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Cartilage tissue lacks an intrinsic capacity for self-regeneration due to slow matrix turnover, a limited supply of mature chondrocytes and insufficient vasculature. Although cartilage tissue engineering has achieved some success using agarose as a scaffolding material, major challenges of agarose-based cartilage repair, including non-degradability, poor tissue–scaffold integration and limited processing capability, have prompted the search for an alternative biomaterial. In this study, silk fiber–hydrogel composites (SF–silk hydrogels) made from silk microfibers and silk hydrogels were investigated for their potential use as a support material for engineered cartilage. We demonstrated the use of 100% silk-based fiber–hydrogel composite scaffolds for the development of cartilage constructs with properties comparable to those made with agarose. Cartilage constructs with an equilibrium modulus in the native tissue range were fabricated by mimicking the collagen fiber and proteoglycan composite architecture of native cartilage using biocompatible, biodegradable silk fibroin from Bombyx mori . Excellent chondrocyte response was observed on SF–silk hydrogels, and fiber reinforcement resulted in the development of more mechanically robust constructs after 42 days in culture compared to silk hydrogels alone. Thus, we demonstrate the versatility of silk fibroin as a composite scaffolding material for use in cartilage tissue repair to create functional cartilage constructs that overcome the limitations of agarose biomaterials, and provide a much-needed alternative to the agarose standard. [ABSTRACT FROM AUTHOR]

The formation and assembly of diverse tissue building blocks is considered a promising bottom-up approach for the construction of complex three-dimensional tissues. Patterned shape-changing materials were investigated as an innovative method to form and harvest free-standing tissue modules with preserved spatial organization and cell–cell connections. Arrays of micro-scale surface-attached hydrogels made of a thermoresponsive polymer were used as cell culture supports to fabricate tissue modules of defined geometric shape. Upon stimulation, these hydrogels swelled anisotropically, resulting in significant expansion of the culture surface and subsequent expulsion of the intact tissue modules. By varying the network crosslink density, the surface strain was modulated and a strain threshold for tissue module release was identified. This mechanical mechanism for rapid tissue module harvest was found to require inter- and intra-cellular tension. These results suggest that the cell–matrix adhesions are disrupted by the incompatibility of surface expansion with tissue module cohesion and stiffness, thus providing a novel method of forming and harvesting tissue building blocks by a mechanism independent of the thermal stimulus that induces the biomaterial shape change. [ABSTRACT FROM AUTHOR]

Hydrogels closely resemble the extracellular matrix (ECM) and can support cell proliferation while new tissue is formed, making them materials of choice as tissue engineering scaffolds. However, their sometimes-poor mechanical properties can hinder their application. The addition of meshes of nanofibers embedded in their matrix forms a composite that draws from the advantages of both components. Given that these materials are still in the early stages of development, there is a lack of uniformity across methods for characterizing their mechanical properties. Here, we propose a simple metric to enable comparisons between materials. The fibrous constituent improves the mechanical properties of the hydrogel, while the biocompatibility and functionality of the gels are maintained or even improved. [ABSTRACT FROM AUTHOR]

The similarity between the extracellular matrix of soft tissue and hydrogels, characterized by high-water-content viscoelastic polymeric networks, has been sustaining the advancement of hydrogels for tissue engineering and regenerative medicine (TERM) purposes. Current research on hydrogels has focused on introducing cell-adhesive peptides to promote cell adhesion and spreading, a critical applicability limitation. Here we report the development of gellan gum (GG) spongy-like hydrogels with ameliorated mechanical performance and flexibility in relation to hydrogels, using a simple and cost-effective method. Most importantly, these materials allow the entrapment of different cell types representing mesenchymal, epidermal and osteoblastic phenotypes that spread within the three-dimensional microstructure. This effect was associated with microstructural rearrangements characterized by pore wall thickening and pore size augmentation, and lower water content than precursor hydrogels. These properties significantly affected protein adsorption once cell adhesion was inhibited in the absence of serum. Spongy-like hydrogels are not adhesive for endothelial cells; however, this issue was surpassed by a pre-incubation with a cell-adhesive protein, as demonstrated for other substrates but not for traditional hydrogels. The proposed cell-compatible GG-based structures avoid time-consuming and expensive strategies that have been used to include cell-adhesive features in traditional hydrogels. This, associated with their off-the-shelf availability in an intermediary dried state, represents unique and highly relevant features for diverse TERM applications. [ABSTRACT FROM AUTHOR]

Development of a xenogeneic biological liver support is important in providing a bridge to transplantation or liver regeneration, thus helping to overcome the chronic shortage of liver donors. Among the critical factors in developing biological liver support are the creation of in vivo mimetic micro liver tissue (mLT), especially mLTs containing liver-specific ultrastructure, and an encapsulation method that can package massive numbers of cells while providing immune-protection from the host immune system. We describe here the development of mLTs that include liver microarchitecture and their in situ encapsulation in hydrogel composites. Concave microwells and the tri-culture of three types of primary liver cells were applied for the construction of mLTs showing excellent liver functions and long-term (>1 month) viability in vitro. Large quantities of rat mLTs were encapsulated in collagen-alginate composites, implanted into hepatic failure mice and sustained their survival during regeneration of the remaining liver. The proposed liver support system offers xenogeneic hepatic assistance by mimicking native liver microarchitecture and providing immune-protection without the need for complicated devices or processes, and as such represents a promising system for recovery of organ function. [ABSTRACT FROM AUTHOR]

Abstract: Alginate hydrogels are commonly used in biomedical applications such as scaffolds for tissue engineering, drug delivery, and as a medium for cell immobilisation. Multivalent cations are often employed to create physical crosslinks between carboxyl and hydroxyl moieties on neighbouring polysaccharide chains, creating hydrogels with a range of mechanical properties. This work describes the manufacture and characterisation of sodium alginate hydrogels using the divalent cations Mg2+, Ca2+ and Sr2+ to promote gelation via non-covalent crosslinks. Gelation time and Young׳s modulus are characterised as a function of cation and alginate concentrations. The implications of this work towards the use of environmental elasticity to control stem cell differentiation are discussed. [Copyright &y& Elsevier]

Abstract: Three-dimensional (3D) tissue-engineered tumor models have the potential to bridge the gap between monolayer cultures and patient-derived xenografts for the testing of nanoparticle (NP)-based cancer therapeutics. In this study, a hydrogel-derived prostate cancer (PCa) model was developed for the in vitro evaluation of doxorubicin (Dox)-loaded polymer NPs (Dox-NPs). The hydrogels were synthesized using chemically modified hyaluronic acid (HA) carrying acrylate groups (HA-AC) or reactive thiols (HA-SH). The crosslinked hydrogel networks exhibited an estimated pore size of 70–100 nm, similar to the spacing of the extracellular matrices (ECM) surrounding tumor tissues. LNCaP PCa cells entrapped in the HA matrices formed distinct tumor-like multicellular aggregates with an average diameter of 50 μm after 7 days of culture. Compared to cells grown on two-dimensional (2D) tissue culture plates, cells from the engineered tumoroids expressed significantly higher levels of multidrug resistance (MDR) proteins, including multidrug resistance protein 1 (MRP1) and lung resistance-related protein (LRP), both at the mRNA and the protein levels. Separately, Dox-NPs with an average diameter of 54 ± 1 nm were prepared from amphiphilic block copolymers based on poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) bearing pendant cyclic ketals. Dox-NPs were able to diffuse through the hydrogel matrices, penetrate into the tumoroid and be internalized by LNCaP PCa cells through caveolae-mediated endocytosis and macropinocytosis pathways. Compared to 2D cultures, LNCaP PCa cells cultured as multicellular aggregates in HA hydrogel were more resistant to Dox and Dox-NPs treatments. Moreover, the NP-based Dox formulation could bypass the drug efflux function of MRP1, thereby partially reversing the resistance to free Dox in 3D cultures. Overall, the engineered tumor model has the potential to provide predictable results on the efficacy of NP-based cancer therapeutics. [Copyright &y& Elsevier]

The article focuses on researches based on hydrogel materials. Topics discussed include methods of preparing hydrogel materials, their chemical properties and applications of hydrogel materials in medical device coating, tissue engineering and drug delivery. The mechanical characterization techniques for hydrogel materials including universal test frame, indentation and frequency based testing have also been discussed.

Abstract: Non-viral gene delivery holds great promise for promoting tissue regeneration, and offers a potentially safer alternative than viral vectors. Great progress has been made to develop biodegradable polymeric vectors for non-viral gene delivery in 2D culture, which generally involves isolating and modifying cells in vitro, followed by subsequent transplantation in vivo. Scaffold-mediated gene delivery may eliminate the need for the multiple-step process in vitro, and allows sustained release of nucleic acids in situ. Hydrogels are widely used tissue engineering scaffolds given their tissue-like water content, injectability and tunable biochemical and biophysical properties. However, previous attempts on developing hydrogel-mediated non-viral gene delivery have generally resulted in low levels of transgene expression inside 3D hydrogels, and increasing hydrogel stiffness further decreased such transfection efficiency. Here we report the development of biodegradable polymeric vectors that led to efficient gene delivery inside poly(ethylene glycol) (PEG)-based hydrogels with tunable matrix stiffness. Photocrosslinkable gelatin was maintained constant in the hydrogel network to allow cell adhesion. We identified a lead biodegradable polymeric vector, E6, which resulted in increased polyplex stability, DNA protection and achieved sustained high levels of transgene expression inside 3D PEG-DMA hydrogels for at least 12 days. Furthermore, we demonstrated that E6-based polyplexes allowed efficient gene delivery inside hydrogels with tunable stiffness ranging from 2 to 175 kPa, with the peak transfection efficiency observed in hydrogels with intermediate stiffness (28 kPa). The reported hydrogel-mediated gene delivery platform using biodegradable polyplexes may serve as a local depot for sustained transgene expression in situ to enhance tissue engineering across broad tissue types. [Copyright &y& Elsevier]

Abstract: A wide variety of hydrogels have been explored as 3D culture platforms and for applications in tissue engineering. Hydrogels formed from natural extracellular matrix (ECM) proteins readily support the formation of vasculature in vitro, but only a handful of hydrogels composed of synthetic materials have shown anything comparable. This relative lack of synthetic material options has hindered efforts to better understand how ECM cues direct vascularization. We developed a biosynthetic hydrogel consisting of polyethylene glycol diacrylamide conjugated to macromolecular type-I collagen. Through their acrylamide-based crosslinks, these materials allow for independent control of physical properties and bulk ligand concentration. These hydrogels exhibited hydrolytic stability, but the collagen component retained its susceptibility to enzymatic remodeling. Photoencapsulation of endothelial cells and fibroblasts within this hydrogel material and their subsequent co-culture led to the formation of capillary vessel-like networks with well-defined hollow lumens. Capillary formation was prevented by inhibiting matrix metalloproteinase (MMP) activity, recapitulating the MMP-dependence of vascularization observed in natural hydrogels. These findings validate the utility of this material platform to decipher how the ECM regulates capillary morphogenesis and to support the formation of vascularized tissue constructs for potential applications in regenerative medicine. [Copyright &y& Elsevier]

Abstract: In this study, an injectable calcium silicate (CS)/sodium alginate (SA) hybrid hydrogel was prepared using a novel material composition design. CS was incorporated into an alginate solution and internal in situ gelling was induced by the calcium ions directly released from CS with the addition of d-gluconic acid δ-lactone (GDL). The gelling time could be controlled, from about 30s to 10min, by varying the amounts of CS and GDL added. The mechanical properties of the hydrogels with different amounts of CS and GDL were systematically analyzed. The compressive strength of 5% CS/SA hydrogels was higher than that of 10% CS/SA for the same amount of GDL. The swelling behaviors of 5% CS/SA hydrogels with different contents of GDL were therefore investigated. The swelling ratios of the hydrogels decreased with increasing GDL, and 5% CS/SA hydrogel with 1% GDL swelled by only less than 5%. Scanning electron microscopy (SEM) observation of the scaffolds showed an optimal interconnected porous structure, with the pore size ranging between 50 and 200μm. Fourier transform infrared spectroscopy and SEM showed that the CS/SA composite hydrogel induced the formation of hydroxyapatite on the surface of the materials in simulated body fluid. In addition, rat bone mesenchymal stem cells (rtBMSCs) cultured in the presence of hydrogels and their ionic extracts were able to maintain the viability and proliferation. Furthermore, the CS/SA composite hydrogel and its ionic extracts stimulated rtBMSCs to produce alkaline phosphatase, and its ionic extracts could also promote angiogenesis of human umbilical vein endothelial cells. Overall, all these results indicate that the CS/SA composite hydrogel efficiently supported the adhesion, proliferation and differentiation of osteogenic and angiogenic cells. Together with its porous three-dimensional structure and injectable properties, CS/SA composite hydrogel possesses great potential for bone regeneration and tissue engineering applications. [Copyright &y& Elsevier]

Abstract: Our objective was to characterize the elasticity of hydrogel formulations intended to mimic physical properties that cells and tissues experience in vivo. Using atomic force microscopy (AFM), we tested a variety of concentrations in a variety of biomaterials, including agarose, alginate, the collagens, fibrin, hyaluronic acid, kerateine, laminin, Matrigel, polyacrylamide, polyethylene glycol diacrylate (PEGDA) and silicone elastomer (polydimethylsiloxane). Manipulations of the concentration of biomaterials were detectable in AFM measurements of elasticity (Young′s modulus, E), and E tended to increase with increased concentration. Depending on the biomaterials chosen, and their concentrations, generation of tunable biocompatible hydrogels in the physiologic range is possible. [Copyright &y& Elsevier]

Highlights: [•] Physical and chemical basis for collagen triple helix stabilization. [•] Higher-order assembly of collagen like peptides. [•] Self-assembly of CLP–polymer conjugates for drug delivery. [•] CLP containing hydrogels for tissue engineering. [ABSTRACT FROM AUTHOR]

Abstract: Islet transplantation offers a promising treatment for type 1 diabetes (T1D). However, a major hurdle in this treatment is the rapid loss of functional islets during culture and after transplantation. The liver site, currently utilized for transplantation, is suboptimal for achieving long-term insulin independence due to a rapid islet loss followed by a chronic decline in islet function after transplantation. Herein, we report a synthetic saccharide–peptide (SP) hydrogel that allows suspending islets in liquid and injecting for in situ polymerization without forming islet clumps, indicating its potential in extrahepatic islet transplantation. In vitro, rat islets in SP hydrogel maintained a 3D structure and high glucose-stimulated insulin release similar to that observed in freshly isolated islets for 4 weeks, while control islets cultured in suspension lost their 3D structure and insulin release responses by 2 weeks. Biocompatibility of SP hydrogel was shown by the absence of cytokine mRNA activation in peripheral blood mononuclear cells (PBMCs) exposed to hydrogel in vitro and by the absence of cellular infiltrates in and around the hydrogel implanted subcutaneously. Syngeneic Lewis rat islets transplanted in SP hydrogel in various extrahepatic sites stained strongly for insulin, and more effectively reversed diabetes than unencapsulated islets when transplanted in an omental pocket. In conclusion, the SP hydrogel is non-cytotoxic and supports normal islet structure and function both in vitro and in vivo. Specifically, the ability of the hydrogel to separate individual islets after transplantation is important for maintaining their function in vivo. This important property, combined with the versatility and biocompatibility, makes our SP hydrogel a promising synthetic scaffold that can facilitate transplantation of organized heterogeneous cells to preserve their micro-structure and function. [Copyright &y& Elsevier]

Abstract: Damage to cartilage caused by injury or disease can lead to pain and loss of mobility, diminishing one''s quality of life. Because cartilage has a limited capacity for self-repair, tissue engineering strategies, such as cells encapsulated in synthetic hydrogels, are being investigated as a means to restore the damaged cartilage. However, strategies to date are suboptimal in part because designing degradable hydrogels is complicated by structural and temporal complexities of the gel and evolving tissue along multiple length scales. To address this problem, this study proposes a multi-scale mechanical model using a triphasic formulation (solid, fluid, unbound matrix molecules) based on a single chondrocyte releasing extracellular matrix molecules within a degrading hydrogel. This model describes the key players (cells, proteoglycans, collagen) of the biological system within the hydrogel encompassing different length scales. Two mechanisms are included: temporal changes of bulk properties due to hydrogel degradation, and matrix transport. Numerical results demonstrate that the temporal change of bulk properties is a decisive factor in the diffusion of unbound matrix molecules through the hydrogel. Transport of matrix molecules in the hydrogel contributes both to the development of the pericellular matrix and the extracellular matrix and is dependent on the relative size of matrix molecules and the hydrogel mesh. The numerical results also demonstrate that osmotic pressure, which leads to changes in mesh size, is a key parameter for achieving a larger diffusivity for matrix molecules in the hydrogel. The numerical model is confirmed with experimental results of matrix synthesis by chondrocytes in biodegradable poly(ethylene glycol)-based hydrogels. This model may ultimately be used to predict key hydrogel design parameters towards achieving optimal cartilage growth. [Copyright &y& Elsevier]

Abstract: Objectives: The clinical translation of stem cell-based Regenerative Endodontics demands further development of suitable injectable scaffolds. Puramatrix™ is a defined, self-assembling peptide hydrogel which instantaneously polymerizes under normal physiological conditions. Here, we assessed the compatibility of Puramatrix™ with dental pulp stem cell (DPSC) growth and differentiation. Methods: DPSC cells were grown in 0.05–0.25% Puramatrix™. Cell viability was measured colorimetrically using the WST-1 assay. Cell morphology was observed in 3D modeling using confocal microscopy. In addition, we used the human tooth slice model with Puramatrix™ to verify DPSC differentiation into odontoblast-like cells, as measured by expression of DSPP and DMP-1. Results: DPSC survived and proliferated in Puramatrix™ for at least three weeks in culture. Confocal microscopy revealed that cells seeded in Puramatrix™ presented morphological features of healthy cells, and some cells exhibited cytoplasmic elongations. Notably, after 21 days in tooth slices containing Puramatrix™, DPSC cells expressed DMP-1 and DSPP, putative markers of odontoblastic differentiation. Significance: Collectively, these data suggest that self-assembling peptide hydrogels might be useful injectable scaffolds for stem cell-based Regenerative Endodontics. [Copyright &y& Elsevier]

Abstract: Hydrogels are presently under investigation as matrices for the controlled release of bioactive molecules, in particular pharmaceutical proteins, and for the encapsulation of living cells. For these applications, it is often required that the gels degrade under physiological conditions. This means that the originally three-dimensional structure has to disintegrate preferably in harmless products to ensure a good biocompatibility of the hydrogel. In this overview, different chemical and physical crosslinking methods used for the design of biodegradable hydrogels are summarized and discussed. Chemical crosslinking is a highly versatile method to create hydrogels with good mechanical stability. However, the crosslinking agents used are often toxic compounds, which have been extracted from the gels before they can be applied. Moreover, crosslinking agents can give unwanted reactions with the bioactive substances present in the hydrogel matrix. Such adverse effects are avoided with the use of physically crosslinked gels. [Copyright &y& Elsevier]

Abstract: Inorganic–organic hydrogels based on methacrylated star polydimethylsiloxane (PDMSstar-MA) and diacrylated poly(ethylene glycol) (PEG-DA) macromers were prepared via solvent-induced phase separation (SIPS). The macromers were combined in a dichloromethane precursor solution and sequentially photopolymerized, dried and hydrated. The chemical and physical properties of the hydrogels were further tailored by varying the number average molecular weight (Mn) of PEG-DA (Mn =3.4k and 6k gmol−1) as well as the weight percent ratio of PDMSstar-MA (Mn =7k gmol−1) to PEG-DA from 0:100 to 20:80. Compared to analogous hydrogels fabricated from aqueous precursor solutions, SIPS produced hydrogels with a macroporous morphology, a more even distribution of PDMSstar-MA, increased modulus and enhanced degradation rates. The morphology, swelling ratio, mechanical properties, bioactivity, non-specific protein adhesion, controlled introduction of cell adhesion, and cytocompatibility of the hydrogels were characterized. As a result of their tunable properties, this library of hydrogels is useful to study material-guided cell behavior and ultimate tissue regeneration. [Copyright &y& Elsevier]

Abstract: The extracellular matrix is continually remodelled by the action of various enzymes such as heparanase, which specifically targets heparan sulfate (HS) and is found in human platelets at high levels. The activity of heparin-containing hydrogels following incubation with platelet extract (PE) was investigated in order to simulate the responses that might occur when the hydrogels, as tissue engineered scaffolds, come in contact with blood products at the site of an injury. The heparanase activity of PE on heparin, used as a model of HS, was confirmed by the decrease in molecular weight. PE treatment diminished heparin''s anticoagulation property but increased its FGF-2 signalling activity, suggesting that the PE''s heparanase activity cleaves at the 3-O-sulfated glucosamine to produce large fragments that can signal cell receptors. The dual effect observed when poly(vinyl alcohol)/heparin co-hydrogels were incubated with PE supports the hypothesis of platelets having the capacity to limit anticoagulation and thus promote blood clot formation, which may be critical in the process of tissue repair. [Copyright &y& Elsevier]

Abstract: Three-dimensional scaffolds like hydrogels can be used for cell and drug delivery and have become a major research focus in tissue engineering. Presently, we investigated the regenerative potency of platelet-rich plasma (PRP) combined with a chondrocyte/hydrogel composite scaffold in the repair of articular cartilage defects using a rabbit model. Primary isolated joint chondrocytes from the trachlear groove of rabbit were cultured in hydrogels as follows; hydrogel (2900Pa or 5900Pa)+chondrocytes and hydrogel+chondrocytes+PRP for in vitro analysis and in vivo implantation. The 5900Pa hydrogel markedly increased cellular viability and development in a time-dependent manner. Furthermore, the hydrogels attenuated the expression of SOX-9, aggrecan, and type II collagen. PRP-containing hydrogels produced an immediate increase in mRNA levels of cannabinoid receptor (CB)1 and CB2, compared with control and PRP-free hydrogels. Osteochondral defects were enhanced recovery with formation of cartilage and perichondrium in the 5900Pa hydrogel+chondrocytes+PRP. Hydrogel may provide a suitable environment for proliferation and maturation of joint chondrocytes in relation to the gelation density and bioactive sources like PRP resulting in improvement for cartilage regeneration. [Copyright &y& Elsevier]

During the last decades, great efforts have been devoted to design polymers for reducing the toxicity, increasing the absorption, and improving the release profile of drugs. Advantage has been also taken from the inherent multivalency of polymers and dendrimers for the incorporation of diverse functional molecules of interest in targeting and diagnosis. In addition, polymeric hydrogels with the ability to encapsulate drugs and cells have been developed for drug delivery and tissue engineering applications. In the long road to this successful story, pharmaceutical sciences have been accompanied by parallel advances in synthetic methodologies allowing the preparation of precise polymeric materials with enhanced properties. In this context, the introduction of the click concept by Sharpless and coworkers in 2001 focusing the attention on modularity and orthogonality has greatly benefited polymer synthesis, an area where reaction efficiency and product purity are significantly challenged. The purpose of this Expert Review is to discuss the impact of click chemistry in the preparation and functionalization of polymers, dendrimers, and hydrogels of interest in drug delivery. [ABSTRACT FROM AUTHOR]

Abstract: Recent advances in pluripotent stem cell research have provided investigators with potent sources of cardiogenic cells. However, tissue engineering methodologies to assemble cardiac progenitors into aligned, 3-dimensional (3D) myocardial tissues capable of physiologically relevant electrical conduction and force generation are lacking. In this study, we introduced 3D cell alignment cues in a fibrin-based hydrogel matrix to engineer highly functional cardiac tissues from genetically purified mouse embryonic stem cell-derived cardiomyocytes (CMs) and cardiovascular progenitors (CVPs). Procedures for CM and CVP derivation, purification, and functional differentiation in monolayer cultures were first optimized to yield robust intercellular coupling and maximize velocity of action potential propagation. A versatile soft-lithography technique was then applied to reproducibly fabricate engineered cardiac tissues with controllable size and 3D architecture. While purified CMs assembled into a functional 3D syncytium only when supplemented with supporting non-myocytes, purified CVPs differentiated into cardiomyocytes, smooth muscle, and endothelial cells, and autonomously supported the formation of functional cardiac tissues. After a total culture time similar to period of mouse embryonic development (21 days), the engineered cardiac tissues exhibited unprecedented levels of 3D organization and functional differentiation characteristic of native neonatal myocardium, including: 1) dense, uniformly aligned, highly differentiated and electromechanically coupled cardiomyocytes, 2) rapid action potential conduction with velocities between 22 and 25cm/s, and 3) significant contractile forces of up to 2mN. These results represent an important advancement in stem cell-based cardiac tissue engineering and provide the foundation for exploiting the exciting progress in pluripotent stem cell research in the future tissue engineering therapies for heart disease. [Copyright &y& Elsevier]

Abstract: A strategy has been designed to develop hyaluronic acid (HA) hydrogel for in vivo bone augmentation using minimal invasive technique. A mild synthetic procedure was developed to prepare aldehyde modified HA by incorporating an amino-glycerol side chain via amidation reaction and selective oxidation of the pendent group. This modification, upon mixing with hydrazide modified HA formed hydrazone-crosslinked hydrogel within 30s that was stable at physiological pH. In vitro experiments showed no cytotoxicity of hydrogel with the controlled release of active bone morphogenic protein-2 (BMP-2). In vivo evaluation of this gel as a BMP-2 carrier was performed by injecting gels over the rat calvarium and showed bone formation in 8weeks in correlation with the amount of BMP-2 loaded (0, 1 and 30μg) within the gel. Furthermore, hydrogels with 30μg of BMP-2 induced less bone formation upon subcutaneous injection in comparison with subperiosteal implantation. Histological examination showed newly formed bone with a high expression of osteocalcin, osteopontin and with angiogenic bone marrow when higher BMP-2 concentration was employed. Our result suggests that novel HA hydrogels could be used as a BMP-2 carrier and can promote bone augmentation for potential orthopedic applications. [Copyright &y& Elsevier]

Abstract: To effectively repair or replace damaged tissues, it is necessary to design scaffolds with tunable structural and biomechanical properties that closely mimic the host tissue. In this paper, we describe a newly synthesized photocrosslinkable interpenetrating polymer network (IPN) hydrogel based on gelatin methacrylate (GelMA) and silk fibroin (SF) formed by sequential polymerization, which possesses tunable structural and biological properties. Experimental results revealed that IPNs, where both the GelMA and SF were independently crosslinked in interpenetrating networks, demonstrated a lower swelling ratio, higher compressive modulus and lower degradation rate as compared to the GelMA and semi-IPN hydrogels, where only GelMA was crosslinked. These differences were likely caused by a higher degree of overall crosslinking due to the presence of crystallized SF in the IPN hydrogels. NIH-3T3 fibroblasts readily attached to, spread and proliferated on the surface of IPN hydrogels, as demonstrated by F-actin staining and analysis of mitochondrial activity (MTT). In addition, photolithography combined with lyophilization techniques was used to fabricate three-dimensional micropatterned and porous microscaffolds from GelMA–SF IPN hydrogels, furthering their versatility for use in various microscale tissue engineering applications. Overall, this study introduces a class of photocrosslinkable, mechanically robust and tunable IPN hydrogels that could be useful for various tissue engineering and regenerative medicine applications. [Copyright &y& Elsevier]

Abstract: The limitations of the current clinical options for valve replacements have inspired the development of enabling technologies to create a tissue engineered heart valve (TEHV). Poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds permit greater biological and biomechanical customization than do non-woven mesh scaffold technologies. However, the material characterization of PEGDA hydrogels has been predominantly limited to compression and tension, as opposed to bending. Since large flexural deformations result in points of maximum stress in native valves as well as TEHVs, it is crucial to evaluate any potential scaffold material in this mode. The effect of formulation parameters on the bending mechanics of cell-seeded PEGDA hydrogels were investigated with a custom designed bending tester. Three molecular weights (3.4, 6, and 8kDa) and three weight fractions (5%, 10%, and 15%, w/v) were subjected to three-point bending tests and the flexural stiffness was calculated. Manipulating the composition of the hydrogels resulted in flexural stiffnesses comparable with native tissues (15–220kPa) with varied mesh sizes and swelling ratios. Hydrogels containing encapsulated valve cells, methacrylated heparin (Hep-MA), or both were substantially less stiff than acellular hydrogels. In conclusion, PEGDA hydrogels are an attractive potential scaffold system for TEHVs because they are not only cytocompatible and modifiable but can also withstand bending deformations. These studies are the first to explore the encapsulation of valvular interstitial cells in pure PEGDA hydrogels as well as to investigate the bending properties of PEGDA gels. [Copyright &y& Elsevier]

Abstract: In this study, injectable hydrogels were prepared by horseradish peroxidase-mediated co-crosslinking of dextran–tyramine (Dex–TA) and heparin–tyramine (Hep–TA) conjugates and used as scaffolds for cartilage tissue engineering. The swelling and mechanical properties of these hydrogels can be easily controlled by the Dex–TA/Hep–TA weight ratio. When chondrocytes were incorporated in these gels, cell viability and proliferation were highest for gels with a 50/50 weight ratio of Dex–TA/Hep–TA. Moreover, these hydrogels induced an enhanced production of chondroitin sulfate and a more abundant presence of collagen as compared to Dex–TA hydrogels. The results indicate that injectable Dex–TA/Hep–TA hydrogels are promising scaffolds for cartilage regeneration. [Copyright &y& Elsevier]

Abstract: Natural polysaccharides are attractive compounds with which to build scaffolds for bone and cartilage tissue engineering. Here we tested two non-standard ones, HE800 and GY785, for the two-dimensional (2-D) and three-dimensional (3-D) culture of osteoblasts (MC3T3-E1) and chondrocytes (C28/I2). These two glycosaminoglycan-like marine exopolysaccharides were incorporated into an injectable silylated hydroxypropylmethylcellulose-based hydrogel (Si-HPMC) that has already shown its suitability for bone and cartilage tissue engineering. Results showed that, similarly to hyaluronic acid (HA) (the control), HE800 and GY785 significantly improved the mechanical properties of the Si-HPMC hydrogel and induced the attachment of MC3T3-E1 and C28/I2 cells when these were cultured on top of the scaffolds. Si-HPMC hydrogel containing 0.67% HE800 exhibited the highest compressive modulus (11kPa) and allowed the best cell dispersion, especially of MC3T3-E1 cells. However, these cells did not survive when cultured in 3-D within hydrogels containing HE800, in contrast to C28/I2 cells. The latter proliferated in the microenvironment or concentrically depending on the nature of the hydrogel. Among all the constructs tested the Si-HPMC hydrogels containing 0.34% HE800 or 0.67% GY785 or 0.67% HA presented the most interesting features for cartilage tissue engineering applications, since they offered the highest compressive modulus (9.5–11kPa) while supporting the proliferation of chondrocytes. [Copyright &y& Elsevier]

Abstract: In this study, a new technique that controls individual cell orientation using nanofiber meshes within three-dimensional (3D) hydrogels is reported. Highly aligned and fragile electrospun nanofibers (average diameter 500 nm) were manufactured into portable and handleable meshes with average line density of 45 nanofibers per 100 μm and thickness ranging between 0.5 and 3.0 μm. Through a facile and reproducible fabrication process, the nanofiber meshes can be incorporated into 3D hydrogels via a bottom-up, layer-by-layer assembly process, resulting in macroscopic and highly organized scaffolds. The nanofibers dictated the orientation of the cytoskeleton of individual cells in a very precise manner, allowing altering of the orientation of a cell population throughout the thickness of the hydrogel. Addition of nanofibers affected cell phenotype and protein synthesis. This nanofiber-cell-hydrogel composite enables replication of the cellular and matrix architecture found in many natural tissues, offering a novel protocol for electrospun nanofibers in regenerative medicine and bioengineering. From the Clinical Editor: A novel protocol for highly organized nanofiber meshes incorporated into 3D hydrogels can be used to direct the overlying cell population cytoskeleton direction, phenotype, and protein synthesis. Nanospun matrices offers a significant advancement for controlled tissue bioengineering and regenerative medicine applications. [Copyright &y& Elsevier]

Abstract: Two series of poly(ether–ester)-based bis-functional macromers terminated with acrylate groups and a well-defined number of ester bonds were synthesized. One series had a chain of 1, 3 or 5 ester bonds at both ends of the central poly(ethylene glycol) block (molecular weight, about 1000), while the other had an alternating structure of oligo(ethylene glycol) each of them linked to two ester bonds, in which 6 or 10 ester bonds were incorporated equally in the macromer molecules and the total molecular weight was adjusted by about 1000. Irradiation of all poly(ether–ester) macromers mixed with camphorquinone resulted in the formation of gels. Gel yield increased and hydrophilic properties of the gels produced decreased with irradiation time. The elastic modulus of the gels decreased with the number of ester bonds. Upon incubation in a PBS solution (pH 8.04), all gels were gradually degraded with time. At 3weeks of incubation, the degradation ratio increased linearly with the number of ester bonds per unit of molecular weight of the macromers. The order of in vivo degradation rates determined from weight loss was similar to that of the in vitro study. Thus, these poly(ether–ester) macromers may be useful for biodegradable biomaterials or tissue engineering scaffolds. [Copyright &y& Elsevier]

Abstract: Soft tissue fillers are rapidly gaining popularity for aesthetic improvements or repair of adipose tissue deficits. Several injectable biopolymers have been investigated for this purpose, but often show rapid resorption or limited adipogenesis and do not mimic the native adipose extracellular matrix (ECM). We have generated an injectable adipose matrix scaffold by efficiently removing both the cellular and lipid contents of human lipoaspirate. The decellularized material retained the complex composition of peptides and glycosaminoglycans found in native adipose ECM. This matrix can be further processed by solubilizing the extracted ECM to generate a thermally responsive hydrogel that self-assembles upon subcutaneous injection. This hydrogel also supports the growth and survival of patient matched adipose-derived stem cells in vitro. The development of an injectable hydrogel from human lipoaspirate represents a minimally invasive option for adipose tissue engineering in terms of both the collection of source material and delivery of the scaffold. [Copyright &y& Elsevier]

Abstract: We present use of a synthetic, injectable matrix metalloproteinase (MMP)- responsive, bioactive hydrogel as an in situ forming scaffold to deliver thymosin β4 (Tβ4), a pro-angiogenic and pro-survival factor, along with vascular cells derived from human embryonic stem cells (hESC) in ischemic injuries to the heart in a rat model. The gel was found to substitute the degrading extracellular matrix in the infarcted myocardium of rats and to promote structural organization of native endothelial cells, while some of the delivered hESC-derived vascular cells formed de novo capillaries in the infarct zone. Magnetic resonance imaging (MRI) revealed that the microvascular grafts effectively preserved contractile performance 3 d and 6 wk after myocardial infarction, attenuated left ventricular dilation, and decreased infarct size as compared to infarcted rats treated with PBS injection as a control (3 d ejection fraction, + ∼7%, P < 0.001; 6 wk ejection faction, + ∼12%, P < 0.001). Elevation in vessel density was observed in response to treatment, which may be due in part to elevations in human (donor)-derived cytokines EGF, VEGF and HGF (1 d). Thus, a clinically relevant matrix for dual delivery of vascular cells and drugs may be useful in engineering sustained tissue preservation and potentially regenerating ischemic cardiac tissue. [ABSTRACT FROM AUTHOR]

Abstract: Injectable scaffolds made of biodegradable biomaterials can stabilize a myocardial infarct and promote cardiac repair. Here, we describe the synthesis of a new, temperature-sensitive, aliphatic polyester hydrogel (HG) conjugated with vascular endothelial growth factor (VEGF) and evaluate its effects on cardiac recovery after a myocardial infarction (MI). Seven days after coronary ligation in rats, PBS, HG, or HG mixed or conjugated with VEGF (HG + VEGF or HG-VEGF, respectively) was injected around the infarct (n = 8–11/group). Function was evaluated by echocardiography at multiple time points. Pressure–volume measurements were taken and infarct morphometry and blood vessel density were assessed at 35 days after injection. HG-VEGF provided localized, sustained VEGF function. Compared with outcomes in the PBS group, fractional shortening, ventricular volumes, preload recruitable stroke work, and end-systolic elastance were all preserved (p < 0.05) in the HG and HG + VEGF groups, and further preserved in the HG-VEGF group. Conjugated VEGF also produced the highest blood vessel density (p < 0.05). The infarct thinned and dilated after PBS injection, but was smaller and thicker in hearts treated with HG (p < 0.05). Our temperature-sensitive HG attenuated adverse cardiac remodeling and improved ventricular function when injected after an MI. VEGF delivery enhanced these effects when the VEGF was conjugated to the HG. [ABSTRACT FROM AUTHOR]

Abstract: Elastin-like polypeptides (ELPs) have found utility in tissue engineering applications, not only because they are biocompatible, biodegradable, and non-immunogenic, but also because their amino acid sequence and molecular weight can be precisely controlled at the genetic or synthetic level, affording exquisite control over final protein functionality. This review presents a basic overview of ELP properties and modifications that are relevant to tissue engineering, as well as a discussion of the application of ELPs to cartilage, intervertebral disc, vascular graft, liver, ocular, and cell sheet engineering. [ABSTRACT FROM AUTHOR]

Abstract: Synthetic nanostructures based on self-assembling systems that aim to mimic natural extracellular matrix are now being used as substrates in tissue engineering applications. Peptides are excellent starting materials for the self-assembly process as they can be readily synthesised both chemically and biologically. P11-4 is an 11 amino acid peptide that undergoes triggered self-assembly to form a self-supporting hydrogel. It exists as unimers of random coil conformations in water above pH 7.5 but at low pH adopts an antiparallel β-sheet conformation. It also self-assembles under physiological conditions in a concentration-dependent manner. Here we describe an unimer P11-4 production system and the use of a simple site-directed mutagenesis approach to generate a series of other P11-family peptide expression vectors. We have developed an efficient purification strategy for these peptide biomaterials using a simple procedure involving chemical cleavage with cyanogen bromide then repeated filtration, lyophilisation and wash steps. We report peptide-fusion protein yields of ca. 4.64 g/L and we believe the highest reported recovery of a recombinant self-assembling peptide at 203 mg/L of pure recombinant P11-4. This peptide forms a self-supporting hydrogel under physiological conditions with essentially identical physico-chemical properties to the chemically synthesised peptide. Critically it also displays excellent cytocompatibility when tested with primary human dermal fibroblasts. This study demonstrates that high levels of a series of recombinant self-assembling peptides can be purified using a simple process for applications as scaffolds in tissue engineering. [Copyright &y& Elsevier]

Abstract: A critical element in the formation of scaffolds for tissue engineering is the introduction of concentration gradients of bioactive molecules. We explored the use of poly(ethylene glycol) (PEG) microspheres fabricated via a thermally induced phase separation to facilitate the creation of gradients in scaffolds. PEG microspheres were produced with different densities (buoyancies) and centrifuged to develop microsphere gradients. We previously found that the time to gelation following phase separation controlled the size of microspheres in the de-swollen state, while crosslink density affected swelling following buffer exchange into PBS. The principle factors used here to control microsphere densities were the temperature at which the PEG solutions were reacted following phase separation in aqueous sodium sulfate solutions and the length of the incubation period above the ‘cloud point’. Using different temperatures and incubation times, microspheres were formed that self-assembled into gradients upon centrifugation. The gradients were produced with sharp interfaces or gradual transitions, with up to 5 tiers of different microsphere types. For proof-of-concept, concentration gradients of covalently immobilized proteins were also assembled. PEG microspheres containing heparin were also fabricated. PEG-heparin microspheres were incubated with fluorescently labeled protamine and used to form gradient scaffolds. The ability to form gradients in microspheres may prove to be useful to achieve better control over the kinetics of protein release from scaffolds or to generate gradients of immobilized growth factors. [ABSTRACT FROM AUTHOR]

Abstract: Bioactive hydrogels formed by Michael-type addition reactions of end-functionalized poly(ethylene glycol) macromers with cysteine-containing peptides have been described as extracellular matrix mimetics and tissue engineering scaffolds. Although these materials have shown favorable behavior in vivo in tissue repair, we sought to develop materials formulations that would be more rapidly responsive to cell-induced enzymatic remodeling. In this study, protease-sensitive peptides that have increased kcat values were characterized and evaluated for their effects on gel degradability. Biochemical properties for soluble peptides and hydrogels were examined for matrix metalloproteinase (MMP)-1 and MMP-2. The most efficient peptide substrates in some cases overlap and in other cases differ between the two enzymes tested, and a range of kcat values was obtained. For each enzyme, hydrogels formed using the peptides with higher kcat values degraded faster than a reference with lower kcat. Fibroblasts showed increased cell spreading and proliferation when cultured in 3D hydrogels with faster degrading peptides, and more cell invasion from aortic ring segments embedded in the hydrogels was observed. These faster degrading gels should provide matrices that are easier for cells to remodel and lead to increased cellular infiltration and potentially more robust healing in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Tissue engineering requires the use of polymeric scaffolds that mimic many roles of extracellular matrices (ECM) in the body. Controlling cell–scaffold interactions is one of the most critical parameters for regulating cell phenotype in tissue engineering, and a peptide with the sequence of RGD has been widely exploited for this purpose. We hypothesized that the spacer arm length of adhesion ligands coupled to synthetic ECMs could be vital for regulation of cell–scaffold interactions. We prepared alginate gels modified with RGD peptides containing varying spacer arm lengths and cultured primary human fibroblasts either on the gels (2-D) or within the gels (3-D). The spacer arm length of the RGD peptides significantly influenced the adhesion and proliferation of fibroblasts in both the 2-D and 3-D studies. We found that a minimum number of four glycine units in the spacer arm was essential for enhanced adhesion and growth of the cells in vitro. An optimal spacer arm length of the RGD peptides was also necessary for minimizing cellular stress responses as determined by analyzing expression of heat shock proteins and Bcl-2 in cultured cells. This approach to controlling cell phenotype using adhesion peptides with various spacer arm lengths could be useful for designing novel scaffolds in tissue engineering applications. [Copyright &y& Elsevier]

Abstract: The cellular microenvironment plays an integral role in improving the function of microengineered tissues. Control of the microarchitecture in engineered tissues can be achieved through photopatterning of cell-laden hydrogels. However, despite high pattern fidelity of photopolymerizable hydrogels, many such materials are not cell-responsive and have limited biodegradability. Here, we demonstrate gelatin methacrylate (GelMA) as an inexpensive, cell-responsive hydrogel platform for creating cell-laden microtissues and microfluidic devices. Cells readily bound to, proliferated, elongated, and migrated both when seeded on micropatterned GelMA substrates as well as when encapsulated in microfabricated GelMA hydrogels. The hydration and mechanical properties of GelMA were demonstrated to be tunable for various applications through modification of the methacrylation degree and gel concentration. The pattern fidelity and resolution of GelMA were high and it could be patterned to create perfusable microfluidic channels. Furthermore, GelMA micropatterns could be used to create cellular micropatterns for in vitro cell studies or 3D microtissue fabrication. These data suggest that GelMA hydrogels could be useful for creating complex, cell-responsive microtissues, such as endothelialized microvasculature, or for other applications that require cell-responsive microengineered hydrogels. [Copyright &y& Elsevier]

Abstract: Native tissues contain space-filling dendritic pore networks, such as vasculature, for the efficient distribution of oxygen and nutrients; however, it is not yet possible to create tissue-engineered scaffolds with dendritic porosity. Fibers are also important structural features of native tissues because they provide sites for cell anchorage, promote cell guidance and contribute to mechanical stability. Here, we have developed a “crystal templating” technique, which is simple and inexpensive, for fabricating polymer scaffolds with space-filling dendritic pore networks and fibrillar microtopography. To do this, we grow dendritic urea crystals in solution cast films of hyaluronic acid (HA), photocrosslink the HA around the crystal network to lock in the dendritic configuration, and dissolve the crystals to obtain empty pores. During in situ crystal growth the HA biopolymer is phase separated from the long narrow urea crystals and shaped into a fibrillar microstructure. The porous fibrillar HA scaffolds created by crystal templating may be applicable as regenerative patches for skin and other tissues. [Copyright &y& Elsevier]

Abstract: Hydrogel nanofiber mats based on thermoresponsive multiblock poly(ester urethane)s comprising poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), and poly(ε-caprolactone) (PCL) segments were fabricated by electrospinning. The hydrogel nanofiber mats were more water absorbent under cold conditions and shrunk when exposed to higher temperatures. The rate of protein release could be controlled by changing the temperature of the nanofiber environment. Cell culture studies on the nanofiber mats were carried out using human dermal fibroblasts, and healthy cell morphology was observed. The adherent viable cells were quantified by MTS after rinsing in excess buffer solution. The results showed that these nanofiber scaffolds supported excellent cell adhesion, comparable with the pure PCL nanofibers. The increased hydrophilicity of these hydrogel nanofiber mats led to a more rapid hydrolytic degradation, compared with the pure PCL nanofiber mats. These hydrogel nanofiber scaffolds could potentially be used as thermoresponsive biodegradable supporting structures for skin tissue engineering applications. [Copyright &y& Elsevier]

Abstract: Polysaccharide hybrids consisting of hyaluronic acid (HA) grafted with a dextran–tyramine conjugate (Dex–TA) were synthesized and investigated as injectable biomimetic hydrogels for cartilage tissue engineering. The design of these hybrids (denoted as HA-g-Dex–TA) is based on the molecular structure of proteoglycans present in the extracellular matrix of native cartilage. Hydrogels of HA-g-Dex–TA were rapidly formed within 2 min via enzymatic crosslinking of the tyramine residues in the presence of horseradish peroxidase and hydrogen peroxide. The gelation time, equilibrium swelling and storage modulus could be adjusted by varying the degree of substitution of tyramine residues and polymer concentration. Bovine chondrocytes incorporated in the HA-g-Dex–TA hydrogels remained viable, as shown by the Live–dead assay. Moreover, enhanced chondrocyte proliferation and matrix production were observed in the HA-g-Dex–TA hydrogels compared to Dex–TA hydrogels. These results suggest that HA-g-Dex–TA hydrogels have a high potential as injectable scaffolds for cartilage tissue engineering. [Copyright &y& Elsevier]

Abstract: 6-Methacryloyl-α-d-galactopyranose (MG) was synthesized, and characterized by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectrometry, and single-crystal X-ray diffraction. A series of interpenetrating polymer network (IPN) hydrogels was fabricated by simultaneously photocuring MG crosslinked by poly(ethylene glycol) diacrylate and chemically crosslinking type I collagen with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. The successful incorporation of the glycopolymer, polymer MG, into collagen hydrogel was confirmed by FTIR and solid-state 13C NMR. The optical characteristics of the IPN hydrogels are comparable to those of human corneas. The tensile strength and modulus of the hydrogels are enhanced by incorporation of polymer MG in comparison to that of the control collagen hydrogel. Biodegradation results indicated that polymer MG enhanced the stability of the composite hydrogels against collagenase. In vitro results demonstrated that the IPN hydrogel supported the adhesion and proliferation of human corneal epithelial cells and outperformed human cornea in blocking bacteria adhesion. Taken together, the IPN hydrogel might be a promising material for use in corneal lamellar keratoplasty. [Copyright &y& Elsevier]

Abstract: This study was conducted to reveal the chondrogenesis of mesenchymal stem cells that had been genetically modified with the green fluorescence protein (GFP) gene and then co-cultured with chondrocytes in vitro and in vivo. Subsequent mixing of chondrocytes in the hydrogel constructs induced increased chondrogenic differentiation of the transfected hMSCs. The proliferation and differentiation of MSCs that were transfected with the GFP gene and co-cultured with chondrocytes (1:1 and 1:3) or chondrocytes alone were evaluated by a live/dead assay, MTT assay, GAG & DNA assay, RT-PCR, real time-PCR, and histological and immunochemical analysis in vitro and in vivo. Real-time PCR revealed that the expression of aggrecan and COMP by genetically modified hMSCs co-cultured with chondrocytes was 2 or 3 times greater than that of genetically modified MSCs alone. Moreover, the expression of collagen type II was more than 3.5 times greater than that of genetically modified MSCs alone. 3-D hydrogel constructs co-cultured with chondrocytes and genetically modified MSCs showed a significantly higher number of specific lacunae phenotypes at the end of the 4 week study, regardless of whether they were co-cultured in the presence of chondrocytes. These findings indicate that co-culture with chondrocytes and genetically modified MSCs can be used to engineer well designed implants for the formation of neocartilage by transplanted genetically modified MSCs. [Copyright &y& Elsevier]

Abstract: There has been an increasing interest in understanding how the mechanical properties of the microenvironment influence stem cell fate. We describe studies of the proliferation and differentiation of neural stem cells (NSCs) encapsulated within three-dimensional scaffolds – alginate hydrogels – whose elastic moduli were varied over two orders of magnitude. The rate of proliferation of neural stem cells decreased with increase in the modulus of the hydrogels. Moreover, we observed the greatest enhancement in expression of the neuronal marker β-tubulin III within the softest hydrogels, which had an elastic modulus comparable to that of brain tissues. To our knowledge, this work represents the first demonstration of the influence of modulus on NSC differentiation in three-dimensional scaffolds. Three-dimensional scaffolds that control stem cell fate would be broadly useful for applications in regenerative medicine and tissue engineering. [Copyright &y& Elsevier]

Abstract: Multicellular spheroids provide a new three-dimensional (3D) level of control over morphology and function of ex vivo cultured tissues. They also represent a valuable experimental technique for drug discovery and cell biology. Nevertheless, the dependence of many cellular processes on the cluster diameter remains unclear. To provide a tool for the systematic evaluation of such dependences, we introduce here inverted colloidal crystal (ICC) scaffolds. Uniformly sized pores in ICC cell matrixes afford a high yield production of controlled size spheroids in standard 96 well-plates. Transparent hydrogel matrix and ship-in-bottle effect also allows for convenient monitoring of cell processes by traditional optical techniques. Different developmental stages of 46.5–151.6μm spheroids from HepG2 hepatocytes with vivid morphological similarities to liver tissue (bile canaliculi) were observed. The liver-specific functions of HepG2 cells were systematically investigated and compared for spheroids of different diameters as well as 2D cultures. Clear trends of albumin production and CYP450 activity were observed; diffusion processes and effect of cellular aggregation on metabolic activity were identified to be the primary contributors to the size dependence of the liver functions in HepG2 spheroids in ICC scaffolds. Since the aggregation of cells into clusters is a universal biological process, these findings and scaffolds can be applied to many other relevant cell types. [Copyright &y& Elsevier]

Abstract: A major hurdle to surmount in bone-tissue engineering is ensuring a sufficient oxygen supply to newly forming tissue to avoid cell death or delayed development of osteogenic features. We hypothesized that an oxygen-enriched hydrogel scaffold would enhance tissue-engineered bone formation in vivo. To test this, we used a well-characterized mesenchymal stem cell (MSC) line, Tet-off BMP2 MSC, whose cells were engineered to express recombinant human bone morphogenetic protein-2. Cells were suspended in hydrogel supplemented with perfluorotributylamine (PFTBA) and implanted subcutaneously in an ectopic site, a radial bone defect, or a lumbar paravertebral muscle (mouse model of spinal fusion) in C3H/HeN mice. For controls, we used cells suspended in the same gel without PFTBA. In the ectopic site, there were significant increases in bone formation (2.5-fold increase), cell survival, and osteocalcin activity in the PFTBA-supplemented groups. PFTBA supplementation significantly increased structural parameters of bone in radial bone defects and triggered a significant 1.4-fold increase in bone volume in the spinal fusion model. We conclude that synthetic oxygen carrier supplementation of tissue-engineered implants enhances ectopic bone formation and yields better bone quality and volume in bone-repair and spinal fusion models, probably due to increased cell survival. [Copyright &y& Elsevier]

Abstract: Modifications of poly(2-hydroxyethyl methacrylate) (PHEMA) with cholesterol and laminin have been developed to design scaffolds that promote cell–surface interaction. Cholesterol-modified superporous PHEMA scaffolds have been prepared by the bulk radical copolymerization of 2-hydroxyethyl methacrylate (HEMA), cholesterol methacrylate (CHLMA) and the cross-linking agent ethylene dimethacrylate (EDMA) in the presence of ammonium oxalate crystals to introduce interconnected superpores in the matrix. With the aim of immobilizing laminin (LN), carboxyl groups were also introduced to the scaffold by the copolymerization of the above monomers with 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA). Subsequently, the MCMEMA moiety in the resulting hydrogel was hydrolyzed to [2-(methacryloyloxy)ethoxy]acetic acid (MOEAA), and laminin was immobilized via carbodiimide and N-hydroxysulfosuccinimide chemistry. The attachment, viability and morphology of mesenchymal stem cells (MSCs) were evaluated on both nonporous and superporous laminin-modified as well as laminin-unmodified PHEMA and poly(2-hydroxyethyl methacrylate-co-cholesterol methacrylate) P(HEMA–CHLMA) hydrogels. Neat PHEMA and laminin-modified PHEMA (LN–PHEMA) scaffolds facilitated MSC attachment, but did not support cell spreading and proliferation; the viability of the attached cells decreased with time of cultivation. In contrast, MSCs spread and proliferated on P(HEMA–CHLMA) and LN-P(HEMA–CHLMA) hydrogels. [Copyright &y& Elsevier]

Abstract: The potential of mesenchymal stem cells (MSCs) as a viable cell source for cartilage repair hinges on the development of engineered scaffolds that support adequate cartilage tissue formation. Evolving networks (hydrogels with mesh sizes that change over time due to crosslink degradation) may provide the control needed to enhance overall tissue formation when compared to static scaffolds. In this study, MSCs were photoencapsulated in combinations of hydrolytically and enzymatically degradable hyaluronic acid (HA) hydrogels to investigate the tunability of these hydrogels and the influence of network evolution on neocartilage formation. In MSC-laden HA hydrogels, compressive mechanical properties increased when degradation complemented extracellular matrix deposition and decreased when degradation was too rapid. In addition, dynamic hydrogels that started at a higher wt% and decreased to a lower wt% were not equivalent to static hydrogels that started at the higher or lower wt%. Specifically, evolving 2wt% hydrogels (2wt% degrading to 1wt%) expressed up-regulation of type II collagen and aggrecan, and exhibited increased glycosaminoglycan content over non-evolving 2 and 1wt% hydrogels. Likewise, mechanical properties and size maintenance were superior in the dynamic system compared to the static 2wt% and 1wt% hydrogels, respectively. Thus, hydrogels with dynamic properties may improve engineered tissues and help translate tissue engineering technology to clinical application. [Copyright &y& Elsevier]

Abstract: To engineer complex tissues, it is necessary to create hybrid scaffolds with micropatterned structural and biomechanical properties, which can closely mimic the intricate body tissues. The current report describes the synthesis of a novel photocrosslinkable interpenetrating polymeric network (IPN) of collagen and hyaluronic acid (HA) with precisely controlled structural and biomechanical properties. Both collagen and HA are present in crosslinked form in IPNs, and the two networks are entangled with each other. IPNs were also compared with semi-IPNs (SIPN), in which only collagen was in network form and HA chains were entangled in the collagen network without being photocrosslinked. Scanning electron microscopy images revealed that IPNs are denser than SIPNs, which results in their molecular reinforcement. This was further confirmed by rheological experiments. Because of the presence of the HA crosslinked network, the storage modulus of IPNs was almost two orders of magnitude higher than SIPNs. The degradation of the collagen–HA IPNs was slower than the SIPNs because of the presence of the crosslinked HA network. Increasing concentration of HA further altered the properties among IPNs. Cytocompatibility of IPNs was confirmed by Schwann cell and dermal fibroblasts adhesion and proliferation studies. We also fabricated patterned scaffolds with regions of IPNs and SIPNs within a bulk hydrogel, resulting in zonal distribution of crosslinking densities, viscoelasticities, water content and pore sizes at the micro- and macro-scales. With the ability to fine-tune the scaffold properties by performing structural modifications and to create patterned scaffolds, these hydrogels can be employed as potential candidates for regenerative medicine applications. [Copyright &y& Elsevier]

Abstract: The drawbacks of poloxamer hydrogel, such as dissolving quickly in aqueous solution, have limited its biomedical application. In order to improve the stability of hydrogel, a novel system was developed by combining the reversible thermo-sensitive property of poloxamer 407 and the thiol-ene reactivity between the acrylate and thiol groups. It was found that the sol–gel transition of the acrylate/thiol modified poloxamer 407 mixture could be achieved at body temperature even with a low concentration of 17.5 wt.%. Meanwhile, the reaction between the acrylate and thiol modified poloxamer 407s occurred spontaneously in mimic physiological conditions, thus the hydrogel with crosslinking structure was formed. As a result, the stability of the crosslinked hydrogel was enhanced remarkably, and the release time of the drug from the crosslinked hydrogel was about 4.0 times as long as that from the poloxamer 407 hydrogel. In vitro and in vivo experiments revealed that the biocompatibilities of the modified poloxamer 407 hydrogel were similar to that of poloxamer 407. These results indicate that the modified poloxamer 407s have potential applications in controlled drug release, tissue engineering and cell encapsulation etc. [Copyright &y& Elsevier]