Implantable Therapeutic Reservoir Systems for Diverse Clinical Applications in Large Animal Models

Regenerative medicine approaches, specifically stem cell technologies, have demonstrated significant potential to treat a diverse array of pathologies. However, such approaches have resulted in a modest clinical benefit, which may be attributed to poor cell retention/survival at the disease site. A delivery system that facilitates regional and repeated delivery to target tissues can provide enhanced clinical efficacy of cell therapies when localized delivery of high doses of cells is required. In this study, a new regenerative reservoir platform (Regenervoir) is described for use in large animal models, with relevance to cardiac, abdominal, and soft tissue pathologies. Regenervoir incorporates multiple novel design features essential for clinical translation, with a focus on scalability, mechanism of delivery, fixation to target tissue, and filling/refilling with a therapeutic cargo, and is demonstrated in an array of clinical applications that are easily translated to human studies. Regenervoir consists of a porous reservoir fabricated from a single material, a flexible thermoplastic polymer, capable of delivering cargo via fill lines to target tissues. A radiopaque shear thinning hydrogel can be delivered to the therapy reservoir and multiple fixation methods (laparoscopic tacks and cyanoacrylate bioadhesive) can be used to secure Regenervoir to target tissues through a minimally invasive approach.In this study, a new regenerative reservoir platform (Regenervoir) is described for use in large animal models that are easily translated to human studies, with relevance to cardiac, abdominal, and soft tissue pathologies. Regenervoir incorporates multiple novel design features essential for clinical translation, with a focus on scalability, mechanism of delivery, fixation, and filling/refilling with a therapeutic cargo.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155890/1/adhm202000305.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155890/2/adhm202000305_am.pd

Beta-alanine supplementation improves isometric, but not isotonic or isokinetic strength endurance in recreationally strength-trained young men

Background: β-alanine (BA) supplementation may be ergogenic during high intensity exercise, primarily due to the buffering of hydrogen cations, although the effects of beta-alanine supplementationon strength endurance are equivocal. Aim: To determine the effects of 4 weeks of beta-alanine supplementation on skeletal muscle endurance using a battery of performance tests. Methods: This study employed a parallel group, repeated measures, randomised, double-blinded and placebo controlled design. Twenty recreationally strength-trained healthy males completed tests of isotonic strength endurance (repeated bench and leg press), along with tests of isometric and isokinetic endurance conducted using an isokinetic dynamometer. Tests were performed before and after a 4 week intervention, comprising an intake of 6.4g.day-1 10 of BA (n = 9) or placebo (maltodextrin, n = 11). Results: Time-to-exhaustion during the isometric endurance test improved by ~17% in the BA group (p 0.1) were shown for any of the performance variables in the isokinetic test (peak torque, fatigue index, total work) nor for the total number of repetitions performed in the isotonic endurance tests (leg or bench press). Conclusions: Four weeks of BA supplementation (6.4 g.day-1 15 ) improved isometric, but not isokinetic or isotonic endurance performance

Thermal elevations of orthopaedic procedures: A bone cell perspective

Thermal elevations experienced by bone during orthopaedic procedures, such as cutting and drilling, exothermal reactions from bone cement, and thermal therapies such as tumour ablation, can result in thermal damage leading to death of bone cells (osteocytes, osteoblasts, osteoclasts and mesenchymal stem cells). Osteocytes are believed to act as orchestrators of bone remodeling by recruiting osteoclasts and activating nearby osteoblasts to control resorption and bone growth. Postsurgical healing of bone tissue is essential to ensure the success of an operation, and as such, a thorough biological understanding of the thermally induced responses of bone cells, in particular osteocytes, to clinically relevant thermal elevations is required. The global aim of this thesis is to discern the direct effects of clinically relevant temperatures on bone cell damage, mineralisation capacity and gene expression responses to initiate remodelling, using both in vitro and in vivo models. The complex hierarchical structure of bone tissue renders the characterisation of thermal elevations arising from surgical procedures a particularly challenging task. As such, the precise temperatures experienced within the bone, particularly by embedded osteocytes, have never been explored. The first study presented in this thesis used a combination of experimental thermal imaging technology and multi-scale computational models to predict temperatures in bone tissue and cells with respect to various cutting parameters. These predicted temperatures (45 - 60°C for 30 - 60 seconds) informed the experimental studies of subsequent Chapters, which investigated the effects of such thermal elevations on bone cells. Experimental methods were developed to determine thermally induced necrosis and apoptosis, mineralisation capacity and remodelling gene expression responses of bone cells in vitro. These studies demonstrated that thermally induced necrosis, apoptosis and viability were dependent on the degree of temperature elevation, the duration of exposure and the phenotype of the cell. Specifically, osteocyte-like MLO-Y4 cells were shown to be more resilient to heat-treatment than osteoblast-like MC3T3-E1 cells (as indicated by percentage apoptosis, necrosis, viability and population size), which may reflect the sensory role of native osteocytes in vivo, detecting damage and signalling to neighbouring cells to regulate bone healing. Additionally, it was shown that severe thermal elevations for clinically relevant durations enhanced mineralised matrix production by osteoprogenitors. Furthermore, a link between osteocyte thermal damage, and bone remodelling responses was identified, whereby heat treated MLO-Y4s elicited a remodelling response by activating nearby MLO-Y4s to initiate pro-osteoclastic and pro-osteoblastic gene expression, as well as directly inducing osteogenic differentiation of nearby pre-osteoblastic MSCs. Finally, a novel in vivo animal model established the vital role of native osteocytes in initiating the thermally induced responses in vivo, whereby heat treatment to 47°C and 60°C induced significant tissue death and osteocyte apoptosis, as well as pro-osteoclastic and pro-osteoblastic gene expression, and that these responses were dependent on the severity of heat treatment. Thus, the studies presented in this thesis provide a novel biological understanding of thermally induced cellular responses in bone tissue at temperatures relevant to orthopaedic cutting procedures. The information elucidated from this body of work will inform the future development of orthopaedic cutting techniques that can minimise thermal elevations in bone tissue in order to optimise postsurgical healing, but can also inform the development of thermally active materials and thermal therapies for treatment of bone pathologies

An injectable alginate/extra cellular matrix (ECM) hydrogel towards acellular treatment of heart failure

As treatments for myocardial infarction (MI) continue to improve, the population of people suffering from heart failure (HF) is rising significantly. Novel treatment strategies aimed at achieving long-term functional stabilisation and improvement in heart function post MI include the delivery of biomaterial hydrogels and myocardial matrix-based therapies to the left ventricle wall. Individually alginate hydrogels and myocardial matrix-based therapies are at the most advanced stages of commercial/clinical development for this potential treatment option. However, despite these individual successes, the potential synergistic effect gained by combining the two therapies remains unexplored. This study serves as a translational step in evaluating the minimally invasive delivery of dual acting alginate-based hydrogels to the heart. We have successfully developed new production methods for hybrid alginate/extracellular matrix (ECM) hydrogels. We have identified that the high G block alginate/ECM hybrid hydrogel has appropriate rheological and mechanical properties (1.6 KPa storage modulus, 29 KPa compressive modulus and 14 KPa dynamic modulus at day 1) and can be delivered using a minimally invasive delivery device. Furthermore, we have determined that these novel hydrogels are not cytotoxic and are capable of enhancing the metabolic activity of dermal fibroblasts in vitro (p < 0.01). Overall these results suggest that an effective minimally invasive HF treatment option could be achieved by combining alginate and ECM particles.AMCARE project funded by European Union’s ‘Seventh Framework’ Programme for research, technological development and demonstration under Grant Agreement no. NMP3-SME-2013-604531.peer-reviewed2019-12-0

An in vitro Model to Explore how Intermittent Actuation Alters Cellular Activity during the Foreign Body Response

The long-term performance of medical implants is hindered by the foreign body response (FBR). This protective mechanism is intended to isolate potentially harmful objects from the body. Ultimately, inflammatory cytokines such as TGF-β1 activate myofibroblasts, which deposit a hypo-permeable, collagen-rich fibrotic capsule (FC). This is problematic for implants designed for therapy delivery as the FC impedes molecular exchange. We previously reported that intermittent pneumatic actuation (IA) of an implanted reservoir can significantly reduce FC thickness in a 14-day rat model1. Recently, we impressively demonstrated that insulin diffusion kinetics were maintained at baseline levels for the entire eight-week study in a mouse model when implanted reservoirs received IA2. We hypothesise that actuation-induced tissue strain can modulate local inflammatory pathways: to investigate this, we examine cellular interactions that modulate collagen production under IA in vitro. Myofibroblasts (WPMY-1) were seeded onto reservoirs and cultured for 14 days under IA. Cell viability was evaluated using metabolic activity and cytotoxicity assays. Collagen (Sircol Soluble Collagen assay) and TGF-β1 (ELISA) production were compared to non-actuated controls. Analytical models of device strain were developed based on Von Kármán plate theory. We observed that IA did not negatively affect cell viability. Interestingly, IA induced a significant reduction (p=0.0463) in TGF-β1 production, followed by a significant reduction in collagen production after 9 (p=0.0132) and 14 (p=0.0038) days actuation compared to non-actuated controls. Models predict maximum radial and tangential strains of 3.9% during IA. We have demonstrated the compelling potential of IA to modulate the FBR, optimising the local implant environment for therapy diffusion1,2. In vitro, we have uncovered alterations in the TGF-β1 pathway leading to a reduction in collagen production by WPMY-1 cells exposed to IA. Ongoing work is investigating a wider range of inflammatory markers, and correlating strain predictions to cell and tissue responses to fully elucidate the mechanism of IA mediated FBR modulation.</p

A methylcellulose and collagen based temperature responsive hydrogel promotes encapsulated stem cell viability and proliferation in vitro

With the number of stem cell-based therapies emerging on the increase, the need for novel and efficient delivery technologies to enable therapies to remain in damaged tissue and exert their therapeutic benefit for extended periods, has become a key requirement for their translation. Hydrogels, and in particular, thermoresponsive hydrogels, have the potential to act as such delivery systems. Thermoresponsive hydrogels, which are polymer solutions that transform into a gel upon a temperature increase, have a number of applications in the biomedical field due to their tendency to maintain a liquid state at room temperature, thereby enabling minimally invasive administration and a subsequent ability to form a robust gel upon heating to physiological temperature. However, various hurdles must be overcome to increase the clinical translation of hydrogels as a stem cell delivery system, with barriers including their low tensile strength and their inadequate support of cell viability and attachment. In order to address these issues, a methylcellulose based hydrogel was formulated in combination with collagen and beta glycerophosphate, and key development issues such as injectability and sterilisation processes were examined. The polymer solution underwent thermogelation at similar to 36 degrees C as determined by rheological analysis, and when gelled, was sufficiently robust to resist significant disintegration in the presence of phosphate buffered saline (PBS) while concomitantly allowing for diffusion of methylene blue dye solution into the gel. We demonstrate that human mesenchymal stem cells (hMSCs) encapsulated within the gel remained viable and showed raised levels of dsDNA at increasing time points, an indication of cell proliferation. Mechanical testing showed the &amp;quot;injectability&amp;quot;, i.e. force required for delivery of the polymer solution through devices such as a syringe, needle or catheter. Sterilisation of the freeze-dried polymer wafer via gamma irradiation showed no adverse effects on the formed hydrogel characteristics. Taken together, these results indicate the potential of this gel as a clinically translatable delivery system for stem cells and therapeutic molecules in vivo

Thermally induced osteocyte damage initiates a remodelling signaling cascade

Thermal elevations experienced by bone during orthopaedic procedures, such as cutting and drilling, exothermal reactions from bone cement, and thermal therapies such as tumor ablation, can result in thermal damage leading to death of native bone cells (osteocytes, osteoblasts, osteoclasts and mesenchymal stem cells). Osteocytes are believed to be the orchestrators of bone remodeling, which recruit nearby osteoclast and osteoblasts to control resorption and bone growth in response to mechanical stimuli and physical damage. However, whether heat-induced osteocyte damage can directly elicit bone remodelling has yet to be determined. This study establishes the link between osteocyte thermal damage and the remodeling cascade. We show that osteocytes directly exposed to thermal elevations (47 degrees C for 1 minute) become significantly apoptotic and alter the expression of osteogenic genes (Opg and Cox2). The Rankl/Opg ratio is consistently down-regulated, at days 1, 3 and 7 in MLO-Y4s heat-treated to 47 degrees C for 1 minute. Additionally, the pro-osteoblastogenic signaling marker Cox2 is significantly up-regulated in heat-treated MLO-Y4s by day 7. Furthermore, secreted factors from heat-treated MLO-Y4s administered to MSCs using a novel co-culture system are shown to activate pre-osteoblastic MSCs to increase production of the pro-osteoblastic differentiation marker, alkaline phosphatase (day 7, 14), and calcium deposition (day 21). Most interestingly, an initial pro-osteoclastogenic signaling response (increase Rankl and Rankl/Opg ratio at day 1) followed by later stage pro-osteoblastogenic signaling (down-regulation in Rankl and the Rankl/Opg ratio and an up-regulation in Opg and Cox2 by day 7) was observed in non-heat-treated MLO-Y4s in co-culture when these were exposed to the biochemicals produced by heat-treated MLO-Y4s. Taken together, these results elucidate the vital role of osteocytes in detecting and responding to thermal damage by means of thermally induced apoptosis followed by a cascade of remodelling responses.Funding from the National University of Ireland, Galway Fellowship Scheme, the National University of Ireland Travelling Scholarships in Engineering, the European Research Council (ERC) (under grant no. 258992;BONEMECHBIO)

A methylcellulose and collagen based temperature responsive hydrogel promotes encapsulated stem cell viability and proliferation in vitro

With the number of stem cell-based therapies emerging on the increase, the need for novel and efficient delivery technologies to enable therapies to remain in damaged tissue and exert their therapeutic benefit for extended periods, has become a key requirement for their translation. Hydrogels, and in particular, thermoresponsive hydrogels, have the potential to act as such delivery systems. Thermoresponsive hydrogels, which are polymer solutions that transform into a gel upon a temperature increase, have a number of applications in the biomedical field due to their tendency to maintain a liquid state at room temperature, thereby enabling minimally invasive administration and a subsequent ability to form a robust gel upon heating to physiological temperature. However, various hurdles must be overcome to increase the clinical translation of hydrogels as a stem cell delivery system, with barriers including their low tensile strength and their inadequate support of cell viability and attachment. In order to address these issues, a methylcellulose based hydrogel was formulated in combination with collagen and beta glycerophosphate, and key development issues such as injectability and sterilisation processes were examined. The polymer solution underwent thermogelation at similar to 36 degrees C as determined by rheological analysis, and when gelled, was sufficiently robust to resist significant disintegration in the presence of phosphate buffered saline (PBS) while concomitantly allowing for diffusion of methylene blue dye solution into the gel. We demonstrate that human mesenchymal stem cells (hMSCs) encapsulated within the gel remained viable and showed raised levels of dsDNA at increasing time points, an indication of cell proliferation. Mechanical testing showed the &amp;quot;injectability&amp;quot;, i.e. force required for delivery of the polymer solution through devices such as a syringe, needle or catheter. Sterilisation of the freeze-dried polymer wafer via gamma irradiation showed no adverse effects on the formed hydrogel characteristics. Taken together, these results indicate the potential of this gel as a clinically translatable delivery system for stem cells and therapeutic molecules in vivo

Thermally induced osteocyte damage initiates a remodelling signaling cascade

Thermal elevations experienced by bone during orthopaedic procedures, such as cutting and drilling, exothermal reactions from bone cement, and thermal therapies such as tumor ablation, can result in thermal damage leading to death of native bone cells (osteocytes, osteoblasts, osteoclasts and mesenchymal stem cells). Osteocytes are believed to be the orchestrators of bone remodeling, which recruit nearby osteoclast and osteoblasts to control resorption and bone growth in response to mechanical stimuli and physical damage. However, whether heat-induced osteocyte damage can directly elicit bone remodelling has yet to be determined. This study establishes the link between osteocyte thermal damage and the remodeling cascade. We show that osteocytes directly exposed to thermal elevations (47 degrees C for 1 minute) become significantly apoptotic and alter the expression of osteogenic genes (Opg and Cox2). The Rankl/Opg ratio is consistently down-regulated, at days 1, 3 and 7 in MLO-Y4s heat-treated to 47 degrees C for 1 minute. Additionally, the pro-osteoblastogenic signaling marker Cox2 is significantly up-regulated in heat-treated MLO-Y4s by day 7. Furthermore, secreted factors from heat-treated MLO-Y4s administered to MSCs using a novel co-culture system are shown to activate pre-osteoblastic MSCs to increase production of the pro-osteoblastic differentiation marker, alkaline phosphatase (day 7, 14), and calcium deposition (day 21). Most interestingly, an initial pro-osteoclastogenic signaling response (increase Rankl and Rankl/Opg ratio at day 1) followed by later stage pro-osteoblastogenic signaling (down-regulation in Rankl and the Rankl/Opg ratio and an up-regulation in Opg and Cox2 by day 7) was observed in non-heat-treated MLO-Y4s in co-culture when these were exposed to the biochemicals produced by heat-treated MLO-Y4s. Taken together, these results elucidate the vital role of osteocytes in detecting and responding to thermal damage by means of thermally induced apoptosis followed by a cascade of remodelling responses