Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment

Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment.     

基于天然水凝胶的生物材料在骨组织工程中的应用*

天然水凝胶是指原材料来自于天然生物材料的水凝胶。由于这种天然的聚合物含有构成生物体的天然成分,与天然组织具有生物学和化学相似性,而受到特别关注。天然水凝胶由于其与细胞外基质高度的相似性被认为是骨组织工程中优良的仿生基质材料。而针对天然水凝胶机械性能差、成骨诱导性能弱等缺陷,通常需要对天然水凝胶进行改性、引入其他材料或生物活性因子,以此来获得更适用于骨组织工程支架材料。对近年来基于天然水凝胶的生物材料在骨组织工程的应用,与其不同的应用形式(可注射水凝胶、多孔水凝胶支架、3D生物打印水凝胶支架等)进行了概述,以期对这类基于天然水凝胶的生物材料在未来骨组织工程中的应用提供参考。     

Optimization of 3D hydrogel microenvironment for enhanced hepatic functionality of primary human hepatocytes

Although primary human hepatocytes (PHHs) are the gold standard in drug efficacy and metabolism studies, long-term survival of PHHs and maintenance of their hepatic function are still challenging. In this study, we focused on the effect of the initial microenvironment on upregulation and long-term preservation of hepatic function of PHHs encapsulated within biodegradable hydrogel systems. PHHs were encapsulated in RGD-functionalized hybrid hydrogels with various degrees of degradability, and their hepatic functionality was analyzed. Regardless of the hydrogel elastic modulus, the combination with nondegradable hydrogels had a predominantly negative effect on the prompt engraftment of PHHs, whereas a degradable hydrogel with intermediate initial degradability was most effective in maintaining hepatic function. Efficient network formation by PHHs and cocultured cells, along with the control of hydrogel degradation, governed the hepatic functionality at an early stage and upon long-term cultivation. Under optimized conditions, expression of genes involved in biological processes such as focal adhesions, cell survival, cytoskeleton formation, and extracellular matrix interactions was significantly higher than that in a control with relatively delayed initial degradation. Thus, we suggest that the orchestrated control of initial cellular remodeling may play an important role in the maintenance of hepatic function in a three-dimensional PHH culture.     

An approach to modulate degradation and mesenchymal stem cell behavior in poly(ethylene glycol) networks

A simple, sequential approach for creation of hydrolytically degradable poly(ethylene glycol) (PEG) hydrogels has been developed and characterized. The chemistry involves an initial step growth polymerization reaction between PEG-diacrylate and dithiothreitol (DTT) to form acrylate-terminated (-PEG-DTT-)n PEG chains, followed by photocross-linking to form a hydrogel network. Varying the extent of step growth polymerization prior to photocross-linking allowed for control over the equilibrium swelling ratio, degradation, and erosion of PEG hydrogels. Hydrogel degradability had a significant effect on behavior of human mesenchymal stem cells (hMSCs) encapsulated within PEG hydrogels, both in the presence and absence of an RGDSP cell adhesion ligand. In particular, enhanced network degradability resulted in enhanced hMSC viability and spreading during in vitro culture. Comparison of degradable and nondegradable hydrogels with similar physical properties (e.g., equilibrium swelling ratio) demonstrated that hMSC viability and spreading were dependent on network degradability. This study demonstrates that hydrolytically degradable PEG hydrogels can be formed via a sequential step growth polymerization and photocross-linking process and the resulting materials may serve as promising matrices for 3-dimensional stem cell culture and tissue engineering applications.     

Protein and mineral composition of osteogenic extracellular matrix constructs generated with a flow perfusion bioreactor

This study investigated the temporal composition of an osteogenic extracellular matrix construct generated by culturing mesenchymal stem cells in an electrospun biodegradable poly(ε-caprolactone) fiber mesh scaffold within a flow perfusion bioreactor. Constructs of different extracellular matrix maturities were analyzed for their protein and mineral composition at several culture durations by liquid chromatography-tandem mass spectrometry, scanning electron microscopy, energy-dispersive X-ray diffraction, X-ray diffraction, and calcium and phosphate assays. The analysis revealed that at short culture durations the cells deposited cellular adhesion proteins as a prerequisite protein network for further bone formation. At the later culture durations, the extracellular matrix was composed of collagen 1, hydroxyapatite, matrix remodeling proteins, and regulatory proteins. These results suggest that the later culture duration constructs would allow for improved bone regeneration due to the ability to mineralize and the capabilities for future remodeling.     

Peptide Hydrogelation and Cell Encapsulation for 3D Culture of MCF-7 Breast Cancer Cells

Three-dimensional (3D) cell culture plays an invaluable role in tumor biology by providing in vivo like microenviroment and responses to therapeutic agents. Among many established 3D scaffolds, hydrogels demonstrate a distinct property as matrics for 3D cell culture. Most of the existing pre-gel solutions are limited under physiological conditions such as undesirable pH or temperature. Here, we report a peptide hydrogel that shows superior physiological properties as an in vitro matrix for 3D cell culture. The 3D matrix can be accomplished by mixing a self-assembling peptide directly with a cell culture medium without any pH or temperature adjustment. Results of dynamic rheological studies showed that this hydrogel can be delivered multiple times via pipetting without permanently destroying the hydrogel architecture, indicating the deformability and remodeling ability of the hydrogel. Human epithelial cancer cells, MCF-7, are encapsulated homogeneously in the hydrogel matrix during hydrogelation. Compared with two-dimensional (2D) monolayer culture, cells residing in the hydrogel matrix grow as tumor-like clusters in 3D formation. Relevant parameters related to cell morphology, survival, proliferation, and apoptosis were analyzed using MCF-7 cells in 3D hydrogels. Interestingly, treatment of cisplatin, an anti-cancer drug, can cause a significant decrease of cell viability of MCF-7 clusters in hydrogels. The responses to cisplatin were dose- and time-dependent, indicating the potential usage of hydrogels for drug testing. Results of confocal microscopy and Western blotting showed that cells isolated from hydrogels are suitable for downstream proteomic analysis. The results provided evidence that this peptide hydrogel is a promising 3D cell culture material for drug testing.     

Synthetic biomimetic hydrogels incorporated with ephrin-A1 for therapeutic angiogenesis

Eph receptors and ephrin ligands are essential for vascular development and angiogenic remodeling. In this work, we developed biomimetic poly(ethylene glycol)-diacrylate hydrogels incorporated with ephrin-A1 and examined their angiogenic properties. Ephrin-A1 was covalently immobilized on the surface of hydrogels by chemical modification and photopolymerization. Ephrin-A1 immobilized on hydrogels was found to retain its capacity to stimulate endothelial cell adhesion in a dose-dependent manner as similar findings were observed on polystyrene culture wells pre-adsorbed with ephrin-A1. Cell adhesion stimulated by ephrin-A1 was abolished by treatment with soluble RGDS and anti-alpha(v)beta3 integrin but not anti-alpha(v)beta5 integrin antibodies, suggesting that ephrin-A1 activates cell adhesion through alpha(v)beta3 integrins. Also, surface immobilized ephrin-A1 was found to induce endothelial tubule formation with luminal diameters ranging 5-30 microm on hydrogels. The results of these studies demonstrate that pro-angiogenic properties of ephrin-A1 are preserved in hydrogels and suggest potential applications of this hydrogel system in regenerative medicine and tissue engineering.     

Hydrogels as extracellular matrix mimics for 3D cell culture

Methods for culturing mammalian cells ex vivo are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Two‐dimensional culture has been the paradigm for typical in vitro cell culture; however, it has been demonstrated that cells behave more natively when cultured in three‐dimensional environments. Permissive, synthetic hydrogels and promoting, natural hydrogels have become popular as three‐dimensional cell culture platforms; yet, both of these systems possess limitations. In this perspective, we discuss the use of both synthetic and natural hydrogels as scaffolds for three‐dimensional cell culture as well as synthetic hydrogels that incorporate sophisticated biochemical and mechanical cues as mimics of the native extracellular matrix. Ultimately, advances in synthetic–biologic hydrogel hybrids are needed to provide robust platforms for investigating cell physiology and fabricating tissue outside of the organism. Biotechnol. Bioeng. 2009;103: 655–663. © 2009 Wiley Periodicals, Inc.     

Viability of human mesenchymal stem cells seeded on crosslinked entropy-elastic gelatin-based hydrogels

Biomimetic polymer network systems with tailorable properties based on biopolymers represent a class of degradable hydrogels that provides sequences for protein adsorption and cell adhesion. Such materials show potential for in vitro MSC proliferation as well as in vivo applications and were obtained by crosslinking different concentrations of gelatin using varying amounts of ethyl lysine diisocyanate in the presence of a surfactant in pH 7.4 PBS solution. Material extracts, which were tested for cytotoxic effects using L929 mouse fibroblasts, were non-toxic. The hydrogels were seeded with human bone marrow-derived MSCs and supported viable MSCs for the incubation time of 9 d. Preadsorption of fibronectin on materials improved this biofunctionality.     

Critical role of filamin-binding LIM protein 1 (FBLP-1)/migfilin in regulation of bone remodeling

Bone remodeling is a complex process that must be precisely controlled to maintain a healthy life. We show here that filamin-binding LIM protein 1 (FBLP-1, also known as migfilin), a kindlin- and filamin-binding focal adhesion protein, is essential for proper control of bone remodeling. Genetic inactivation of FBLIM1 (the gene encoding FBLP-1) in mice resulted in a severe osteopenic phenotype. Primary FBLP-1 null bone marrow stromal cells (BMSCs) exhibited significantly reduced extracellular matrix adhesion and migration compared with wild type BMSCs. Loss of FBLP-1 significantly impaired the growth and survival of BMSCs in vitro and decreased the number of osteoblast (OB) progenitors in bone marrow and OB differentiation in vivo. Furthermore, the loss of FBLP-1 caused a dramatic increase of osteoclast (OCL) differentiation in vivo. The level of receptor activator of nuclear factor κB ligand (RANKL), a key regulator of OCL differentiation, was markedly increased in FBLP-1 null BMSCs. The capacity of FBLP-1 null bone marrow monocytes (BMMs) to differentiate into multinucleated OCLs in response to exogenously supplied RANKL, however, was not different from that of WT BMMs. Finally, we show that a loss of FBLP-1 promotes activating phosphorylation of ERK1/2. Inhibition of ERK1/2 activation substantially suppressed the increase of RANKL induced by the loss of FBLP-1. Our results identify FBLP-1 as a key regulator of bone homeostasis and suggest that FBLP-1 functions in this process through modulating both the intrinsic properties of OB/BMSCs (i.e., BMSC-extracellular matrix adhesion and migration, cell growth, survival, and differentiation) and the communication between OB/BMSCs and BMMs (i.e., RANKL expression) that controls osteoclastogenesis.     

Hydrogel‐based three‐dimensional cell culture for organ‐on‐a‐chip applications

Recent studies have reported that three‐dimensionally cultured cells have more physiologically relevant functions than two‐dimensionally cultured cells. Cells are three‐dimensionally surrounded by the extracellular matrix (ECM) in complex in vivo microenvironments and interact with the ECM and neighboring cells. Therefore, replicating the ECM environment is key to the successful cell culture models. Various natural and synthetic hydrogels have been used to mimic ECM environments based on their physical, chemical, and biological characteristics, such as biocompatibility, biodegradability, and biochemical functional groups. Because of these characteristics, hydrogels have been combined with microtechnologies and used in organ‐on‐a‐chip applications to more closely recapitulate the in vivo microenvironment. Therefore, appropriate hydrogels should be selected depending on the cell types and applications. The porosity of the selected hydrogel should be controlled to facilitate the movement of nutrients and oxygen. In this review, we describe various types of hydrogels, external stimulation‐based gelation of hydrogels, and control of their porosity. Then, we introduce applications of hydrogels for organ‐on‐a‐chip. Last, we also discuss the challenges of hydrogel‐based three‐dimensional cell culture techniques and propose future directions. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:580–589, 2017     

Pectin-based injectable biomaterials for bone tissue engineering

A variety of natural polymers and proteins are considered to be 3D cell culture structures able to mimic the extracellular matrix (ECM) to promote bone tissue regeneration. Pectin, a natural polysaccharide extracted from the plant cell walls and having a chemical structure similar to alginate, provides interesting properties as artificial ECM. In this work, for the first time, pectin, modified with an RGD-containing oligopeptide or not, is used as an ECM alternative to immobilize cells for bone tissue regeneration. The viability, metabolic activity, morphology, and osteogenic differentiation of immobilized MC3T3-E1 preosteoblats demonstrate the potential of this polysaccharide to keep immobilized cells viable and differentiating. Preosteoblasts immobilized in both types of pectin microspheres maintained a constant viability up to 29 days and were able to differentiate. The grafting of the RGD peptide on pectin backbone induced improved cell adhesion and proliferation within the microspheres. Furthermore, not only did cells grow inside but also they were able to spread out from the microspheres and to organize themselves in 3D structures producing a mineralized extracellular matrix. These promising results suggest that pectin can be proposed as an injectable cell vehicle for bone tissue regeneration.     

光交联水凝胶在组织工程中的研究进展

由于生物相容性、可降解性、与天然细胞外基质结构的相似性,水凝胶成为组织工程的研究热点与重点。基于原位形成和可注射性、与现有加工技术(3D打印、静电纺丝)的兼容性,光交联水凝胶在组织工程领域广泛应用。综述了近年来光交联水凝胶在组织工程领域的研究进展,包括其在软骨组织、骨组织、脂肪组织、牙周组织和皮肤组织方面的研究思路及应用进展,以期为后续光交联水凝胶作为组织工程支架的研究提供参考。     

Synthesis and evaluation of novel biodegradable hydrogels based on poly(ethylene glycol) and sebacic acid as tissue engineering scaffolds

Novel biodegradable poly(ethylene glycol) (PEG) based hydrogels, namely, PEG sebacate diacrylate (PEGSDA) were synthesized, and their properties were evaluated. Chemical structures of these polymers were confirmed by Fourier transform infrared and proton nuclear magnetic resonance (1H NMR) spectroscopy. After photopolymerization, the dynamic shear modulus of the hydrogels was up to 0.2 MPa for 50% PEGSDA hydrogel, significantly higher than conventional hydrogels such as PEG diacrylate (PEGDA). The swelling ratios of these macromers were significantly lower than PEGDA. The in vitro degradation study demonstrated that these hydrogels were biodegradable with weight losses about 66% and 32% for 25% and 50% PEGSDA after 8 weeks of incubation in phosphate-buffered saline at 37 degrees C. In vitro biocompatibility was assessed using cultured rat bone marrow stromal cells (MSCs) in the presence of unreacted monomers or degradation products. Unlike conventional PEGDA hydrogels, PEGSDA hydrogel without RGD peptide modification induced MSC cell adhesion similar to tissue culture polystyrene. Finally, complex three-dimensional structures of PEGSDA hydrogels using solid free form technique were fabricated and their structure integrity was better maintained than PEGDA hydrogels. These hydrogels may find use as scaffolds for tissue engineering applications.     

Design of biomimetic cell-interactive substrates using hyaluronic acid hydrogels with tunable mechanical properties

Hyaluronic acid (HA) is a natural polysaccharide abundant in biological tissues with excellent potential for constructing synthetic extracellular matrix analogues. In this work, we established a simple and dependable approach to prepare hyaluronic acid-based hydrogels with controlled stiffness and cell recognition properties for use as cell-interactive substrates. This approach relied on a new procedure for the synthesis of methacrylate-modified HA macromers (HA-MA) and, on photorheometry allowing real time monitoring of gelation during photopolymerization. We showed in this way the ability to obtain gels that encompass the range of physiologically relevant elastic moduli while still maintaining the recognition properties of HA by specific cell surface receptors. These hydrogels were prepared from HA macromers having a degree of methacrylation <0.5, which allows to minimize compromising effects on the binding affinity of HA to its cell receptors due to high substitution on the one hand, and to achieve nearly 100% conversion of the methacrylate groups on the other. When the HA hydrogels were immobilized on glass substrates, it was observed that the attachment and the spreading of a variety of mammalian cells rely on CD44 and its coreceptor RHAMM. The attachment and spreading were also shown to be modulated by the elastic properties of the HA matrix. All together, these results highlight the biological potential of these HA hydrogel systems and the needs of controlling their chemical and physical properties for applications in cell culture and tissue engineering.     

Laminin Peptide-Immobilized Hydrogels Modulate Valve Endothelial Cell Hemostatic Regulation

Valve endothelial cells (VEC) have unique phenotypic responses relative to other types of vascular endothelial cells and have highly sensitive hemostatic functions affected by changes in valve tissues. Furthermore, effects of environmental factors on VEC hemostatic function has not been characterized. This work used a poly(ethylene glycol) diacrylate (PEGDA) hydrogel platform to evaluate the effects of substrate stiffness and cell adhesive ligands on VEC phenotype and expression of hemostatic genes. Hydrogels of molecular weights (MWs) 3.4, 8, and 20 kDa were polymerized into platforms of different rigidities and thiol-modified cell adhesive peptides were covalently bound to acrylate groups on the hydrogel surfaces. The peptide RKRLQVQLSIRT (RKR) is a syndecan-1 binding ligand derived from laminin, a trimeric protein and a basement membrane matrix component. Conversely, RGDS is an integrin binding peptide found in many extracellular matrix (ECM) proteins including fibronectin, fibrinogen, and von Willebrand factor (VWF). VECs adhered to and formed a stable monolayer on all RKR-coated hydrogel-MW combinations. RGDS-coated platforms supported VEC adhesion and growth on RGDS-3.4 kDa and RGDS-8 kDa hydrogels. VECs cultured on the softer RKR-8 kDa and RKR-20 kDa hydrogel platforms had significantly higher gene expression for all anti-thrombotic (ADAMTS-13, tissue factor pathway inhibitor, and tissue plasminogen activator) and thrombotic (VWF, tissue factor, and P-selectin) proteins than VECs cultured on RGDS-coated hydrogels and tissue culture polystyrene controls. Stimulated VECs promoted greater platelet adhesion than non-stimulated VECs on their respective culture condition; yet stimulated VECs on RGDS-3.4 kDa gels were not as responsive to stimulation relative to the RKR-gel groups. Thus, the syndecan binding, laminin-derived peptide promoted stable VEC adhesion on the softer hydrogels and maintained VEC phenotype and natural hemostatic function. In conclusion, utilization of non-integrin adhesive peptide sequences derived from basement membrane ECM may recapitulate balanced VEC function and may benefit endothelialization of valve implants.     

Acyl-gelatins for cell-hybrid biomaterials: preparation of gelatins with high melting point and affinity for hydrophobic surfaces

In the development of cell-hybrid biomaterials, the functional activity of cells depends on the selective binding of cells to artificial ligands on the biomaterials. The extracellular matrix (ECM) is the most important ligand for cell activity. ECM is known to contain collagen, one of whose constituents is gelatin. Although natural gelatin has good cell attachment properties, the melting point of gelatin hydrogel is lower than body temperature. Thus, non-chemically cross-linked gelatin hydrogel is not a biomaterial that is used for prostheses. In the present study, we report the preparation of acyl-gelatin hydrogels with high melting point (>37 degrees C) and high affinity for hydrophobic surfaces for easy handling for transportation and adhesion activities on the hydrophobic surfaces. In addition, the doubling time of endothelial cells on the coated cell culture plate was faster than that of natural gelatin owing to the higher adhesion activity of acyl-gelatin. The results clearly demonstrated that the acyl-gelatin acted as an interface that enabled cell adhesion to artificial materials surfaces.     

Cell engineering biointerface focusing on cytocompatibility using phospholipid polymer with an isomeric oligo(lactic acid) segment

Initial contact between a biological environment and a biomaterial ultimately decides the in vivo performance. Therefore, the fabrication of a delicate biointerface is important because it can be utilized as a platform for novel biomaterials. For the preparation of advanced biomedical devices such as biochips, nanoparticles, and cell engineering devices, the surface properties may be modified by the design of polymeric biomaterials. Anomalous phospholipid polymers with an isomeric oligo(lactic acid) segment were designed and evaluated as a biointerface. The phospholipid polymer containing 2-methacryloyloxyethyl phosphorylcholine was easily copolymerized with isomeric oligo(lactic acid) macromonomers, and the obtained polymer could easily form thin coating membranes as biointerfaces. The oligo(lactic acid) involves three kinds of isomers: dl-, d-, and l-forms. The favorable characteristic on the surface provides regulation of cell-material interactions on the biointerface. The oligo(lactic acid) segment could form hydrophobic domains, which were considered to be located on the interface, to enhance protein adsorption and cell adhesion. The most favorable characteristics on the biointerface were dual functions of cytocompatibility by the phospholipid polymer and cell adhesion property by the oligo(lactic acid) segment. In this study, we focused on the biological responses such as protein adsorption and cell adhesion by change in the oligo(lactic acid) component. The cell viability on the confluent stage was evaluated in terms of metabolic activity.     

Response of zonal chondrocytes to extracellular matrix-hydrogels

We investigated the biological response of chondrocytes isolated from different zones of articular cartilage and their cellular behaviors in poly (ethylene glycol)-based (PEG) hydrogels containing exogenous type I collagen, hyaluronic acid (HA), or chondroitin sulfate (CS). The cellular morphology was strongly dependent on the extracellular matrix component of hydrogels. Additionally, the exogenous extracellular microenvironment affected matrix production and cartilage specific gene expression of chondrocytes from different zones. CS-based hydrogels showed the strongest response in terms of gene expression and matrix accumulation for both superficial and deep zone chondrocytes, but HA and type I collagen-based hydrogels demonstrated zonal-dependent cellular responses.     

The extracellular matrix and cell surface, mediators of cell interactions in chicken gastrulation

This article reviews the factors that are involved in cell-cell and cell-matrix interactions during chicken gastrulation. The chemical nature of the extracellular matrix, the structure, composition, cellular origin and remodeling of the basement membrane, and the nature of the cell surfaces are successively analyzed in relation to a variety of cell biological processes, such as cell-to-cell and cell-to-substrate adhesion, specific binding to biological macromolecules and to receptors of the cell surface, promotion of cell migration and positioning, transmembrane triggering of intracellular events, and modulation of cell shape. These processes are the cellular basis of morphogenesis in the chicken blastoderm.