The In Vivo functionality of collagen-based scaffolds for orthopaedic tissue repair

Bone and articular cartilage are incredibly tough tissues with the ability to withstand repetitive stress throughout an individual’s lifetime. Unfortunately, their ability to heal after injury is finite, resulting in impaired function and degeneration. Despite considerable advances in modern surgical management, such as the use of auto- and allografting, the associated limitations with these approaches has motivated the development of alternative therapeutic interventions to replace or repair bone and cartilage, including tissue-engineered (TE) biomaterials. In relation to bone, a major obstacle to the in vivo use of biomaterials is ensuring an adequate blood supply to meet the metabolic demands of seeded o r invading cells following implantation. In the context of producing superior bone graft substitutes, the overall aim of this research, therefore, was to address the problem of vascularising TE biomaterials by improving the blood supply to collagen-based bone graft substitutes using two distinct vascularisation strategies; in vitro pre-vascularisation of scaffolds (Chapter 2) and an endochondral ossification-based vascularisation strategy (Chapters 3 and 4). In addition, the ability of a novel, multi-layered, cell-free collagen scaffold, that combines hydroxyapatite for bone repair and hyaluronic acid for cartilage repair, to enhance osteochondral repair was also investigated (Chapter 5). Within the Tissue Engineering Research Group in the RCSI, a series of collagenbased scaffolds have been developed for tissue regeneration. The composition and structure of these biocompatible biodegradable biomaterials has been tailored to provide biological, architectural, and mechanical cues to influence cell infiltration, differentiation and matrix synthesis in a variety of tissues. Throughout this thesis, these scaffolds served as templates for tissue formation in conjunction with seeded cells (Chapters 2, 3 and 4) o r as cell-free implants (Chapter 5). Mesenchymal stem cells (MSCs), with their multi-lineage differentiation potential, were used to establish conditions capable of promoting enhanced vascularisation of these scaffolds fo r bone repair using two distinct methods. In Chapter 2, human umbilical vein endothelial cells (HUVECs) alone, o r in combination with human MSCs, were used to create in vitro micro-vascular networks within collagen-chondroitin sulphate (CCS) scaffolds. These pre-vascularised constructs were shown to significantly enhance bone repair in a critical-sized rat calvarial defect, while the MSCs in the coculture group had an immunomodulatory effect on the host response. As an alternative method to in vitro pre-vascularisation, attention has recently focused on reproducing aspects of embryological skeletal development and fracture healing via endochondral ossification (ECO) to promote blood vessel invasion of TE constructs in vivo. In Chapter 3, it was demonstrated that MSCs seeded onto two distinct collagen-based scaffolds, a collagen-hyaluronic acid scaffold (CHyA) previously optimised for cartilage formation and a collagen-hydroxyapatite scaffold (CHA) previously optimised for bone formation, could be induced to follow an in vitro process similar to ECO. These constructs synthesised cartilage-specific matrix and were driven towards hypertrophy, resulting in the secretion of the proangiogenic growth factor VEGF, as well as mineralisation in vitro. Chapter 4 investigated the ability of this system to promote healing and bone formation in a critical-sized rat calvarial defect. Compared to traditional intramembranous ossification (IMO)-based approaches that aim to induce direct osteogenic differentiation in vitro, it was found that ECO-based constructs were capable of superior bone formation, construct remodelling and vascularisation in vivo. Despite a host of repair techniques used to treat chondral and osteochondral injuries, including cell-based and cell-free TE strategies, the generation of durable hyaline-like articular cartilage with appropriate functional properties remains to be achieved clinically. Chapter 5 of this thesis evaluated a multi-layered scaffold, based on seamlessly combining the CHyA and CHA scaffolds, which were investigated independently in Chapters 3 and 4, as a cell-free strategy fo r osteochondral defect repair. This biomimetic scaffold was found to facilitate cellular infiltration and region specific differentiation, resulting in the regeneration of tissue with several key features of native osteochondral tissue, thus confirming its potential as an off-theshelf device fo r direct implantation, overcoming several of the drawbacks associated with current treatments used in orthopaedic clinical practice. Collectively, Collectively, this study emphasises the significant in vivo capacity of both cell-seeded and cell-free collagen-based scaffolds developed in the RCSI Tissue Engineering Research Group to promote bone and cartilage regeneration using established animal models and highlights the clinical potential of these TE constructs fo r orthopaedic tissue repair applications

Long-term controlled delivery of rhBMP-2 from collagen-hydroxyapatite scaffolds for superior bone tissue regeneration.

The clinical utilization of recombinant human bone morphogenetic protein 2 (rhBMP-2) delivery systems for bone regeneration has been associated with very severe side effects, which are due to the non-controlled and non-targeted delivery of the growth factor from its collagen sponge carrier post-implantation which necessitates supraphysiological doses. However, rhBMP-2 presents outstanding regenerative properties and thus there is an unmet need for a biocompatible, fully resorbable delivery system for the controlled, targeted release of this protein. With this in mind, the purpose of this work was to design and develop a delivery system to release low rhBMP-2 doses from a collagen-hydroxyapatite (CHA) scaffold which had previously been optimized for bone regeneration and recently demonstrated significant healing in vivo. In order to enhance the potential for clinical translation by minimizing the design complexity and thus upscaling and regulatory hurdles of the device, a microparticle and chemical functionalization-free approach was chosen to fulfill this aim. RhBMP-2 was combined with a CHA scaffold using a lyophilization fabrication process to produce a highly porous CHA scaffold supporting the controlled release of the protein over the course of 21days while maintaining in vitro bioactivity as demonstrated by enhanced alkaline phosphatase activity and calcium production by preosteoblasts cultured on the scaffold. When implanted in vivo, these materials demonstrated increased levels of healing of critical-sized rat calvarial defects 8weeks post-implantation compared to an empty defect and unloaded CHA scaffold, without eliciting bone anomalies or adjacent bone resorption. These results demonstrate that it is possible to achieve bone regeneration using 30 times less rhBMP-2 than INFUSE®, the current clinical gold standard; thus, this work represents the first step of the development of a rhBMP-2 eluting material with immense clinical potential

Controlled release of vascular endothelial growth factor from spray-dried alginate microparticles in collagen-hydroxyapatite scaffolds for promoting vascularization and bone repair.

A major limitation with current tissue-engineering approaches is creating functionally vascularized constructs that can successfully integrate with the host; this often leads to implant failure, due to avascular necrosis. In order to overcome this, the objective of the present work was to develop a method to incorporate growth factor-eluting alginate microparticles (MPs) into freeze-dried, collagen-based scaffolds. A collagen-hydroxyapatite (CHA) scaffold, previously optimized for bone regeneration, was functionalized for the sustained delivery of an angiogenic growth factor, vascular endothelial growth factor (VEGF), with the aim of facilitating angiogenesis and enhancing bone regeneration. VEGF was initially encapsulated in alginate MPs by spray-drying, producing particles of \u3c 10 µm in diameter. This process was found to effectively encapsulate and control VEGF release while maintaining its stability and bioactivity post-processing. These VEGF-MPs were then incorporated into CHA scaffolds, leading to homogeneous distribution throughout the interconnected scaffold pore structure. The scaffolds were capable of sustained release of bioactive VEGF for up to 35 days, which was proficient at increasing tubule formation by endothelial cells in vitro. When implanted in vivo in a rat calvarial defect model, this scaffold enhanced vessel formation, resulting in increased bone regeneration compared to empty-defect and VEGF-free scaffolds. This biologically functionalized scaffold, composed entirely of natural-based materials, may offer an ideal platform to promote angiogenesis and tissue regeneration. Copyright © 2015 John Wiley \u26 Sons, Ltd

A DHODH inhibitor increases p53 synthesis and enhances tumor cell killing by p53 degradation blockage

ML, CD, IvL, GP, TM, SD, MS, APF, CT, DL, MAH, KL and SL: project grants from the Swedish Research Council, the Swedish Cancer Society and the Swedish Childhood Cancer Foundation. MHi and JC: Cancer Research UK (C8/A6613). MC, EP and WE: Wellcome Trust (073915). MN and BV: projects MEYS-NPS-LO1413 and GACR P206/12/G151. EMC, MP, MMS, ZF and PG: Norwegian Cancer Society (182735, 732200) and Helse Vest (911884, 911789). RB and SC: NIH (R01 CA95684), the Leukemia and Lymphoma Society and the Waxman Foundation. NW, AH, Ad’H: Cancer Research UK (C21383/A6950) and Engineering and Physical Sciences Research Council Doctoral Training Program. JL and YZ: Cancer Research UK (C240/A15751). MH and BW: SARomics Biostructures ABUY, KF: DDDP SciLife, Sweden. LJ, MHa, RS and A-LG: CBCS, Sweden. VP: SciLife fellowship. AT: Breast Cancer Research Scotland.The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect.Publisher PDFPeer reviewe

Development of collagen–hydroxyapatite scaffolds incorporating PLGA and alginate microparticles for the controlled delivery of rhBMP-2 for bone tissue engineering

The spatiotemporally controlled delivery of the pro-osteogenic factor rhBMP-2 would overcome most of the severe secondary effects linked to the products delivering this protein for bone regeneration. With this in mind, the aim of the present work was to develop a controlled rhBMP-2 release system using collagen-hydroxyapatite (CHA) scaffolds, which had been previously optimized for bone regeneration, as delivery platforms to produce a device with enhanced capacity for bone repair. Spray-drying and emulsion techniques were used to encapsulate bioactive rhBMP-2 in alginate and PLGA microparticles, with a high encapsulation efficiency. After incorporation of these microparticles into the scaffolds, rhBMP-2 was delivered in a sustained fashion for up to 28days. When tested in vitro with osteoblasts, these eluting materials showed an enhanced pro-osteogenic effect. From these results, an optimal rhBMP-2 eluting scaffold composition was selected and implanted in critical-sized calvarial defects in a rat model, where it demonstrated an excellent healing capacity in vivo. These platforms have an immense potential in the field of tissue regeneration; by tuning the specific therapeutic molecule to the tissue of interest and by utilizing different collagen-based scaffolds, similar systems can be developed for enhancing the healing of a diverse range of tissues and organs.</p

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care