No abstract

Although it is widely accepted that specific intracellular receptor proteins are involved in the oestrogenic regulation of gene expression and growth in reproductive tissues, the precise nature of the regulation is poorly understood. Among the unresolved issues are the distribution and dynamics of the oestrogen receptor protein (oestrophilin) in target tissues in the presence and absence of oestrogens and antioestrogens. The use of radiolabelled and unlabelled receptor ligands to detect and measure oestrogen receptors in tissues has been complicated by the presence of other intracellular steroid-binding proteins and by the low concentration of receptors in responsive tissues. We report here the development of an immunocytochemical procedure that is suitable for localizing oestrophilin directly in frozen tissue sections or cells from human and several non-human sources. When monoclonal antibodies to oestrophilin were used to detect receptor in various oestrogen-sensitive tissues, specific staining was confined to the nucleus of all stained cells, suggesting that both cytosol and nuclear forms of the receptor protein may reside in the nuclear compartment.

Biomaterial scaffolds have been extensively used to deliver growth factors to induce new bone formation. The pharmacokinetics of growth factor delivery has been a critical regulator of their clinical success. This review will focus on the surface interactions that control the non-covalent incorporation of growth factors into scaffolds and the mechanisms that control growth factor release from clinically relevant biomaterials. We will focus on the delivery of recombinant human bone morphogenetic protein-2 from materials currently used in the clinical practice, but also suggest how general mechanisms that control growth factor incorporation and release delineated with this growth factor could extend to other systems. A better understanding of the changing mechanisms that control growth factor release during the different stages of preclinical development could instruct the development of future scaffolds for currently untreatable injuries and diseases.

No abstract

Natural proteins perform a variety of functions in biological systems, including actuation, catalysis, structural support, and molecular sequestering. The variety of natural protein functions suggest that they could serve as valuable and versatile building blocks for synthesis of functional materials. Based on this premise, several investigators have developed schemes to include functional proteins into hydrogel networks to take advantage of their structural, catalytic, and ligand binding properties. Natural proteins such as collagen [1] and elastin [2] have been used to develop materials with tailored structural and mechanical properties, while catalysis by enzymes (e.g., glucose oxidase [3] ) has been used to build smart, environmentally-responsive hydrogel networks. The ability of proteins to selectively bind ligands has been used to develop materials that change their degree of physical or chemical crosslinking in the presence of biological antigens, [4,5] small molecule drugs, [6] and carbohydrates, [7][8][9] resulting in environmentallyresponsive biomaterials. In each of these previous studies a nanometer-scale function associated with a specific protein was exploited to build a material that performed an intriguing macroscopic function. A function that has not been explored as extensively in materials science is the ability of proteins to undergo complex conformational changes. Proteins change conformations in response to a broad range of stimuli, including light, pH, and the binding of biological molecules. Over 200 conformational changes are well-characterized, [10] representing a vast unexplored databank of molecular building blocks for design of dynamic materials. Recent studies have demonstrated the utility of protein conformational changes in nano-scale engineered systems, including cooperative nanometer-scale motors [11] and molecular shuttles, [12] suggesting that dynamic protein molecules could also be a useful component of 3-dimensional materials. To that end, we recently reported an approach that used a dynamic protein as a partial cross-linker in a chemically cross-linked hydrogel network, and these hydrogels changed their volume in the presence of a specific protein-binding molecule.[13] Here we describe assembly of protein-based, dynamic materials using a novel photochemical approach. This approach allows for high concentrations of protein to be functionally included into a network in response to light, and the resulting materials undergo striking volume changes upon protein-ligand binding. Photochemical assembly also enables spatial control over the location of dynamic proteins in a hydrogel network, which is likely to be important in potential materials science and engineering applications.

The objective of this clinical study was to test if blood from osteoarthritis (OA) patients (n = 105) could be processed by a device system to form an autologous protein solution (APS) with preferentially increased concentrations of anti-inflammatory cytokines compared to inflammatory cytokines. To address this objective, APS was prepared from patients exhibiting radiographic evidence of knee OA. Patient metrics were collected including: demographic information, medical history, medication records, and Knee Injury and Osteoarthritis Outcome Score (KOOS) surveys. Cytokine and growth factor concentrations in whole blood and APS were measured using enzyme-linked immunosorbent assays. Statistical analyses were used to identify relationships between OA patient metrics and cytokines. The results of this study indicated that anti-inflammatory cytokines were preferentially increased compared to inflammatory cytokines in APS from 98% of OA patients. APS contained high concentrations of anti-inflammatory proteins including 39,000 ± 20,000 pg/ml IL-1ra, 21,000 ± 5,000 pg/ml sIL-1RII, 2,100 ± 570 pg/ml sTNF-RI, and 4,200 ± 1,500 pg/ml sTNF-RII. Analysis of the 82 patient metrics indicated that no single patient metric was strongly correlated (R2 > .7) with the key cytokine concentrations in APS. Therefore, APS can be prepared from a broad range of OA patients.

Hydrogels have been commonly used as model systems for 3-dimensional (3-D) cell biology, as they have material properties that resemble natural extracellular matrices (ECMs), and their cellinteractive properties can be readily adapted in order to address a particular hypothesis. Natural and synthetic hydrogels have been used to gain fundamental insights into virtually all aspects of cell behavior, including cell adhesion, migration, and differentiated function. However, cell responses to complex 3-D environments are difficult to adequately explore due to the large number of variables that must be controlled simultaneously. Here we describe an adaptable, automated approach for 3-D cell culture within hydrogel arrays. Our initial results demonstrate that the hydrogel network chemistry (both natural and synthetic), cell type, cell density, cell adhesion ligand density, and degradability within each array spot can be systematically varied to screen for environments that promote cell viability in a 3-D context. In a testbed application we then demonstrate that a hydrogel array format can be used to identify environments that promote viability of HL-1 cardiomyocytes, a cell line that has not been cultured previously in 3-D hydrogel matrices. Results demonstrate that the fibronectin-derived cell adhesion ligand RGDSP improves HL-1 viability in a dose-dependent manner, and that the effect of RGDSP is particularly pronounced in degrading hydrogel arrays. Importantly, in the presence of 70µM RGDSP, HL-1 cardiomyocyte viability does not decrease even after 7 days of culture in PEG hydrogels. Taken together, our results indicate that the adaptable, array-based format developed in this study may be useful as an enhanced throughput platform for 3-D culture of a variety of cell types.

Development of hydrogel materials that respond to specific stimuli has been of significant interest in the design of modern functional materials. A variety of previous studies have used the ligand‐binding capability of proteins to design hydrogels that change their crosslinking density in response to stimuli. However, these materials generally undergo relatively small dynamic response, with limited control over response characteristics. This manuscript describes an alternative approach that exploits the ability of proteins to undergo nanometer‐scale conformational changes in response to stimuli. We report a class of novel protein‐based hydrogel materials that undergo tunable, reversible dynamic responses with a wide dynamic range (volume decreases to ∼25–90% of initial volume). These materials also undergo tunable, reversible changes in optical transparency (optical transparency decreases to ∼35–100% of initial optical transparency), and this phenomenon is used as a mechanism for label‐free biosensing. The materials are generated by photo‐crosslinking of an engineered version of the protein calmodulin flanked on each end with poly(ethylene glycol)‐diacrylate (PEGDA) moieties. The mechanism for dynamic changes derives from calmodulin's well‐characterized “hinge motion”‐upon‐ligand binding. Variations in network parameters in these hydrogels, including the molecular weight between cross‐links and the cross‐linking density, result in systematic tuning of material responsiveness. The influence of network parameters on hydrogel dynamics reported here may serve as a guide for the design of other protein‐based, responsive materials assembled using similar principles, and these materials may be a useful platform for design of biosensors, actuators, and drug delivery systems.