An injectable degradable porous polymer scaffold for tissue engineering and drug delivery

Cell transplantation on biodegradable scaffolds is an established approach in tissue engineering to the problem of the regeneration of diseased or damaged tissues. As cells grow and organise themselves, they secrete their own extracellular matrix, while the polymer degrades into natural metabolites resulting in eventual natural tissue replacement. Polymeric materials used for these scaffolds must satisfy a number of requirements. These include defined cell-interactive properties, porosity, biodegradability, mechanical and controlled release properties. To date, scaffolds have been designed to conform to these requirements. However, the need to perform defined three-dimensional structures requires prior knowledge of the dimensions of the defect or cavity to be filled. Furthermore the general use of toxic solvents in the processing of these scaffolds prevents the incorporation of biological agents and cells during fabrication. Therefore, poor transportation of cells through the scaffolds can result in low cell seeding efficiencies. Finally such scaffolds require an invasive operation for transplantation of the material. In contrast a number of injectable materials have been proposed and investigated. The transformation from liquid pre-cursor to gel in such systems can, however, require cell harmful trigger signals such as UV exposure or pH changes. Furthermore, these injectable gels lack a porous structure preventing effective cell migration and restricting tissue formation and vascularisation tothe barrier of diffusion for signalling and nutrient molecules. The work in this thesis presents a scaffold that is both injectable and conforms to the requirements of water-insoluble porous scaffolds. This starts with the synthesis of a biotinylated poly (lactic acid)-poly (ethylene glycol) (PLA-PEG) copolymer. The polymer is degradable, protein resistant and cell interactive when used in conjunction with biotinylated cell adhesive peptides. The biotin unit tethered to the PEG-PLA also provides the polymer with self-assembling properties when used in conjunction with avidin. In contrast to alternative injectable materials, the scaffold presented in this thesis is porous. This porosity is necessary for tissue ingrowth and vascularization. Therefore, before progressing on to the manufacture of the scaffold, a systematic study of two cell types involved in vascularisation was carried out over defined pore features. These studies revealed that cell behaviour over pore features was related to cell type, cell density and pore size. This had significant implications for the injectable scaffold in development because proposed advantages were delivery of a variety of cell types, controlled porous structure, and efficient cell seeding. Microparticles were then manufactured from the PLA-PEG-biotin using a single emulsion manufacturing process. Surface Plasmon Resonance (SPR) confirmed that these microparticles would bind efficiently to avidin. The condition for optimum self-assembling of particles was then determined using aggregation studies. These studies showed that a critical quantity of avidin was required for microparticles to aggregate together. The ability to aggregate particles of different sizes leads to the potential for controlling scaffold porosity. Rheological testing showed that the scaffold's mechanical properties could be tailored to that of the tissue in which regeneration is required. The self-assembly of microparticles was also demonstrated to form complex three-dimensional scaffolds without the use of toxic solvents. Scaffolds prepared in simulated tissues maintained shape upon injection. Scaffolds were then self-assembled with cells entrapped within them. Cell viability within the self-assembling scaffolds was confirmed by Alamar Blue assays. In vivo studies have demonstrated that cell-scaffold composites permit tissue ingrowth and thus readily undergo vascularisation. The novel molecular-interaction mechanism of self-assembly of these scaffolds differentiates this material from other injectable systems. The formation of porous scaffolds within a cavity or a soft-tissue could be a pre-requisite for tissue remodelling using new cell sources that are dependent on vascularisation and tissue ingrowth. The basic component of the scaffold is a biodegradable microparticle that presents a protein resistant surface with biotinylated moieties. Therefore, standard controlled release technologies and biotin-avidin mediated surface engineering can be combined with the self-assembly to form biomimetic scaffolds that stimulate integrin-mediated cell adhesion and then release growth factors

An injectable degradable porous polymer scaffold for tissue engineering and drug delivery

Cell transplantation on biodegradable scaffolds is an established approach in tissue engineering to the problem of the regeneration of diseased or damaged tissues. As cells grow and organise themselves, they secrete their own extracellular matrix, while the polymer degrades into natural metabolites resulting in eventual natural tissue replacement. Polymeric materials used for these scaffolds must satisfy a number of requirements. These include defined cell-interactive properties, porosity, biodegradability, mechanical and controlled release properties. To date, scaffolds have been designed to conform to these requirements. However, the need to perform defined three-dimensional structures requires prior knowledge of the dimensions of the defect or cavity to be filled. Furthermore the general use of toxic solvents in the processing of these scaffolds prevents the incorporation of biological agents and cells during fabrication. Therefore, poor transportation of cells through the scaffolds can result in low cell seeding efficiencies. Finally such scaffolds require an invasive operation for transplantation of the material. In contrast a number of injectable materials have been proposed and investigated. The transformation from liquid pre-cursor to gel in such systems can, however, require cell harmful trigger signals such as UV exposure or pH changes. Furthermore, these injectable gels lack a porous structure preventing effective cell migration and restricting tissue formation and vascularisation tothe barrier of diffusion for signalling and nutrient molecules. The work in this thesis presents a scaffold that is both injectable and conforms to the requirements of water-insoluble porous scaffolds. This starts with the synthesis of a biotinylated poly (lactic acid)-poly (ethylene glycol) (PLA-PEG) copolymer. The polymer is degradable, protein resistant and cell interactive when used in conjunction with biotinylated cell adhesive peptides. The biotin unit tethered to the PEG-PLA also provides the polymer with self-assembling properties when used in conjunction with avidin. In contrast to alternative injectable materials, the scaffold presented in this thesis is porous. This porosity is necessary for tissue ingrowth and vascularization. Therefore, before progressing on to the manufacture of the scaffold, a systematic study of two cell types involved in vascularisation was carried out over defined pore features. These studies revealed that cell behaviour over pore features was related to cell type, cell density and pore size. This had significant implications for the injectable scaffold in development because proposed advantages were delivery of a variety of cell types, controlled porous structure, and efficient cell seeding. Microparticles were then manufactured from the PLA-PEG-biotin using a single emulsion manufacturing process. Surface Plasmon Resonance (SPR) confirmed that these microparticles would bind efficiently to avidin. The condition for optimum self-assembling of particles was then determined using aggregation studies. These studies showed that a critical quantity of avidin was required for microparticles to aggregate together. The ability to aggregate particles of different sizes leads to the potential for controlling scaffold porosity. Rheological testing showed that the scaffold's mechanical properties could be tailored to that of the tissue in which regeneration is required. The self-assembly of microparticles was also demonstrated to form complex three-dimensional scaffolds without the use of toxic solvents. Scaffolds prepared in simulated tissues maintained shape upon injection. Scaffolds were then self-assembled with cells entrapped within them. Cell viability within the self-assembling scaffolds was confirmed by Alamar Blue assays. In vivo studies have demonstrated that cell-scaffold composites permit tissue ingrowth and thus readily undergo vascularisation. The novel molecular-interaction mechanism of self-assembly of these scaffolds differentiates this material from other injectable systems. The formation of porous scaffolds within a cavity or a soft-tissue could be a pre-requisite for tissue remodelling using new cell sources that are dependent on vascularisation and tissue ingrowth. The basic component of the scaffold is a biodegradable microparticle that presents a protein resistant surface with biotinylated moieties. Therefore, standard controlled release technologies and biotin-avidin mediated surface engineering can be combined with the self-assembly to form biomimetic scaffolds that stimulate integrin-mediated cell adhesion and then release growth factors

Copper Oxide Nanoparticle Diameter Mediates Serum-Sensitive Toxicity in BEAS-2B Cells

Copper oxide (CuO) nanoparticles (NPs) are abundant in manufacturing processes, but they are an airway irritant. In vitro pulmonary toxicity of CuO NPs has been modeled using cell lines such as human bronchial epithelial cell line BEAS-2B. In 2D in vitro culture, BEAS-2B undergoes squamous differentiation due to the presence of serum. Differentiation is part of the repair process of lung cells in vivo that helps to preserve the epithelial lining of the respiratory tract. Herein, the effects of serum on the hydrodynamic diameter, cellular viability, cellular differentiation, and cellular uptake of 5 and 35 nm CuO NPs are investigated, and the mean cell area is used as the differentiation marker for BEAS-2B cells. The results demonstrate that the hydrodynamic diameter decreases with the addition of serum to the culture medium. Serum also increases the mean cell area, and only affects dose-dependent cytotoxicity of 35 nm CuO NPs, while simultaneously having no effect on intracellular Cu2+. This study presents evidence that both NP size and the presence of serum in culture media influence the relative viability of BEAS-2B cells following CuO NP exposure and highlights a critical need for carefully designed experiments and accurately reported conditions

Mapping the immune environment in clear cell renal carcinoma by single-cell genomics

Clear cell renal cell carcinoma (ccRCC) is one of the most immunologically distinct tumor types due to high response rate to immunotherapies, despite low tumor mutational burden. To characterize the tumor immune microenvironment of ccRCC, we applied single-cell-RNA sequencing (SCRS) along with T-cell-receptor (TCR) sequencing to map the transcriptomic heterogeneity of 25,688 individual CD4

New cytotoxic dammarane type saponins from Ziziphus spina-christi

Cancer is the world's second-leading cause of death. Drug development efforts frequently focus on medicinal plants since they are a valuable source of anticancer medications. A phytochemical investigation of the edible Ziziphus spina-christi (F. Rhamnaceae) leaf extract afforded two new dammarane type saponins identified as christinin E and F (1, 2), along with the known compound christinin A (3). Different cancer cell lines, such as lung cancer (A549), glioblastoma (U87), breast cancer (MDA-MB-231), and colorectal carcinoma (CT-26) cell lines, were used to investigate the extracted compounds' cytotoxic properties. Our findings showed significant effects on all the tested cell lines at varying concentrations (1, 5, 10, and 20 µg/mL). The three compounds exhibited potent activity at low concentrations (&lt; 10 μg/mL), as evidenced by their low IC50 values. To further investigate the complex relationships between these identified cancer-relevant biological targets and to identify critical targets in the pathogenesis of the disease, we turned to network pharmacology and in silico-based investigations. Following this, in silico-based analysis (e.g., inverse docking, ΔG calculation, and molecular dynamics simulation) was performed on the structures of the isolated compounds to identify additional potential targets for these compounds and their likely interactions with various signalling pathways relevant to this disease. Based on our findings, Z. spina-christi's compounds showed promise as potential anti-cancer therapeutic leads in the future.</p

Force-Bioreactor for Assessing Pharmacological Therapies for Mechanobiological Targets

Tissue fibrosis is a major health issue that impacts millions of people and is costly to treat. However, few effective anti-fibrotic treatments are available. Due to their central role in fibrotic tissue deposition, fibroblasts and myofibroblasts are the target of many therapeutic strategies centered primarily on either inducing apoptosis or blocking mechanical or biochemical stimulation that leads to excessive collagen production. Part of the development of these drugs for clinical use involves in vitro prescreening. 2D screens, however, are not ideal for discovering mechanobiologically significant compounds that impact functions like force generation and other cell activities related to tissue remodeling that are highly dependent on the conditions of the microenvironment. Thus, higher fidelity models are needed to better simulate in vivo conditions and relate drug activity to quantifiable functional outcomes. To provide guidance on effective drug dosing strategies for mechanoresponsive drugs, we describe a custom force-bioreactor that uses a fibroblast-seeded fibrin gels as a relatively simple mimic of the provisional matrix of a healing wound. As cells generate traction forces, the volume of the gel reduces, and a calibrated and embedded Nitinol wire deflects in proportion to the generated forces over the course of 6 days while overhead images of the gel are acquired hourly. This system is a useful in vitro tool for quantifying myofibroblast dose-dependent responses to candidate biomolecules, such as blebbistatin. Administration of 50 μM blebbistatin reliably reduced fibroblast force generation approximately 40% and lasted at least 40 h, which in turn resulted in qualitatively less collagen production as determined via fluorescent labeling of collagen

Targeting mitochondrial responses to intra-articular fracture to prevent posttraumatic osteoarthritis

We tested whether inhibiting mechanically responsive articular chondrocyte mitochondria after severe traumatic injury and preventing oxidative damage represent a viable paradigm for posttraumatic osteoarthritis (PTOA) prevention. We used a porcine hock intra-articular fracture (IAF) model well suited to human-like surgical techniques and with excellent anatomic similarities to human ankles. After IAF, amobarbital or N-acetylcysteine (NAC) was injected to inhibit chondrocyte electron transport or downstream oxidative stress, respectively. Effects were confirmed via spectrophotometric enzyme assays or glutathione/glutathione disulfide assays and immunohistochemical measures of oxidative stress. Amobarbital or NAC delivered after IAF provided substantial protection against PTOA at 6 months, including maintenance of proteoglycan content, decreased histological disease scores, and normalized chondrocyte metabolic function. These data support the therapeutic potential of targeting chondrocyte metabolism after injury and suggest a strong role for mitochondria in mediating PTOA