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Nonunion fractures and large bone defects are significant targets for osteochondral tissue engineering strategies. A major hurdle in the use of these therapies is the foreign body response of the host. Herein, we report the development of a bone tissue engineering scaffold with the ability to release anti-inflammatory drugs, in the hope of evading this response. Porous, sintered scaffolds composed of poly(D,L-lactic acid-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) were prepared with and without the anti-inflammatory drug diclofenac sodium. Analysis of drug release over time demonstrated a profile suitable for the treatment of acute inflammation with ∼80% of drug released over the first 4 days and a subsequent release of around 0.2% per day. Effect of drug release was monitored using an in vitro osteoblast inflammation model, comprised of mouse primary calvarial osteoblasts stimulated with proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Levels of inflammation were monitored by cell viability and cellular production of nitric oxide (NO) and prostaglandin E2 (PGE2). The osteoblast inflammation model revealed that proinflammatory cytokine addition to the medium reduced cell viability to 33%, but the release of diclofenac sodium from scaffolds inhibited this effect with a final cell viability of ∼70%. However, releasing diclofenac sodium at high concentrations had a toxic effect on the cells. Proinflammatory cytokine addition led to increased NO and PGE2 production; diclofenac-sodium-releasing scaffolds inhibited NO release by ∼64% and PGE2 production by ∼52%, when the scaffold was loaded with the optimal concentration of drug. These observations demonstrate the potential use of PLGA/PEG scaffolds for localized delivery of anti-inflammatory drugs in bone tissue engineering applications.

Injectable scaffolds are of interest in the field of regenerative medicine because of their minimally invasive mode of delivery. For tissue repair applications, it is essential that such scaffolds have the mechanical properties, porosity and pore diameter to support the formation of new tissue. In the current study, porous poly(dl-lactic acid-co-glycolic acid) (PLGA) microspheres were fabricated with an average size of 84 ± 24 μm for use as injectable cell carriers. Treatment with ethanolic sodium hydroxide for 2 min was observed to increase surface porosity without causing the microsphere structure to disintegrate. This surface treatment also enabled the microspheres to fuse together at 37 °C to form scaffold structures. The average compressive strength of the scaffolds after 24 h at 37 °C was 0.9 ± 0.1 MPa, and the average Young’s modulus was 9.4 ± 1.2 MPa. Scaffold porosity levels were 81.6% on average, with a mean pore diameter of 54 ± 38 μm. This study demonstrates a method for fabricating porous PLGA microspheres that form solid porous scaffolds at body temperature, creating an injectable system capable of supporting NIH-3T3 cell attachment and proliferation in vitro., Acta Biomaterialia, 10 (12), ISSN:1742-7061, ISSN:1878-7568

Objectives/Hypothesis: To develop a porous, biodegradable scaffold for mastoid air-cell regeneration. Study Design: In vitro development of a temperature-sensitive poly(DL-lactic acid-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG) scaffold tailored for this application. Methods: Human mastoid bone microstructure and porosity were investigated using micro-computed tomography. PLGA/PEG-alginate scaffolds were developed, and scaffold porosity was assessed. Human bone marrow mesenchymal stem cells (hBM-MSCs) were cultured on the scaffolds in vitro. Scaffolds were loaded with ciprofloxacin, and release of ciprofloxacin over time in vitro was assessed. Results: Porosity of human mastoid bone was measured at 83% with an average pore size of 1.3 mm. PLGA/PEG-alginate scaffold porosity ranged from 43% to 78% depending on the alginate bead content. The hBM-MSCs proliferate on the scaffolds in vitro, and release of ciprofloxacin from the scaffolds was demonstrated over 7 to 10 weeks. Conclusions: The PLGA/PEG-alginate scaffolds developed in this study demonstrate similar structural features to human mastoid bone, support cell growth, and display sustained antibiotic release. These scaffolds may be of potential clinical use in mastoid air-cell regeneration. Further in vivo studies to assess the suitability of PLGA/PEG-alginate scaffolds for this application are required. Key Words: Scaffold, poly(DL-lactic acid-co-glycolic acid), alginate, mastoid, ciprofloxacin. Level of Evidence: N/A.

Graphical abstract Schematic figure showing the methods used to prepare Fit-C labelled gel-MA and the different surface treatment approaches used to modify the surface of PdlLGA microspheres with it., Injectable poly (dl-lactic-co-glycolic acid) (PdlLGA) microspheres are promising candidates as biodegradable controlled release carriers for drug and cell delivery applications; however, they have limited functional groups on the surface to enable dense grafting of tissue specific biocompatible molecules. In this study we have evaluated surface adsorption, entrapment and oxygen plasma treatment as three approaches to modify the surfaces of PdlLGA microspheres with gelatine methacrylate (gel-MA) as a biocompatible and photo cross-linkable macromolecule. Time of flight secondary ion mass spectroscopy (TOF SIMS) and X-ray photoelectron spectroscopy (XPS) were used to detect and quantify gel-MA on the surfaces. Fluorescent and scanning electron microscopies (SEM) were used to image the topographical changes. Human mesenchymal stem cells (hMSCs) of immortalised cell line were cultured on the surface of gel-MA modified PdlLGA microspheres and Presto-Blue assay was used to study the effect of different surface modifications on cell proliferation. Data analysis showed that the oxygen plasma treatment approach resulted in the highest density of gel-MA deposition. This study supports oxygen plasma treatment as a facile approach to modify the surface of injectable PdlLGA microspheres with macromolecules such as gel-MA to enhance proliferation rate of injected cells and potentially enable further grafting of tissue specific molecules. Statement of Significance Poly (dl lactic-co-glycolic) acid (PdlLGA) microspheres offer limited functional groups on their surface to enable proper grafting of tissue specific bioactive molecules. To overcome this limitation, previous approaches have suggested using alkaline solutions to introduce active groups to the surface; however, they may compromise surface topography and lose any potential surface patterns. Plasma polymerisation of bioactive monomers has been suggested to enhance surface biocompatibility; however, it is not applicable on low vapour pressure macromolecules such as most extracellular matrix (ECM) proteins and growth factors. This study aims to evaluate three different approaches to modify the surface of PdlLGA microspheres with gelatine-methacrylate (gel-MA) to enable further grafting of cross-linkable biomolecules without compromising the surface topography or the biocompatibility of the system.

Sustained and controlled delivery of growth factors, such as bone morphogenetic protein 2 (BMP-2), from polymer scaffolds has excellent potential for enhancing bone regeneration. The present study investigated the use of novel sintered polymer scaffolds prepared using temperature-sensitive PLGA/PEG particles. Growth factors can be incorporated into these scaffolds by mixing the reconstituted growth factor with the particles prior to sintering. The ability of the PLGA/PEG scaffolds to deliver BMP-2 in a controlled and sustained manner was assessed and the osteogenic potential of these scaffolds was determined in a mouse calvarial defect model. BMP-2 was released from the scaffolds in vitro over 3 weeks. On average, ca. 70% of the BMP-2 loaded into the scaffolds was released by the end of this time period. The released BMP-2 was shown to be active and to induce osteogenesis when used in a cell culture assay. A substantial increase in new bone volume of 55% was observed in a mouse calvarial defect model for BMP-2-loaded PLGA/PEG scaffolds compared to empty defect controls. An increase in new bone volume of 31% was observed for PLGA/PEG scaffolds without BMP-2, compared to empty defect controls. These results demonstrate the potential of novel PLGA/PEG scaffolds for sustained BMP-2 delivery for bone-regeneration applications. Copyright © 2012 John Wiley & Sons, Ltd.

The Publisher's final version can be found by following the DOI link. An injectable poly(DL-lactic-co-glycolic acid) (PLGA) system comprising both porous and protein-loaded microspheres capable of forming porous scaffolds at body temperature was developed for tissue regeneration purposes. Porous and non-porous (lysozyme loaded) PLGA microspheres were formulated to represent ‘low molecular weight’ 22–34 kDa, ‘intermediate molecular weight’ (IMW) 53 kDa and ‘high molecular weight’ 84–109 kDa PLGA microspheres. The respective average size of the microspheres was directly related to the polymer molecular weight. An initial burst release of lysozyme was observed from both microspheres and scaffolds on day 1. In the case of the lysozyme-loaded microspheres, this burst release was inversely related to the polymer molecular weight. Similarly, scaffolds loaded with 1 mg lysozyme/g of scaffold exhibited an inverse release relationship with polymer molecular weight. The burst release was highest amongst IMW scaffolds loaded with 2 and 3 mg/g. Sustained lysozyme release was observed after day 1 over 50 days (microspheres) and 30 days (scaffolds). The compressive strengths of the scaffolds were found to be inversely proportional to PLGA molecular weight at each lysozyme loading. Surface analysis indicated that some of the loaded lysozyme was distributed on the surfaces of the microspheres and thus responsible for the burst release observed. Overall the data demonstrates the potential of the scaffolds for use in tissue regeneration.

Introduction The median survival of patients with glioblastoma multiforme (astrocytoma grade 4) remains less than 18 months despite radical surgery, radiotherapy and systemic chemotherapy. Surgical implantation of chemotherapy eluting wafers into the resection cavity has been shown to improve length of survival but the current licensed therapy has several drawbacks. This paper investigates in vivo efficacy of a novel drug eluting paste in glioblastoma. Methods Poly(lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG) self-sintering paste was loaded with the chemotherapeutic agent etoposide and delivered surgically into partially resected tumours in a flank murine glioblastoma xenograft model. Results Surgical delivery of the paste was successful and practical, with no toxicity or surgical morbidity to the animals. The paste was retained in the tumour cavity, and preliminary results suggest a useful antitumour and antiangiogenic effect, particularly at higher doses. Bioluminescent imaging was not affected significantly by the presence of the paste in the tumour. Conclusions Chemotherapy loaded PLGA/PEG paste seems to be a promising technology capable of delivering active drugs into partially resected tumours. The preliminary results of this study suggest efficacy with no toxicity and will lead to larger scale efficacy studies in orthotopic glioblastoma models.

The enrichment of substrates/surfaces with selected functional groups, methyl (–CH3), allyl amine (–NH2), allyl alcohol (–OH) and acrylic acid (–COOH), can be used to trigger mesenchymal stem (MSC) cell differentiation into specified lineages, minimising the need for exogenous biological supplementation. We present the successful translation of this research phenomenon to an injectable two phase injectable PLGA system, utilising plasma techniques, for the repair of bone defects. Modified microspheres were characterised using water contact angel (WCA), X-ray Photon Spectroscopy (XPS) and scanning electron microscopy (SEM). When cultured in contact with MSCs in vitro, the ability of the modified particles, within the 2 phase system, to induce differentiation was characterised using quantitative assays for cell viability and histological analysis for key markers of differentiation throughout the entirety of the three dimensional scaffold. Biological analysis proved that selected modified microspheres have the ability to induce MSC osteogenic (-NH2 modified scaffolds) and chondrogenic (–OH modified scaffolds) differentiation throughout the entirety of the formed scaffold. Therefore optimised plasma modification of microspheres is an effective tool for the production of injectable systems for the repair of bone and cartilage defects.

Introduction\ud Polymer-based delivery systems offer innovative intra-cavity administration of drugs, with the potential to better target micro-deposits of cancer cells in brain parenchyma beyond the resected cavity. Here we evaluate clinical utility, toxicity and sustained drug release capability of a novel formulation of poly(lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) microparticles.\ud \ud Methods\ud PLGA/PEG microparticle-based matrices were molded around an ex vivo brain pseudo-resection cavity and analyzed using magnetic resonance imaging and computerized tomography. In vitro toxicity of the polymer was assessed using tumor and endothelial cells and drug release from trichostatin A-, etoposide- and methotrexate-loaded matrices was determined. To verify activity of released agents, tumor cells were seeded onto drug-loaded matrices and viability assessed.\ud \ud Results\ud PLGA/PEG matrices can be molded around a pseudo-resection cavity wall with no polymer-related artifact on clinical scans. The polymer withstands fractionated radiotherapy, with no disruption of microparticle structure. No toxicity was evident when tumor or endothelial cells were grown on control matrices in vitro. Trichostatin A, etoposide and methotrexate were released from the matrices over a 3-4 week period in vitro and etoposide released over 3 days in vivo, with released agents retaining cytotoxic capabilities. PLGA/PEG microparticle-based matrices molded around a resection cavity wall are distinguishable in clinical scanning modalities. Matrices are non-toxic in vitro suggesting good biocompatibility in vivo. Active trichostatin A, etoposide and methotrexate can be incorporated and released gradually from matrices, with radiotherapy unlikely to interfere with release.\ud \ud Conclusion\ud The PLGA/PEG delivery system offers an innovative intra-cavity approach to administer chemotherapeutics for improved local control of malignant brain tumors.

Biodegradable polymer scaffolds have great potential for regenerative medicine applications such as the repair of musculoskeletal tissues. Here, we describe the development of scaffolds that blend hydrogel components with thermoplastic materials, combining the unique properties of both components to create mouldable formulations. This study focuses on the structural and mechanical properties of the composite scaffolds, produced by combining temperature-sensitive poly(DL-lactic acid-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) particles with a hydrogel component [Pluronic F127, fibrin or hyaluronic acid (HyA)]. The composite formulations solidified over time at 37°C, with a significant increase (p > 0.05) in compressive strength observed from 15 min to 2 h at this temperature. The maximum compressive strength was 1.2 MPa for PLGA/PEG-Pluronic F127 scaffolds, 2.4 MPa for PLGA/ PEG-HyA scaffolds and 0.6 MPa for PLGA/PEG-fibrin scaffolds. Porosity for each of the PLGA/PEG-hydrogel formulations tested was between 50 and 51%. This study illustrates the ability to combine this thermoplastic PLGA/PEG system with hydrogels to fabricate composite scaffolds, and demonstrates that altering the particle to hydrogel ratio produces scaffolds with varying mechanical properties. Refereed/Peer-reviewed

Injectable scaffolds are a class of materials that stimulate the regeneration of functional tissue within the body. These materials are attracting interest in regenerative medicine because they allow tissue repair to occur after minimally invasive administration. From a chemistry perspective these materials present new challenges because they must convert from an injectable material to a solid or gel with appropriate kinetics and without damaging surrounding tissues. Furthermore, the material may have to carry living cells or sensitive drug molecules into the body. The demands placed on these materials have stimulated research into novel chemical and physical mechanisms of forming porous structures within aqueous conditions. This review examines the underlying chemistry of a number of classes of injectable scaffolds and sets out challenges for these materials in the future. Refereed/Peer-reviewed

INTRODUCTION: A thermo-setting biomaterial containing a depot of clinically relevant chemotherapeutics applied to the post-surgical resection cavity offers an opportunity for localised control of residual malignant glioma. Here we evaluate mouldable polymer matrices of poly(lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG) microparticles for tailored combination drug release. METHOD: Clinically-approved or experimental chemotherapeutics, alone and in combination, were loaded onto PLGA/PEG microparticle matrices, or encapsulated inside PLGA microspheres. Mono- and dual-therapy release rates were measured by liquid chromatography and stability verified by mass spectrometry. Cytotoxicity of released drugs was assessed using patient-derived invasive margin cells and flank mouse xenografts. RESULTS: Temozolomide (TMZ), etoposide (ETO) and methotrexate were released from PLGA/PEG alone or in dual combinations over 2 weeks in vitro, whilst irinotecan, cilengitide and imatinib released over a similar period as monotherapies. Cytotoxicity was not impaired by polymer interaction. Drug encapsulation in PLGA microspheres dampens the rapid burst release. PLGA/PEG overcomes TMZ instability, resulting in a 10-day release where the AIC active component was measured. In vivo ETO release markedly reduced glioma bioluminescence with longer-term survivors relative to resection-only controls. CONCLUSION: We have developed a PLGA-based platform technology to deliver multiple clinically relevant chemotherapy agents, including TMZ, from a single matrix. Prolonged release kinetics can be achieved by drug encapsulating drugs, potentially filling the 3 week therapy gap that currently exists between surgery and radiotherapy commencement.

The mechanical behaviour of polymer scaffolds plays a vital role in their successful use in bone tissue engineering. The present study utilised novel sintered polymer scaffolds prepared using temperature-sensitive poly(dl-lactic acid-co-glycolic acid)/poly(ethylene glycol) particles. The microstructure of these scaffolds was monitored under compressive strain by image-guided failure assessment (IGFA), which combined synchrotron radiation computed tomography (SR CT) and in situ micro-compression. Three-dimensional CT data sets of scaffolds subjected to a strain rate of 0.01%/s illustrated particle movement within the scaffolds with no deformation or cracking. When compressed using a higher strain rate of 0.02%/s particle movement was more pronounced and cracks between sintered particles were observed. The results from this study demonstrate that IGFA based on simultaneous SR CT imaging and micro-compression testing is a useful tool for assessing structural and mechanical scaffold properties, leading to further insight into structure–function relationships in scaffolds for bone tissue engineering applications.

Sustained and controlled delivery of growth factors, such as bone morphogenetic protein 2 (BMP-2), from polymer scaffolds has excellent potential for enhancing bone regeneration. The present study investigated the use of novel sintered polymer scaffolds prepared using temperature-sensitive PLGA/PEG particles. Growth factors can be incorporated into these scaffolds by mixing the reconstituted growth factor with the particles prior to sintering. The ability of the PLGA/PEG scaffolds to deliver BMP-2 in a controlled and sustained manner was assessed and the osteogenic potential of these scaffolds was determined in a mouse calvarial defect model. BMP-2 was released from the scaffolds in vitro over 3 weeks. On average, ca. 70% of the BMP-2 loaded into the scaffolds was released by the end of this time period. The released BMP-2 was shown to be active and to induce osteogenesis when used in a cell culture assay. A substantial increase in new bone volume of 55% was observed in a mouse calvarial defect model for BMP-2-loaded PLGA/PEG scaffolds compared to empty defect controls. An increase in new bone volume of 31% was observed for PLGA/PEG scaffolds without BMP-2, compared to empty defect controls. These results demonstrate the potential of novel PLGA/PEG scaffolds for sustained BMP-2 delivery for bone-regeneration applications.

Despite advances in surgery, radiation therapy, and chemotherapeutics, patients with malignant glioma have a dismal prognosis. The formations of aberrant tumour vasculature and glioma cell invasion are major obstacles for effective treatment. Angiogenesis is a key event in the progression of malignant gliomas, a process involving endothelial cell proliferation, migration, reorganization of extracellular matrix and tube formation. Such processes are regulated by the homeostatic balance between proangiogenic and antiangiogenic factors, most notably vascular endothelial growth factors (VEGFs) produced by glioma cells. Current strategies targeting VEGF-VEGF receptor signal transduction pathways, though effective in normalizing abnormal tumor vasculature, eventually result in tumor resistance whereby a highly infiltrative and invasive phenotype may be adopted. Here we review recent anti-angiogenic therapy for malignant glioma and highlight implantable devices and nano/microparticles as next-generation methods for chemotherapeutic delivery. Intrinsic and adaptive modes of glioma resistance to anti-angiogenic therapy will be discussed with particular focus on the glioma stem cell paradigm.

Objective To develop a biodegradable, modified-release antibiotic pellet capable of eradicating biofilms as a potential novel treatment for biofilm infections. Design Pellets containing poly(DL-lactic-co-glycolic acid) microparticles, rifampin and clindamycin hydrochloride (3.5%, 7%, or 28% antibiotic by weight), and carrier gel (carboxymethylcellulose or poloxamer 407) were tested in vitro. Drug release was assessed using serial plate transfer testing and high-performance liquid chromatography, and pellets were tested against biofilms in an in vitro model of Staphylococcus aureus biofilm grown on silicone. Results Serial plate transfer testing demonstrated continuing bacterial inhibition for up to 21 days for all pellets studied. High-performance liquid chromatography showed high levels of drug release for 2 to 4 days, with greatly reduced levels subsequently; continued measurable clindamycin (but not rifampin) release for up to 21 days was achieved. Pellets made with poloxamer released higher drug levels for a longer period. Irrespective of the carrier gel used, pellets containing 7% and 28% (but not 3.5%) antibiotic eradicated biofilms successfully. Conclusions Antibiotic pellets can release antibiotics for up to 21 days and are able to eradicate biofilms in an in vitro model. Use of modified-release antibiotic formulations in the middle ear as a treatment for biofilms appears to be a potentially promising new therapy for otitis media with effusion.

Open fractures are at risk of serious infection and, if infected, require several surgical interventions and courses of systemic antibiotics. We investigated a new injectable formulation that simultaneously hardens in vivo to form a porous scaffold for bone repair and delivers antibiotics at high concentrations to the local site of infection. Duration of antimicrobial activity against Staphylococcus aureus was determined using the serial plate transfer test. Ultimate compressive strength and porosity of the material was measured with and without antibiotics. The material was evaluated in vivo in an ovine medial femoral condyle defect model contaminated with S. aureus.. Sheep were sacrificed at either 2 or 13 weeks and the defect and surrounding bone assessed using micro-computed tomography and histology. Antimicrobial activity in vitro persisted for 19-21 days. Sheep with antibiotic-free material and bacteria became infected, while those with antibiotic-containing material and bacteria did not. Similarly, new bone growth was seen in uninoculated animals with plain polymer, and in those with antibiotic polymer with bacteria, but not in sheep with plain polymer and bacteria. The antibiotic-impregnated scaffolds were effective in preventing S. aureus infections whilst supporting bone growth and repair. If translated into clinical practice, this approach might reduce the need for systemic antibiotics.