessment, precluding interventions prior to the development of visible tissue damage. There is an unmet need for solutions that can detect early tissue damage before visual and tactile signs occur. Assessments based on sub-epidermal moisture (SEM) measurements represent an opportunity for robust and objective early detection of PUs, preventing broken skin PUs in more high-risk patients at high-risk anatomical locations. While SEM assessment technology has been validated in computational, bench and tissue phantom models, validation in soft tissue was absent. In this study, we successfully validated the ability of a commercially available SEM assessment device to measure and detect sub-epidermal moisture changes in a novel ex vivo porcine soft tissue model of localised oedema. When controlled and incremental fluid volumes (Phosphate Buffer Solution) were injected into porcine soft tissues, statistically significant differences were found in SEM values between fluid-injected sites, representing an inflammatory oedematous condition, and healthy tissue control sites, as measured by the SEM device. The device provided reproducible readings by detecting localised oedema changes in soft tissues, reflecting the build-up of fluid as small as 1 ml into the underlying tissue. Spatial characterization experiments described the ability of the device technology to differentiate between healthy and oedematous tissue. Our findings validate the use of SEM assessment technology to measure and quantify localized oedema., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Current treatments for repairing articular cartilage defects are limited. However, pro-chondrogenic hydrogels formulated using articular cartilage matrix components (such as hyaluronic acid (HA) and collagen type II (Col II)), offer a potential solution if they could be injected into the defect via minimally invasive arthroscopic procedures, or used as bioinks to 3D print patient-specific customised regenerative scaffolds-potentially combined with cells. However, HA and Col II are difficult to incorporate into injectable/3D printable hydrogels due to poor physicochemical properties. This study aimed to overcome this by developing an articular cartilage matrix-inspired pro-chondrogenic hydrogel with improved physicochemical properties for both injectable and 3D printing (3DP) applications. To achieve this, HA was methacrylated to improve mechanical properties and mixed in a 1:1 ratio with Col I, a Col I/Col II blend or Col II. Col I possesses superior mechanical properties to Col II and so was hypothesised to enhance hydrogel mechanical properties. Rheological analysis showed that the pre-gels had viscoelastic and shear thinning properties. Subsequent physicochemical analysis of the crosslinked hydrogels showed that Col II inclusion resulted in a more swollen and softer polymer network, without affecting degradation time. While all hydrogels exhibited exemplary injectability, only the Col I-containing hydrogels had sufficient mechanical stability for 3DP applications. To facilitate 3DP of multi-layered scaffolds using methacrylated HA (MeHA)-Col I and MeHA-Col I/Col II, additional mechanical support in the form of a gelatin slurry support bath freeform reversible embedding of suspended hydrogels was utilised. Biological analysis revealed that Col II inclusion enhanced hydrogel-embedded MSC chondrogenesis, thus MeHA-Col II was selected as the optimal injectable hydrogel, and MeHA-Col I/Col II as the preferred bioink. In summary, this study demonstrates how tailoring biomaterial composition and physicochemical properties enables development of pro-chondrogenic hydrogels with potential for minimally invasive delivery to injured articular joints or 3DP of customised regenerative implants for cartilage repair., (Creative Commons Attribution license.)

Introduction: Recent decades have seen a move to minimally invasive techniques to manage urolithiasis. Trainees are expected to develop competency in common endourology procedures. Knowledge of ureter mechanics and the theory behind new technologies is important to ensure safe and efficient techniques. We aim to evaluate the exposure to endourology, self-reported competency in common techniques and knowledge of basic ureter biomechanics and technology in training urologists. Methods: An online survey was circulated to all training urologists in the Republic of Ireland. Questions focused on self-reported competency, clinical knowledge, ureter mechanical properties and laser technology. Results: Thirty responses were received with a range of 1-8 years of urology experience (mean=4 years). The respondents reported high levels of exposure to endourology with the majority reporting competency in flexible ureterorenoscopy (FURS) (n=18, 60%) and semi-rigid ureteroscopy (URS) (n=21, 70%). The respondents demonstrated good clinical knowledge but variable knowledge of laser settings, laser thermodynamics and ureter mechanics. Half of the respondents (n=15, 50%) correctly described fragmentation laser settings, with 10 trainees (n=33%) accurately identifying both factors that increase ureteral access sheath (UAS) insertion force. Most of the respondents (n=20, 67%) described the proximal ureter as the site with the greatest compliance, while the site of the greatest force during ureteroscope insertion was correctly identified by 17% (n=5). Conclusion: To our knowledge, this represents the first study evaluating urologist understanding of laser technology and the mechanical properties of the human ureter. Despite trainees reporting high levels of experience in endourology, there is a variable understanding of the principles of laser technology and ureter mechanics. Further research and education are needed with a focus on laser safety, suitable laser settings and the safe limit of insertion forces., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 J Lasers Med Sci.)

Introduction: Attempted tracheal replacement efforts thus far have had very little success. Major limiting factors have been the inability to efficiently re-vascularise and mimic the mechanical properties of native tissue. The major objective of this study was to optimise a previously developed collagen-hyaluronic acid scaffold (CHyA-B), which has shown to facilitate the growth of respiratory cells in distinct regions, as a potential tracheal replacement device. Methods: A biodegradable thermoplastic polymer was 3D-printed into different designs and underwent multi-modal mechanical assessment. The 3D-printed constructs were incorporated into the CHyA-B scaffolds and subjected to in vitro and ex vivo vascularisation. Results: The polymeric backbone provided sufficient strength to the CHyA-B scaffold, with yield loads of 1.31-5.17 N/mm and flexural moduli of 0.13-0.26 MPa. Angiogenic growth factor release (VEGF and bFGF) and angiogenic gene upregulation (KDR, TEK-2 and ANG-1) was detected in composite scaffolds and remained sustainable up to 14 days. Confocal microscopy and histological sectioning confirmed the presence of infiltrating blood vessel throughout composite scaffolds both in vitro and ex vivo . Discussion: By addressing both the mechanical and physiological requirements of tracheal scaffolds, this work has begun to pave the way for a new therapeutic option for large tracheal defects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Khalid, Soriano, Lemoine, Cryan, O’Brien and O’Leary.)

Despite the glimmer of hope provided by the discovery and commercialization of bone morphogenetic protein-2 (BMP-2) as a bone graft substitute, side effects related to the use of supraphysiological doses have hindered its clinical usage. In this study, we compared the osteoinductive potential of BMP-2 homodimer with a heterodimer of BMP-2/7, both delivered via a collagen-hydroxyapatite (CHA) scaffold delivery system, with the aim to reduce the overall therapeutic BMP doses and the associated side-effects. We first show that the incorporation of hydroxyapatite in collagen-based BMP delivery systems is pivotal for achieving efficient BMP sequestration and controlled release. Using an ectopic implantation model, we then showed that the CHA+BMP-2/7 was more osteoinductive than CHA+BMP-2. Further evaluation of the molecular mechanisms responsible for this increased osteoinductivity at an early stage in the regeneration process indicated that the CHA+BMP-2/7 enhanced progenitor cell homing at the implantation site, upregulated the key transcriptomic determinants of bone formation, and increased the production of bone extracellular matrix components. Using fluorescently labelled BMP-2/7 and BMP-2, we demonstrated that the CHA scaffold provided a long-term delivery of both molecules for at least 20 days. Finally, using a rat femoral defect model, we showed that an ultra-low dose (0.5 µg) of BMP-2/7 accelerated fracture healing and performed at a level comparable to 20-times higher BMP-2 dose. Our results indicate that the sustained delivery of BMP-2/7 via a CHA scaffold could bring us a step closer in the quest for the use of physiological growth factor doses in fracture healing. STATEMENT OF SIGNIFICANCE: • Incorporation of hydroxyapatite (HA) in a collagen scaffold dramatically improves bone morphogenic protein (BMP) sequestration via biophysical interactions with BMP, thereby providing more controlled BMP release compared with pristine collagen. • We then investigate the molecular mechanisms responsible for increased osteoinductive potential of a heterodimer BMP-2/7 with is clinically used counterpart, the BMP-2 homodimer. • The superior osteoinductive properties of BMP-2/7 are a consequence of its direct positive effect on progenitor cell homing at the implantation site, which consequently leads to upregulation of cartilage and bone related genes and biochemical markers. • An ultra-low dose of BMP-2/7 delivered via a collagen-HA (CHA) scaffold leads to accelerated healing of a critical femoral defect in rats while a 20-times higher BMP-2 dose was required to achieve comparable results., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Deepak Bushan Raina reports financial support was provided by Maggie-Stephens Foundation. Deepak Bushan Raina reports financial support was provided by Sten K Johnson Foundation. Deepak Bushan Raina reports financial support was provided by Crafoord Foundation. Fergal J O'Brien reports financial support was provided by Health Research Board. Fergal J O'Brien reports financial support was provided by European Research Council. Fergal J O'Brien reports financial support was provided by EU BlueHuman Interreg Atlantic Area Project. Deepak Bushan Raina reports a relationship with Moroxite AB that includes: equity or stocks. Deepak Bushan Raina reports a relationship with Moroxite F AB that includes: equity or stocks. Fergal J O'Brien has patent issued to Locate Bio., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Chronic, nonhealing wounds in the form of diabetic foot ulcers (DFUs) are a major complication for diabetic patients. The inability of a DFU to heal appropriately leads to an open wound with a high risk of infection. Current standards of care fail to fully address either the underlying defective wound repair mechanism or the risk of microbial infection. Thus, it is clear that novel approaches are needed. One such approach is the use of multifunctional biomaterials as platforms to direct and promote wound healing. In this study, a biomimetic, bilayered antimicrobial collagen-based scaffold was developed to deal with the etiology of DFUs. An epidermal, antimicrobial collagen/chitosan film for the prevention of wound infection was combined with a dermal collagen-glycosaminoglycan scaffold, which serves to support angiogenesis in the wound environment and ultimately accelerate wound healing. Biophysical and biological characterization identified an 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide cross-linked bilayered scaffold to have the highest structural stability with similar mechanical properties to products on the market, exhibiting a similar structure to native skin, successfully inhibiting the growth and infiltration of Staphylococcus aureus and supporting the proliferation of epidermal cells on its surface. This bilayered scaffold also demonstrated the ability to support the proliferation of key cell types involved in vascularization, namely, induced pluripotent stem cell derived endothelial cells and supporting stromal cells, with early signs of organization of these cells into vascular structures, showing great promise for the promotion of angiogenesis. Taken together, the results indicate that the bilayered scaffold is an excellent candidate for enhancement of diabetic wound healing by preventing wound infection and supporting angiogenesis.

Objectives: To systematically review the literature to ascertain the upper tract pressures generated during endourology, the relevant influencing variables and clinical implications., Materials and Methods: A systematic review of the MEDLINE, Scopus and Cochrane databases was performed by two authors independently (S.C., N.D.). Studies reporting ureteric or intrarenal pressures (IRP) during semi-rigid ureteroscopy (URS)/flexible ureterorenoscopy (fURS)/percutaneous nephrolithotomy (PCNL)/miniaturized PCNL (mPCNL) in the period 1950-2021 were identified. Both in vitro and in vivo studies were considered for inclusion. Findings were independently screened for eligibility based on content, with disagreements resolved by author consensus. Data were assessed for bias and compiled based on predefined variables., Results: Fifty-two studies met the inclusion criteria. Mean IRP appeared to frequently exceed a previously proposed threshold of 40 cmH 2 O. Semi-rigid URS with low-pressure irrigation (gravity <1 m) resulted in a wide mean IRP range (lowest reported 6.9 cmH 2 O, highest mean 149.5 ± 6.2 cmH 2 O; animal models). The lowest mean observed with fURS without a ureteric access sheath (UAS) was 47.6 ± 4.1 cmH 2 O, with the maximum peak IRP being 557.4 cmH 2 O (in vivo human data). UAS placement significantly reduced IRP during fURS, but did not guarantee pressure control with hand-operated pump/syringe irrigation. Miniaturization of PCNL sheaths was associated with increased IRP; however, a wide mean human IRP range has been recorded with both mPCNL (lowest -6.8 ± 2.2 cmH 2 O [suction sheath]; highest 41.2 ± 5.3 cmH 2 O) and standard PCNL (lowest 6.5 cmH 2 O; highest 41.2 cmH 2 O). Use of continuous suction in mPCNL results in greater control of mean IRP, although short pressure peaks >40 cmH 2 O are not entirely prevented. Definitive conclusions are limited by heterogeneity in study design and results. Postoperative pain and pyrexia may be correlated with increased IRP, however, few in vivo studies correlate clinical outcome with measured IRP., Conclusions: Intrarenal pressure generated during upper tract endoscopy often exceeds 40 cmH 2 O. IRP is multifactorial in origin, with contributory variables discussed. Larger prospective human in vivo studies are required to further our understanding of IRP thresholds and clinical sequelae., (© 2022 The Authors. BJU International published by John Wiley & Sons Ltd on behalf of BJU International.)

A particular challenge to the field of neuroscience involves translating findings from 2D in vitro systems to 3D in vivo environments. Standardized cell culture environments that adequately reflect the properties of the central nervous system (CNS) such as the stiffness, protein composition, and microarchitecture in which to study 3D cell-cell and cell-matrix interactions are generally lacking for in vitro culture systems. In particular, there remains an unmet need for reproducible, low-cost, high-throughput, and physiologically relevant environments comprised of tissue-native matrix proteins for the study of CNS microenvironments in 3D. Advances in the field of biofabrication over the past number of years have facilitated the production and characterization of biomaterial-based scaffolds. Typically developed for tissue engineering applications, they also provide sophisticated environments in which to study cell-cell and cell-matrix interactions and have been used for 3D modeling for a range of tissues. Here, we describe a simple and scalable protocol for the production of biomimetic, highly porous freeze-dried hyaluronic acid scaffolds with tunable microarchitecture, stiffness, and protein composition. Furthermore, we describe several different approaches that can be used to characterize a range of physicochemical properties and how to employ the scaffolds for the 3D culture of sensitive CNS cells in vitro. Finally, we detail several approaches for the study of key cell responses within the 3D scaffold environments. Overall, this protocol describes the manufacture and testing of a biomimetic and tunable macroporous scaffold system for neuronal cell culture applications. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Scaffold manufacture Basic Protocol 2: Scaffold characterization Basic Protocol 3: Cell culture and analysis of neurons in scaffolds., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Damaged or diseased bone can be treated using autografts or a range of different bone grafting biomaterials, however limitations with such approaches has motivated increased interest in developmentally inspired bone tissue engineering (BTE) strategies that seek to recapitulate the process of endochondral ossification (EO) as a means of regenerating critically sized defects. The clinical translation of such strategies will require the engineering of scaled-up, geometrically defined hypertrophic cartilage grafts that can be rapidly vascularised and remodelled into bone in mechanically challenging defect environments. The goal of this study was to 3D bioprint mechanically reinforced cartilaginous templates and to assess their capacity to regenerate critically sized femoral bone defects. Human mesenchymal stem/stromal cells (hMSCs) were incorporated into fibrin based bioinks and bioprinted into polycaprolactone (PCL) frameworks to produce mechanically reinforced constructs. Chondrogenic priming of such hMSC laden constructs was required to support robust vascularisation and graft mineralisation in vivo following their subcutaneous implantation into nude mice. With a view towards maximising their potential to support endochondral bone regeneration, we next explored different in vitro culture regimes to produce chondrogenic and early hypertrophic engineered grafts. Following their implantation into femoral bone defects within transiently immunosuppressed rats, such bioprinted constructs were rapidly remodelled into bone in vivo, with early hypertrophic constructs supporting higher levels of vascularisation and bone formation compared to the chondrogenic constructs. Such early hypertrophic bioprinted constructs also supported higher levels of vascularisation and spatially distinct patterns of new formation compared to BMP-2 loaded collagen scaffolds (here used as a positive control). In conclusion, this study demonstrates that fibrin based bioinks support chondrogenesis of hMSCs in vitro, which enables the bioprinting of mechanically reinforced hypertrophic cartilaginous templates capable of supporting large bone defect regeneration. These results support the use of 3D bioprinting as a strategy to scale-up the engineering of developmentally inspired templates for BTE. STATEMENT OF SIGNIFICANCE: Despite the promise of developmentally inspired tissue engineering strategies for bone regeneration, there are still challenges that need to be addressed to enable clinical translation. This work reports the development and assessment (in vitro and in vivo) of a 3D bioprinting strategy to engineer mechanically-reinforced cartilaginous templates for large bone defect regeneration using human MSCs. Using distinct in vitro priming protocols, it was possible to generate cartilage grafts with altered phenotypes. More hypertrophic grafts, engineered in vitro using TGF-β3 and BMP-2, supported higher levels of blood vessel infiltration and accelerated bone regeneration in vivo. This study also identifies some of the advantages and disadvantages of such endochondral bone TE strategies over the direct delivery of BMP-2 from collagen-based scaffolds., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest associated with this article., (Copyright © 2022. Published by Elsevier Ltd.)

Assessment of cell cytotoxicity following transfection of cells with microRNA (miRNA) is an essential step in the evaluation of basic miRNA functional effects within cells in both 2D and 3D microenvironments. The lactate dehydrogenase (LDH) assay is a colorimetric assay that provides a basic, dependable method for determining cellular cytotoxicity through assessment of the level of plasma membrane damage in a cell population. Here, we describe the overexpression of miRNA in breast cancer cells when cultured in 3D collagen-based biomaterial scaffolds, achieved by Lipofectamine transfection, with subsequent examination of cell cytotoxicity using the LDH assay., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Patient and Public Involvement (PPI) in research aims to improve the quality, relevance and appropriateness of research. PPI has an established role in clinical research where there is evidence of benefit, and where policymakers and funders place continued emphasis on its inclusion. However, for preclinical research, PPI has not yet achieved the same level of integration. As more researchers, including our team, aim to include PPI in preclinical research, the development of an evidence-based approach is important. Therefore, this scoping review aimed to identify and map studies where PPI has been used in preclinical research and develop principles that can be applied in other projects., Methods: A scoping review was conducted to search the literature in Medline (PubMed), EMBASE, CINAHL, PsycInfo and Web of Science Core Collection to identify applied examples of preclinical PPI. Two independent reviewers conducted study selection and data extraction separately. Data were extracted relating to PPI in terms of (i) rationale and aims, (ii) approach used, (iii) benefits and challenges, (iv) impact and evaluation and (v) learning opportunities for preclinical PPI. Findings were reviewed collaboratively by PPI contributors and the research team to identify principles that could be applied to other projects., Results: Nine studies were included in the final review with the majority of included studies reporting PPI to improve the relevance of their research, using approaches such as PPI advisory panels and workshops. Researchers report several benefits and challenges, although evidence of formal evaluation is limited., Conclusion: Although currently there are few examples of preclinical research studies reporting empirical PPI activity, their findings may support those aiming to use PPI in preclinical research. Through collaborative analysis of the scoping review findings, several principles were developed that may be useful for other preclinical researchers., Patient or Public Contribution: This study was conducted as part of a broader project aiming to develop an evidence base for preclinical PPI that draws on a 5-year preclinical research programme focused on the development of advanced biomaterials for spinal cord repair as a case study. A PPI Advisory Panel comprising seriously injured rugby players, clinicians, preclinical researchers and PPI facilitators collaborated as co-authors on the conceptualization, execution and writing of this review, including refining the findings into the set of principles reported here., (© 2022 The Authors. Health Expectations published by John Wiley & Sons Ltd.)

Diabetic foot ulcers (DFU) are chronic wounds sustained by pathological fibroblasts and aberrant extracellular matrix (ECM). Porous collagen-based scaffolds (CS) have shown clinical promise for treating DFUs but may benefit from functional enhancements. Our previous work showed fibroblasts differentiated from induced pluripotent stem cells are an effective source of new ECM mimicking fetal matrix, which notably promotes scar-free healing. Likewise, functionalizing CS with this rejuvenated ECM showed potential for DFU healing. Here, we demonstrate for the first time an approach to DFU healing using biopsied cells from DFU patients, reprogramming those cells, and functionalizing CS with patient-specific ECM as a personalized acellular tissue engineered scaffold. We took a two-pronged approach: 1) direct ECM blending into scaffold fabrication; and 2) seeding scaffolds with reprogrammed fibroblasts for ECM deposition followed by decellularization. The decellularization approach reduced cell number requirements and maintained naturally deposited ECM proteins. Both approaches showed enhanced ECM deposition from DFU fibroblasts. Decellularized scaffolds additionally enhanced glycosaminoglycan deposition and subsequent vascularization. Finally, reprogrammed ECM scaffolds from patient-matched DFU fibroblasts outperformed those from healthy fibroblasts in several metrics, suggesting ECM is in fact able to redirect resident pathological fibroblasts in DFUs towards healing, and a patient-specific ECM signature may be beneficial.

In the clinical treatment of fractures, rhBMP-2 administration is associated with a well-established profile of side-effects, including osteolysis and ectopic bone formation, which are driven by pro-inflammatory processes triggered by the use of high doses. Immunomodulatory strategies could minimize the incidence of side-effects by enabling the use of lower, and safer, rhBMP-2 doses. This study investigated whether interleukin-1 receptor antagonist (IL-1Ra) can enhance the therapeutic efficacy of a low dose of rhBMP-2 in a weight-bearing femoral fracture healing model. Exogenous IL-1Ra, in combination with rhBMP-2, was delivered using a collagen-hydroxyapatite scaffold (CHA) to attenuate IL-1β produced in response to fracture. Femoral defects were treated with CHA scaffolds alone, or loaded with IL-1Ra (2.5 µg), rhBMP-2 (1 µg), IL-1Ra (2.5 µg) in combination with rhBMP-2 (1 µg). Bone healing was assessed over 14 weeks in comparison to control groups, empty defect, and a higher dose of rhBMP-2 (5 µg), which were recently demonstrated to lead to non-union, and successful bridging of the defect, respectively. The combination of IL-1Ra and rhBMP-2 led to significantly faster early bone formation, at both week 4 and 6, compared to a low dose of rhBMP-2 alone. By 14 weeks, the combination of IL-1Ra and a rhBMP-2 promoted full bridging of femurs, which were 3-fold more mechanically reliable compared to the femurs treated with a low dose of rhBMP-2 alone. Taken together, this study demonstrates that IL-1Ra can significantly enhance femoral bone healing when used in combination with a low dose of rhBMP-2. STATEMENT OF SIGNIFICANCE: Enabling the use of lower and safer doses of rhBMP-2, a potent inducer of bone formation, is of clinical relevance in orthopaedic medicine. In this study, the immunomodulatory interleukin-1 receptor antagonist (IL-1Ra) was investigated for its capacity to enhance the therapeutic efficacy of rhBMP-2 when used at lower doses in a weight-bearing femoral fracture healing model. The combination of IL-1Ra and rhBMP-2 led to significantly faster early bone formation, and resulted in more mechanically reliable healed femurs, compared to a low dose of rhBMP-2 alone. This demonstrates for the first time in a rat long bone healing model that IL-1Ra can significantly enhance bone healing when used in combination with a low dose of rhBMP-2., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Bone cells, in particular osteoblasts, are capable of communication with each other during bone growth and homeostasis. More recently it has become clear that they also communicate with other cell-types; including chondrocytes in articular cartilage. One way that this process is facilitated is by interstitial fluid movement within the pericellular and extracellular matrices. This stimulus is also an important mechanical signal in skeletal tissues, and is known to generate shear stresses at the micron-scale (known as fluid flow shear stresses (FFSS)). The primary aim of this study was to develop and characterize an in vitro bone-cartilage crosstalk system, to examine the effect of FFSS on these cell types. Specifically, we evaluated the response of osteoblasts and chondrocytes to FFSS and the effect of FFSS-induced soluble factors from the former, on the latter. This system will ultimately be used to help us understand the role of subchondral bone damage in articular cartilage degeneration. We also carried out a comparison of responses between cell lines and primary murine cells in this work. Our findings demonstrate that primary cells produce a more reliable and reproducible response to FFSS. Furthermore we found that at lower magnitudes , direct FFSS produces anabolic responses in both chondrocytes and osteoblasts, whereas higher levels produce more catabolic responses. Finally we show that exposure to osteoblast-derived factors in conditioned media experiments produced similarly catabolic changes in primary chondrocytes.

Introduction and Hypothesis: The use of polypropylene (PP) mesh for stress urinary incontinence (SUI) surgery has declined because of safety concerns. The aim of this study is to evaluate a biodegradable polycaprolactone (PCL) mesh and a PCL composite mesh tissue engineered with human uterine fibroblasts (HUFs) for SUI surgery by comparing mechanical properties and in vitro biocompatibility to commercially available PP and porcine dermis (PD)., Methods: The mechanical properties of four scaffold materials were evaluated: PCL, PCL-collagen-hyaluronic acid composite, acellular porcine dermal collagen (PD) (Pelvicol™) and polypropylene (Gynecare TVT™ Exact®). HUFs were seeded on separate scaffolds. After 7 and 14 days scaffolds were assessed for metabolic activity and cell proliferation using Alamar Blue, Live/Dead and PicoGreen assays. Soluble collagen production was evaluated using a Sircol assay., Results: PCL and the composite scaffold reached ultimate tensile strength (UTS) values closest to healthy pelvic floor tissue (PCL = 1.19 MPa; composite = 1.13 MPa; pelvic floor = 0.79 MPa; Lei et al. Int Urogynecol J Pelvic Floor Dysfunct. 18(6):603-7, 2007). Cells on PCL showed significantly greater cell viability than PP at day 7 (p < 0.0001). At D14 the composite scaffold showed significantly greater cell viability than PP (p = 0.0006). PCL was the best performing scaffold for soluble collagen production at day 14 (106.1 μg versus 13.04 μg for PP, p = 0.0173)., Conclusions: We have designed a biodegradable PCL mesh and a composite mesh which demonstrate better biocompatibility than PP and mechanical properties closer to that of healthy pelvic floor tissue. This in vitro study provides promising evidence that these two implants should be evaluated in animal and human trials., (© 2022. The Author(s).)

The ability to regenerate damaged cartilage capable of long-term performance in an active joint remains an unmet clinical challenge in regenerative medicine. Biomimetic scaffold biomaterials have shown some potential to direct effective cartilage-like formation and repair, albeit with limited clinical translation. In this context, type II collagen (CII)-containing scaffolds have been recently developed by our research group and have demonstrated significant chondrogenic capacity using murine cells. However, the ability of these CII-containing scaffolds to support improved longer-lasting cartilage repair with reduced calcified cartilage formation still needs to be assessed in order to elucidate their potential therapeutic benefit to patients. To this end, CII-containing scaffolds in presence or absence of hyaluronic acid (HyA) within a type I collagen (CI) network were manufactured and cultured with human mesenchymal stem cells (MSCs) in vitro under chondrogenic conditions for 28 days. Consistent with our previous study in rat cells, the results revealed enhanced cartilage-like formation in the biomimetic scaffolds. In addition, while the variable chondrogenic abilities of human MSCs isolated from different donors were highlighted, protein expression analysis illustrated consistent responses in terms of the deposition of key cartilage extracellular matrix (ECM) components. Specifically, CI/II-HyA scaffolds directed the greatest cell-mediated synthesis and accumulation in the matrices of type II collagen (a principal cartilage ECM component), and reduced deposition of type X collagen (a key protein associated with hypertrophic cartilage formation). Taken together, these results provide further evidence of the capability of these CI/II-HyA scaffolds to direct enhanced and longer-lasting cartilage repair in patients with reduced hypertrophic cartilage formation.