Fibroblast growth factor 7 releasing particles enhance islet engraftment and improve metabolic control following islet transplantation in mice with diabetes.

Transplantation of islets in type 1 diabetes (T1D) is limited by poor islet engraftment into the liver, with two to three donor pancreases required per recipient. We aimed to condition the liver to enhance islet engraftment to improve long-term graft function. Diabetic mice received a non-curative islet transplant (n = 400 islets) via the hepatic portal vein (HPV) with fibroblast growth factor 7-loaded galactosylated poly(DL-lactide-co-glycolic acid) (FGF7-GAL-PLGA) particles; 26-µm diameter particles specifically targeted the liver, promoting hepatocyte proliferation in short-term experiments: in mice receiving 0.1-mg FGF7-GAL-PLGA particles (60-ng FGF7) vs vehicle, cell proliferation was induced specifically in the liver with greater efficacy and specificity than subcutaneous FGF7 (1.25 mg/kg ×2 doses; ~75-µg FGF7). Numbers of engrafted islets and vascularization were greater in liver sections of mice receiving islets and FGF7-GAL-PLGA particles vs mice receiving islets alone, 72 h posttransplant. More mice (six of eight) that received islets and FGF7-GAL-PLGA particles normalized blood glucose concentrations by 30-days posttransplant, versus zero of eight mice receiving islets alone with no evidence of increased proliferation of cells within the liver at this stage and normal liver function tests. This work shows that liver-targeted FGF7-GAL-PLGA particles achieve selective FGF7 delivery to the liver-promoting islet engraftment to help normalize blood glucose levels with a good safety profile.

Highly efficient delivery of functional cargoes by the synergistic effect of GAG binding motifs and cell-penetrating peptides.

Protein transduction domains (PTDs) are powerful nongenetic tools that allow intracellular delivery of conjugated cargoes to modify cell behavior. Their use in biomedicine has been hampered by inefficient delivery to nuclear and cytoplasmic targets. Here we overcame this deficiency by developing a series of novel fusion proteins that couple a membrane-docking peptide to heparan sulfate glycosaminoglycans (GAGs) with a PTD. We showed that this GET (GAG-binding enhanced transduction) system could deliver enzymes (Cre, neomycin phosphotransferase), transcription factors (NANOG, MYOD), antibodies, native proteins (cytochrome C), magnetic nanoparticles (MNPs), and nucleic acids [plasmid (p)DNA, modified (mod)RNA, and small inhibitory RNA] at efficiencies of up to two orders of magnitude higher than previously reported in cell types considered hard to transduce, such as mouse embryonic stem cells (mESCs), human ESCs (hESCs), and induced pluripotent stem cells (hiPSCs). This technology represents an efficient strategy for controlling cell labeling and directing cell fate or behavior that has broad applicability for basic research, disease modeling, and clinical application.

Combined hydrogels that switch human pluripotent stem cells from self-renewal to differentiation.

The ability of materials to define the architecture and microenvironment experienced by cells provides new opportunities to direct the fate of human pluripotent stem cells (HPSCs) [Robinton DA, Daley GQ (2012) Nature 481(7381):295-305]. However, the conditions required for self-renewal vs. differentiation of HPSCs are different, and a single system that efficiently achieves both outcomes is not available [Giobbe GG, et al. (2012) Biotechnol Bioeng 109(12):3119-3132]. We have addressed this dual need by developing a hydrogel-based material that uses ionic de-cross-linking to remove a self-renewal permissive hydrogel (alginate) and switch to a differentiation-permissive microenvironment (collagen). Adjusting the timing of this switch can preferentially steer the HPSC differentiation to mimic lineage commitment during gastrulation to ectoderm (early switch) or mesoderm/endoderm (late switch). As an exemplar differentiated cell type, we showed that directing early lineage specification using this single system can promote cardiogenesis with increased gene expression in high-density cell populations. This work will facilitate regenerative medicine by allowing in situ HPSC expansion to be coupled with early lineage specification within defined tissue geometries.

Sub-Optimal Paternal Diet at the Time of Mating Disrupts Maternal Adaptations to Pregnancy in the Late Gestation Mouse.

Pregnancy represents a stage during which maternal physiology and homeostatic regulation undergo dramatic change and adaptation. The fundamental purpose of these adaptations is to ensure the survival of her offspring through adequate nutrient provision and an environment that is tolerant to the semi-allogenic foetus. While poor maternal diet during pregnancy is associated with perturbed maternal adaptations during pregnancy, the influence of paternal diet on maternal well-being is less clearly defined. We fed C57BL/6 male mice either a control (CD), low protein diet (LPD), a high fat/sugar Western diet (WD) or the LPD or WD supplemented with methyl donors (MD-LPD and MD-WD, respectively) for a minimum of 8 weeks prior to mating with C57BL/6 females. Mated females were culled at day 17 of gestation for the analysis of maternal metabolic, gut, cardiac and bone health. Paternal diet had minimal influences on maternal serum and hepatic metabolite levels or gut microbiota diversity. However, analysis of the maternal hepatic transcriptome revealed distinct profiles of differential gene expression in response to the diet of the father. Paternal LPD and MD-LPD resulted in differential expression of genes associated with lipid metabolism, transcription, ubiquitin conjugation and immunity in dams, while paternal WD and MD-WD modified the expression of genes associated with ubiquitin conjugation and cardiac morphology. Finally, we observed changes in maternal femur length, volume of trabecular bone, trabecular connectivity, volume of the cortical medullar cavity and thickness of the cortical bone in response to the father's diets. Our current study demonstrates that poor paternal diet at the time of mating can influence the patterns of maternal metabolism and gestation-associated adaptations to her physiology.

Hollow colloidosomes prepared using accelerated solvent evaporation.

We demonstrate a new, scalable, simple, and generally applicable two-step method to prepare hollow colloidosomes. First, a high volume fraction oil-in-water emulsion was prepared. The oil phase consisted of CH2Cl2 containing a hydrophobic structural polymer, such as polycaprolactone (PCL) or polystyrene (PS), which was fed into the water phase. The water phase contained poly(vinylalcohol), poly(N-isopropylacrylamide), or a range of cationic graft copolymer surfactants. The emulsion was rotary evaporated to rapidly remove CH2Cl2. This caused precipitation of PCL or PS particles which became kinetically trapped at the periphery of the droplets and formed the shell of the hollow colloidosomes. Interestingly, the PCL colloidosomes were birefringent. The colloidosome yield increased and the polydispersity decreased when the preparation scale was increased. One example colloidosome system consisted of hollow PCL colloidosomes stabilized by PVA. This system should have potential biomaterial applications due to the known biocompatibility of PCL and PVA.

Rapid micropatterning of cell lines and human pluripotent stem cells on elastomeric membranes.

Tissue function during development and in regenerative medicine completely relies on correct cell organization and patterning at micro and macro scales. We describe a rapid method for patterning mammalian cells including human embryonic stem cells (HESCs) and induced pluripotent stem cells (iPSCs) on elastomeric membranes such that micron-scale control of cell position can be achieved over centimeter-length scales. Our method employs surface engineering of hydrophobic polydimethylsiloxane (PDMS) membranes by plasma polymerization of allylamine. Deposition of plasma polymerized allylamine (ppAAm) using our methods may be spatially restricted using a micro-stencil leaving faithful hydrophilic ppAAm patterns. We employed airbrushing to create aerosols which deposit extracellular matrix (ECM) proteins (such as fibronectin and Matrigel™) onto the same patterned ppAAm rich regions. Cell patterns were created with a variety of well characterized cell lines (e.g., NIH-3T3, C2C12, HL1, BJ6, HESC line HUES7, and HiPSC line IPS2). Individual and multiple cell line patterning were also achieved. Patterning remains faithful for several days and cells are viable and proliferate. To demonstrate the utility of our technique we have patterned cells in a variety of configurations. The ability to rapidly pattern cells at high resolution over macro scales should aid future tissue engineering efforts for regenerative medicine applications and in creating in vitro stem cell niches.

Directed differentiation of human embryonic stem cells to interrogate the cardiac gene regulatory network.

The limited ability of the heart to regenerate has prompted development of new systems to produce cardiomyocytes for therapeutics. While differentiation of human embryonic stem cells (hESCs) into cardiomyocytes has been well documented, the process remains inefficient and/or expensive, and progress would be facilitated by better understanding the early genetic events that cause cardiac specification. By maintaining a transgenic cardiac-specific MYH6-monomeric red fluorescent protein (mRFP) reporter hESC line in conditions that promote pluripotency, we tested the ability of combinations of 15 genes to induce cardiac specification. Screening identified GATA4 plus TBX5 as the minimum requirement to activate the cardiac gene regulatory network and produce mRFP(+) cells, while a combination of GATA4, TBX5, NKX2.5, and BAF60c (GTNB) was necessary to generate beating cardiomyocytes positive for cTnI and α-actinin. Including the chemotherapeutic agent, Ara-C, from day 10 of induced differentiation enriched for cTnI/α-actinin double positive cells to 45%. Transient expression of GTNB for 5-7 days was necessary to activate the cardiogenesis through progenitor intermediates in a manner consistent with normal heart development. This system provides a route to test the effect of different factors on human cardiac differentiation and will be useful in understanding the network failures that underlie disease phenotypes.

Tissue engineering in the development of replacement technologies.

The field of tissue engineering is generating new scaffolds, bioreactors and methods for stimulating cells within complex cultures, with the aim of recreating the conditions under which cells form functional tissues. Hitherto, the primary focus of this field has been on clinical applications. However, there are many methods of in vitro tissue engineering that represent new opportunities in 3D cell culture and could be the basis for new replacement methods that either replace the use of a tissue isolated from an animal or the use of a living animal. This chapter presents an overview of tissue engineering and provides tissue-specific examples of recent advances.

Scaffolds containing growth factors and extracellular matrix induce hepatocyte proliferation and cell migration in normal and regenerating rat liver.

BACKGROUND & AIMS: Intrahepatic drug delivery from implantable scaffolds is being developed as a strategy to modulate growth and enhance regeneration at the time of liver resection. In this study we examine the effects of scaffolds containing hepatocyte growth factor, epidermal growth factor, fibroblast growth factor 1, fibroblast growth factor 2, and liver-derived extracellular matrix (L-ECM) when implanted into normal and partially hepatectomized rat livers. METHODS: Scaffolds loaded with combinations of growth factors and L-ECM were implanted into normal livers (controls=L-ECM, polymer or sham) and livers following partial hepatectomy (controls=partial hepatectomy or sham). The primary end points were hepatocyte DNA synthesis and liver tissue penetration into scaffolds. Secondary end points included non-parenchymal cell DNA synthesis, liver weight analysis, liver function, and histological characterisation of the peri-implant parenchyma. RESULTS: Four days after implantation in normal livers, there was significantly more hepatocyte proliferation around growth factor scaffolds than controls. Seven days after implantation, there was significantly more tissue penetration into growth factor scaffolds than control scaffolds. ED-1 and desmin positive cells were present in the pores of scaffolds. Two days after partial hepatectomy, there was significantly more hepatocyte proliferation around scaffold implanted livers than after partial hepatectomy alone. CONCLUSIONS: Growth factors and L-ECM accelerated non-parenchymal cell migration into scaffolds and increased hepatocyte and non-parenchymal cell proliferation around them. These results demonstrate the potential for intrahepatic implantation of scaffolds containing growth factors and L-ECM to modulate growth in the normal and regenerating liver.

Thermally triggered assembly of cationic graft copolymers containing 2-(2-methoxyethoxy)ethyl methacrylate side chains.

Thermoresponsive copolymers continue to attract a great deal of interest in the literature. In particular, those based on ethylene oxide-containing methacrylates have excellent potential for biomaterial applications. Recently, some of us reported a study of thermoresponsive cationic graft copolymers containing poly(N-isopropylacrylamide), PNIPAm, (Liu et al., Langmuir, 24, 7099). Here, we report an improved version of this new family of copolymers. In the present study, we replaced the PNIPAm side chains with poly(2-(2-methyoxyethoxy)ethylmethacrylate), PMeO(2)MA. These new, nonacrylamide containing, cationic graft copolymers were prepared using atom transfer radical polymerization (ATRP) and a macroinitiator. They contained poly(trimethylamonium)-aminoethyl methacrylate and PMeO(2)MA, i.e., PTMA(+)(x)-g-(PMeO(2)MA(n))(y). They were investigated using variable-temperature turbidity, photon correlation spectroscopy (PCS), electrophoretic mobility, and (1)H NMR measurements. For one system, four critical temperatures were measured and used to propose a mechanism for the thermally triggered changes that occur in solution. All of the copolymers existed as unimolecular micelles at 20 °C. They underwent reversible aggregation with heating. The extent of aggregation was controlled by the length of the side chains. TEM showed evidence of micellar aggregates. The thermally responsive behaviors of our new copolymers are compared to those for the cationic PNIPAm graft copolymers reported by Liu et al. Our new cationic copolymers retained their positive charge at all temperatures studied, have high zeta potentials at 37 °C, and are good candidates for conferring thermoresponsiveness to negatively charged biomaterial surfaces.

Combination of injectable multiple growth factor-releasing scaffolds and cell therapy as an advanced modality to enhance tissue neovascularization.

OBJECTIVE: Vasculogenic progenitor cell therapy for ischemic diseases bears great potential but still requires further optimization for justifying its clinical application. Here, we investigated the effects of in vivo tissue engineering by combining vasculogenic progenitors with injectable scaffolds releasing controlled amounts of proangiogenic growth factors. METHODS AND RESULTS: We produced biodegradable, injectable polylactic coglycolic acid-based scaffolds releasing single factors or combinations of vascular endothelial growth factor, hepatocyte growth factor, and angiopoietin-1. Dual and triple combinations of scaffold-released growth factors were superior to single release. In murine hindlimb ischemia models, scaffolds releasing dual (vascular endothelial growth factor and hepatocyte growth factor) or triple combinations improved effects of cord blood-derived vasculogenic progenitors. Increased migration, homing, and incorporation of vasculogenic progenitors into the vasculature augmented capillary density, translating into improved blood perfusion. Most importantly, scaffold-released triple combinations including the vessel stabilizer angiopoietin-1 enhanced the number of perivascular smooth muscle actin(+) vascular smooth muscle cells, indicating more efficient vessel stabilization. CONCLUSIONS: Vasculogenic progenitor cell therapy is significantly enhanced by in vivo tissue engineering providing a proangiogenic and provasculogenic growth factor-enriched microenvironment. Therefore, combined use of scaffold-released growth factors and cell therapy improves neovascularization in ischemic diseases and may translate into more pronounced clinical effects.

Designing topographically textured microparticles for induction and modulation of osteogenesis in mesenchymal stem cell engineering.

Mesenchymal stem cells are the focus of intense research in bone development and regeneration. The potential of microparticles as modulating moieties of osteogenic response by utilizing their architectural features is demonstrated herein. Topographically textured microparticles of varying microscale features are produced by exploiting phase-separation of a readily soluble sacrificial component from polylactic acid. The influence of varying topographical features on primary human mesenchymal stem cell attachment, proliferation and markers of osteogenesis is investigated. In the absence of osteoinductive supplements, cells cultured on textured microparticles exhibit notably increased expression of osteogenic markers relative to conventional smooth microparticles. They also exhibit varying morphological, attachment and proliferation responses. Significantly altered gene expression and metabolic profiles are observed, with varying histological characteristics in vivo. This study highlights how tailoring topographical design offers cell-instructive 3D microenvironments which allow manipulation of stem cell fate by eliciting the desired downstream response without use of exogenous osteoinductive factors.

Label-free molecular imaging of immunological synapses between dendritic and T cells by Raman micro-spectroscopy.

Confocal Raman micro-spectroscopy (CRMS) was used to measure spectral images of immunological synapse formation between dendritic and T cells without using molecular labels or other invasive procedures. The purpose-built inverted CRMS instrument integrated an environmental enclosure and a near-infrared laser to allow measurements on live cells maintained under physiological conditions. The integration of the wide-field fluorescence also enabled viability assays and direct comparison between Raman spectral images and gold-standard immuno-fluorescence images for specific molecules. Raman spectral images of nucleus and proteins were built by fuzzy c-mean clustering method. The Raman images were found to be in good correspondence with the immuno-fluorescence images of DNA and actin. These results indicate that actin is a main contributor to the Raman spectrum of the cytoplasm of dendritic and T cells. While for control cells the Raman spectral images of proteins indicated a more homogeneous distribution of proteins in the cytoplasm of dendritic cells, they indicated a higher accumulation of proteins at the immunological synapses when dendritic cells were pre-treated with laminin. These conclusions were also supported by confocal immuno-fluorescence imaging after cell fixation and labelling. This study demonstrates the potential of CRMS for label-free non-invasive imaging of junctions between live cells. Therefore, this technique may become a useful tool for studying cellular processes in live cells and where non-invasive molecular specific imaging is desirable, such as cell-cell interactions.

Multi-material 3D bioprinting of porous constructs for cartilage regeneration.

The current gold standard for nasal reconstruction after rhinectomy or severe trauma includes transposition of autologous cartilage grafts in conjunction with coverage using an autologous skin flap. Harvesting autologous cartilage requires a major additional procedure that may create donor site morbidity. Major nasal reconstruction also requires sculpting autologous cartilages to form a cartilage framework, which is complex, highly skill-demanding and very time consuming. These limitations have prompted facial reconstructive surgeons to explore different techniques such as tissue engineered cartilage. This work explores the use of multi-material 3D bioprinting with chondrocyte-laden gelatin methacrylate (GelMA) and polycaprolactone (PCL) to fabricate constructs that can potentially be used for nasal reconstruction. In this study, we have investigated the effect of 3D manufacturing parameters including temperature, needle gauge, UV exposure time, and cell carrier formulation (GelMA) on the viability and functionality of chondrocytes in bioprinted constructs. Furthermore, we printed chondrocyte-laden GelMA and PCL into composite constructs to combine biological and mechanical properties. It was found that 20% w/v GelMA was the best concentration for the 3D bioprinting of the chondrocytes without comprising the scaffold's porous structure and cell functionality. In addition, the 3D bioprinted constructs showed neocartilage formation and similar mechanical properties to nasal alar cartilage after a 50-day culture period. Neocartilage formation was also observed in the composite constructs evidenced by the presence of glycosaminoglycans and collagen type II. This study shows the feasibility of manufacturing neocartilage using chondrocytes/GelMA/PCL 3D bioprinted porous constructs which could be applied as a method for fabricating implants for nose reconstruction.

Localized Induction of Gene Expression in Embryonic Stem Cell Aggregates Using Holographic Optical Tweezers to Create Biochemical Gradients.

Three-dimensional (3D) cell models that mimic the structure and function of native tissues are enabling more detailed study of physiological and pathological mechanisms in vitro. We have previously demonstrated the ability to build and manipulate 3D multicellular microscopic structures using holographic optical tweezers (HOTs). Here, we show the construction of a precisely patterned 3D microenvironment and biochemical gradient model consisting of mouse embryoid bodies (mEBs) and polymer microparticles loaded with retinoic acid (RA), embedded in a hydrogel. We demonstrate discrete, zonal expression of the RA-inducible protein Stra8 within mEBs in response to release of RA from polymer microparticles, corresponding directly to the defined 3D positioning of the microparticles using HOTs. These results demonstrate the ability of this technology to create chemical microgradients at definable length scales and to elicit, with fidelity and precision, specific biological responses. This technique can be used in the study of in vitro microenvironments to enable new insights on 3D cell models, their cellular assembly, and the delivery of drug or biochemical molecules for engineering and interrogation of functional and morphogenic responses. Graphical abstract.

Enhanced Cellular Transduction of Nanoparticles Resistant to Rapidly Forming Plasma Protein Coronas.

Nanoparticles (NPs) are increasingly being developed as biomedical platforms for drug/nucleic acid delivery and imaging. However, in biological fluids, NPs interact with a wide range of proteins that form a coating known as protein corona. Coronae can critically influence self-interaction and binding of other molecules, which can affect toxicity, promote cell activation, and inhibit general or specific cellular uptake. Glycosaminoglycan (GAG)-binding enhanced transduction (GET) is developed to efficiently deliver a variety of cargoes intracellularly; employing GAG-binding peptides, which promote cell targeting, and cell penetrating peptides (CPPs) which enhance endocytotic cell internalization. Herein, it is demonstrated that GET peptide coatings can mediate sustained intracellular transduction of magnetic NPs (MNPs), even in the presence of serum or plasma. NP colloidal stability, physicochemical properties, toxicity and cellular uptake are investigated. Using label-free snapshot proteomics, time-resolved profiles of human plasma coronas formed on functionalized GET-MNPs demonstrate that coronae quickly form (<1 min), with their composition relatively stable but evolving. Importantly GET-MNPs present a subtly different corona composition to MNPs alone, consistent with GAG-binding activities. Understanding how NPs interact with biological systems and can retain enhanced intracellular transduction will facilitate novel drug delivery approaches for cell-type specific targeting of new nanomaterials.

Magnetic Retrieval of Encapsulated Beta Cell Transplants from Diabetic Mice Using Dual-Function MRI Visible and Retrievable Microcapsules.

Encapsulated beta cell transplantation offers a potential cure for a subset of diabetic patients. Once transplanted, beta cell grafts can help to restore glycemic control; however, locating and retrieving cells in the event of graft failure may pose a surgical challenge. Here, a dual-function nanoparticle-loaded hydrogel microcapsule is developed that enables graft retrieval under an applied magnetic field. Additionally, this system facilitates graft localization via magnetic resonance imaging (MRI), and graft isolation from the immune system. Iron oxide nanoparticles encapsulated within alginate hydrogel capsules containing viable islets are transplanted and the in vitro and in vivo retrieval of capsules containing nanoparticles functionalized with various ligands are compared. Capsules containing islets co-encapsulated with COOH-coated nanoparticles restore normal glycemia in immunocompetent diabetic mice for at least 6 weeks, can be visualized using MRI, and are retrievable in a magnetic field. Application of a magnetic field for 90 s via a magnetically assisted retrieval device facilitates rapid retrieval of up to 94% (±3.1%) of the transplant volume 24 h after surgical implantation. This strategy aids monitoring of cell-capsule locations in vivo, facilitates graft removal at the end of the transplant lifetime, and may be applicable to many encapsulated cell transplant systems.

Thermoresponsive and photocrosslinkable PEGMEMA-PPGMA-EGDMA copolymers from a one-step ATRP synthesis.

Thermoresponsive and photocrosslinkable polymers can be used as injectable scaffolds in tissue engineering to yield gels in situ with enhanced mechanical properties and stability. They allow easy handling and hold their shapes prior to photopolymerization for clinical practice. Here we report a novel copolymer with both thermoresponsive and photocrosslinkable properties via a facile one-step deactivation enhanced atom transfer radical polymerization (ATRP) using poly(ethylene glycol) methyl ether methylacrylate (PEGMEMA, M(n) = 475) and poly(propylene glycol) methacrylate (PPGMA, M(n) = 375) as monofunctional vinyl monomers and up to 30% of ethylene glycol dimethacrylate (EGDMA) as multifunctional vinyl monomer. The resultant PEGMEMA-PPGMA-EGDMA copolymers have been characterized by gel permeation chromatography (GPC) and 1H NMR analysis, which demonstrate their multivinyl functionality and hyperbranched structures. These water-soluble copolymers show lower critical solution temperature (LCST) behavior at 32 degrees C, which is comparable to poly(N-isopropylacrylamide) (PNIPAM). The copolymers can also be cross-linked by photopolymerization through their multivinyl functional groups. Rheological studies clearly demonstrate that the photocrosslinked gels formed at a temperature above the LCST have higher storage moduli than those prepared at a temperature below the LCST. Moreover, the cross-linking density of the gels can be tuned to tailor their porous structures and mechanical properties by adjusting the composition and concentration of the copolymers. Hydrogels with a broad range of storage moduli from 10 to 400 kPa have been produced.

Photo-cross-linked hydrogels from thermoresponsive PEGMEMA-PPGMA-EGDMA copolymers containing multiple methacrylate groups: mechanical property, swelling, protein release, and cytotoxicity.

Photo-cross-linked hydrogels from thermoresponsive polymers can be used as advanced injectable biomaterials via a combination of physical interaction (in situ thermal gelation) and covalent cross-links (in situ photopolymerization). This can lead to gels with significantly enhanced mechanical properties compared to non-photo-cross-linked thermoresponsive hydrogels. Moreover, the thermally phase-separated gels have attractive advantages over non-thermoresponsive gels because thermal gelation upon injection allows easy handling and holds the shape of the gels prior to photopolymerization. In this study, water-soluble thermoresponsive copolymers containing multiple methacrylate groups were synthesized via one-step deactivation enhanced atom transfer radical polymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA, M(n) = 475), poly(propylene glycol) methacrylate (PPGMA, M(n) = 375), and ethylene glycol dimethacrylate (EGDMA) and were used to form covalent cross-linked hydrogels by photopolymerization. The cross-linking density was found to have a significant influence on the mechanical and swelling properties of the photo-cross-linked gels. Release studies using lysozyme as a model protein demonstrated a sustained release profile that varied dependent on the copolymer composition, cross-linking density, and the temperature. Mouse C2C12 myoblast cells were cultured in the presence of the copolymers at concentrations up to 1 mg/mL. It was found that the majority of the cells remained viable, as assessed by Alamar Blue, lactate dehydrogenase (LDH), and Live/Dead cell viability/cytotoxicity assays. These studies demonstrate that thermoresponsive PEGMEMA-PPGMA-EGDMA copolymers offer potential as in situ photopolymerizable materials for tissue engineering and drug delivery applications through a combination of facile synthesis, enhanced mechanical properties, tunable cross-linking density, low cytotoxicity, and accessible functionality for further structure modifications.

Formulations for delivery of therapeutic proteins.

Advances in biotechnology have now created a capacity to produce therapeutically active proteins on a commercial scale, opening the potential for their application in an array of disease conditions. The process of translation of the variety of different therapeutic proteins into the medicines used in clinics is now occurring. To assist in this translation, new formulations to deliver proteins could play an important role. These new formulations need to more adequately address the pharmacological and therapeutic requirement for each particular protein/peptide and, in that way, either improve present therapies or extend with new entries the current list of protein based medicines used in clinic.