Optimizing the Delivery of mRNA to Mesenchymal Stem Cells for Tissue Engineering Applications.

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.

Dual Glyoxalase-1 and ß-Klotho Gene-Activated Scaffold Reduces Methylglyoxal and Reprograms Diabetic Adipose-Derived Stem Cells: Prospects in Improved Wound Healing.

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 ß-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 ß-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.

Dual delivery gene-activated scaffold directs fibroblast activity and keratinocyte epithelization.

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 ß-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-ß1 (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 ß-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.

A Multifunctional Scaffold for Bone Infection Treatment by Delivery of microRNA Therapeutics Combined With Antimicrobial Nanoparticles.

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.

Mechanical characteristics of the ureter and clinical implications.

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.

Chronic tympanic membrane perforation repair with a collagen-based scaffold: An in vivo model.

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.

Development of miR-26a-activated scaffold to promote healing of critical-sized bone defects through angiogenic and osteogenic mechanisms.

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.

An injectable and 3D printable pro-chondrogenic hyaluronic acid and collagen type II composite hydrogel for the repair of articular cartilage defects.

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.

Dual scaffold delivery of miR-210 mimic and miR-16 inhibitor enhances angiogenesis and osteogenesis to accelerate bone healing.

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.

In vivo ureteroscopic intrarenal pressures and clinical outcomes: a multi-institutional analysis of 120 consecutive patients.

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.

A Good Craftsperson Knows Their Tools: Understanding of Laser and Ureter Mechanics in Training Urologists.

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.

Perceptions and Practice Patterns of Urologists Relating to Intrarenal Pressure During Ureteroscopy: Findings from a Global Cross-Sectional Analysis.

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 H2O) 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.

Development of tissue-engineered tracheal scaffold with refined mechanical properties and vascularisation for tracheal regeneration.

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.

A Biomimetic, Bilayered Antimicrobial Collagen-Based Scaffold for Enhanced Healing of Complex Wound Conditions.

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.

Sustained delivery of a heterodimer bone morphogenetic protein-2/7 via a collagen hydroxyapatite scaffold accelerates and improves critical femoral defect healing.

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.

The Manufacture and Characterization of Biomimetic, Biomaterial-Based Scaffolds for Studying Physicochemical Interactions of Neural Cells in 3D Environments.

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.

Upper urinary tract pressures in endourology: a systematic review of range, variables and implications.

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 cmH2 O. Semi-rigid URS with low-pressure irrigation (gravity <1 m) resulted in a wide mean IRP range (lowest reported 6.9 cmH2 O, highest mean 149.5 ± 6.2 cmH2 O; animal models). The lowest mean observed with fURS without a ureteric access sheath (UAS) was 47.6 ± 4.1 cmH2 O, with the maximum peak IRP being 557.4 cmH2 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 cmH2 O [suction sheath]; highest 41.2 ± 5.3 cmH2 O) and standard PCNL (lowest 6.5 cmH2 O; highest 41.2 cmH2 O). Use of continuous suction in mPCNL results in greater control of mean IRP, although short pressure peaks >40 cmH2 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 cmH2 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.

3D bioprinting of cartilaginous templates for large bone defect healing.

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-ß3 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.

Assessment of Cell Cytotoxicity in 3D Biomaterial Scaffolds Following miRNA Transfection.

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.

Personalized Scaffolds for Diabetic Foot Ulcer Healing Using Extracellular Matrix from Induced Pluripotent Stem-Reprogrammed Patient Cells.

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.