Mary C. Farach-Carson

Hyaluronic acid-based hydrogels: from a natural polysaccharide to complex networks

Hyaluronic acid (HA) is one of natures most versatile and fascinating macromolecules. Being an essential component of the natural extracellular matrix (ECM), HA plays an important role in a variety of biological processes. Inherently biocompatible, biodegradable and non-immunogenic, HA is an attractive starting material for the construction of hydrogels with desired morphology, stiffness and bioactivity. While the interconnected network extends to the macroscopic level in HA bulk gels, HA hydrogel particles (HGPs, microgels or nanogels) confine the network to microscopic dimensions. Taking advantage of various scaffold fabrication techniques, HA hydrogels with complex architecture, unique anisotropy, tunable viscoelasticity and desired biologic outcomes have been synthesized and characterized. Physical entrapment and covalent integration of hydrogel particles in a secondary HA network give rise to hybrid networks that are hierarchically structured and mechanically robust, capable of mediating cellular activities through the spatial and temporal presentation of biological cues. This review highlights recent efforts in converting a naturally occurring polysaccharide to drug releasing hydrogel particles, and finally, complex and instructive macroscopic networks. HA-based hydrogels are promising materials for tissue repair and regeneration.

Three-Dimensional In Vitro Tumor Models for Cancer Research and Drug Evaluation

Cancer occurs when cells acquire genomic instability and inflammation, produce abnormal levels of epigenetic factors/proteins and tumor suppressors, reprogram the energy metabolism and evade immune destruction, leading to the disruption of cell cycle/normal growth. An early event in carcinogenesis is loss of polarity and detachment from the natural basement membrane, allowing cells to form distinct three-dimensional (3D) structures that interact with each other and with the surrounding microenvironment. Although valuable information has been accumulated from traditional in vitro studies in which cells are grown on flat and hard plastic surfaces (2D culture), this culture condition does not reflect the essential features of tumor tissues. Further, fundamental understanding of cancer metastasis cannot be obtained readily from 2D studies because they lack the complex and dynamic cell-cell communications and cell-matrix interactions that occur during cancer metastasis. These shortcomings, along with lack of spatial depth and cell connectivity, limit the applicability of 2D cultures to accurate testing of pharmacologically active compounds, free or sequestered in nanoparticles. To recapitulate features of native tumor microenvironments, various biomimetic 3D tumor models have been developed to incorporate cancer and stromal cells, relevant matrix components, and biochemical and biophysical cues, into one spatially and temporally integrated system. In this article, we review recent advances in creating 3D tumor models employing tissue engineering principles. We then evaluate the utilities of these novel models for the testing of anticancer drugs and their delivery systems. We highlight the profound differences in responses from 3D in vitro tumors and conventional monolayer cultures. Overall, strategic integration of biological principles and engineering approaches will both improve understanding of tumor progression and invasion and support discovery of more personalized first line treatments for cancer patients.

Hyaluronan: a simple polysaccharide with diverse biological functions.

Hyaluronan (HA) is a linear polysaccharide with disaccharide repeats of d-glucuronic acid and N-acetyl-d-glucosamine. It is evolutionarily conserved and abundantly expressed in the extracellular matrix (ECM), on the cell surface and even inside cells. Being a simple polysaccharide, HA exhibits an astonishing array of biological functions. HA interacts with various proteins or proteoglycans to organize the ECM and to maintain tissue homeostasis. The unique physical and mechanical properties of HA contribute to the maintenance of tissue hydration, the mediation of solute diffusion through the extracellular space and the lubrication of certain tissues. The diverse biological functions of HA are manifested through its complex interactions with matrix components and resident cells. Binding of HA with cell surface receptors activates various signaling pathways, which regulate cell function, tissue development, inflammation, wound healing and tumor progression and metastasis. Taking advantage of the inherent biocompatibility and biodegradability of HA, as well as its susceptibility to chemical modification, researchers have developed various HA-based biomaterials and tissue constructs with promising and broad clinical potential. This paper illustrates the properties of HA from a matrix biology perspective by first introducing the principles underlying the biosynthesis and biodegradation of HA, as well as the interactions of HA with various proteins and proteoglycans. It next highlights the roles of HA in physiological and pathological states, including morphogenesis, wound healing and tumor metastasis. A deeper understanding of the mechanisms underlying the roles of HA in various physiological processes can provide new insights and tools for the engineering of complex tissues and tissue models.

Attachment, proliferation, and migration of marrow stromal osteoblasts cultured on biomimetic hydrogels modified with an osteopontin-derived peptide.

We prepared oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels modified with a rat osteopontin-derived peptide (ODP), Asp-Val-Asp-Val-Pro-Asp-Gly-Arg-Gly-Asp-Ser-Leu-Ala-Try-Gly (DVDVPDGRGDSLAYG), as well as Gly-Arg-Gly-Asp-Ser (GRGDS) and investigated the modulation of marrow stromal osteoblast function on the peptide-modified hydrogels. Osteoblast attachment was competitively inhibited by a soluble peptide suggesting that the interaction of osteoblasts with the hydrogel was ligand specific. The proliferation index of osteoblasts relative to the initial seeding density was similar on the hydrogels modified with ODP (1.18+/-0.13) and GRGDS (1.27+/-0.12). However, fibroblasts proliferated faster on GRGDS-modified hydrogels than on ODP-modified hydrogels as evidenced by the proliferation indices of 4.89+/-0.03 and 2.42+/-0.16, respectively. A megacolony migration assay conducted for 3 days with a seeding density of 53,000 cells/cm(2) showed that osteoblasts migrated to a longer distance on ODP-modified hydrogels (0.23+/-0.06 mm/day) than on hydrogels modified with GRGDS (0.15+/-0.02 mm/day). In addition, osteoblasts migrated faster than fibroblasts seeded at the same density on ODP-modified hydrogels (0.15+/-0.11 mm/day). The migration of osteoblasts on the peptide-modified hydrogels was dependent on the peptide concentration of the hydrogels resulting in an increased migration distance with increasing the peptide concentration for the concentrations tested. These results show that OPF-based biomimetic hydrogels hold promise for modulating cell proliferation and migration for specific applications by altering the specific ligand and its concentration in the hydrogels.

Isolation, characterization and immunolocalization of a 53-kDal dentin sialoprotein (DSP).

We isolated a sialic-rich protein from rat dentin extracts and have named it dentin sialoprotein, DSP (formerly called 95K glycoprotein). DSP is rich in aspartic acid, glutamic acid, glycine and serine, but contains no cysteine or phosphate. The 30% carbohydrate content includes about 9% sialic acid and indicates that several N-glycosides and O-glycosides are present. Sedimentation equilibrium analysis gave a M(r) of 52,570. Based on this molecular weight we calculated that DSP contains about 350-amino acids and 75 monosaccharides. With automated Edman degradation the sequence of the first 8-amino acids was shown to be: Ile-Pro-Val-Pro-Gln-Leu-Val-Pro. The initial 3 residues of this sequence are identical to the first 3 in human osteopontin (OPN) and are closely similar to the Leu-Pro-Val sequences of OPN from other species, as well as at the beginning of bone acidic glycoprotein-75 (BAG-75). On Western immunoblots, purified polyclonal antibodies reacted only with DSP in dentin extracts and with none of the proteins from bone. Similarly, immunolocalization experiments showed the presence of DSP in dentin but not in enamel or alveolar bone. Along with immunohistochemical localization data reported elsewhere, these observations suggest that DSP may be an important marker for cells in the odontoblast lineage.

Modeling Ewing sarcoma tumors in vitro with 3D scaffolds

The pronounced biological influence of the tumor microenvironment on cancer progression and metastasis has gained increased recognition over the past decade, yet most preclinical antineoplastic drug testing is still reliant on conventional 2D cell culture systems. Although monolayer cultures recapitulate some of the phenotypic traits observed clinically, they are limited in their ability to model the full range of microenvironmental cues, such as ones elicited by 3D cell–cell and cell–extracellular matrix interactions. To address these shortcomings, we established an ex vivo 3D Ewing sarcoma model that closely mimics the morphology, growth kinetics, and protein expression profile of human tumors. We observed that Ewing sarcoma cells cultured in porous 3D electrospun poly(ε-caprolactone) scaffolds not only were more resistant to traditional cytotoxic drugs than were cells in 2D monolayer culture but also exhibited remarkable differences in the expression pattern of the insulin-like growth factor-1 receptor/mammalian target of rapamycin pathway. This 3D model of the bone microenvironment may have broad applicability for mechanistic studies of bone sarcomas and exhibits the potential to augment preclinical evaluation of antineoplastic drug candidates for these malignancies.

The in vivo performance of plasmonic nanobubbles as cell theranostic agents in zebrafish hosting prostate cancer xenografts

Cell theranostics is a new approach that unites diagnosis, therapy and confirmation (guidance) of the results of therapy in one single process at cell level, thus principally improving both the rapidity and precision of treatment. The ideal theranostic agent will support all three of the above functions in vivo with cellular resolution, allowing individual assessment of disease state and the elimination of diseased cells while leaving healthy cells intact. We have developed and evaluated plasmonic nanobubbles (PNBs) as an in vivo tunable theranostic cellular agent in zebrafish hosting prostate cancer xenografts. PNBs were selectively generated around gold nanoparticles in cancer cells in the zebrafish with short single laser pulses. By varying the energy of the laser pulse, we dynamically tuned the PNB size in a theranostic sequence of two PNBs: an initial small PNB detected a cancer cell through optical scattering, followed by a second bigger PNB, which mechanically ablated this cell without damage to surrounding tissue, while its optical scattering confirmed the destruction of the cell. Thus PNBs supported the diagnosis and guided ablation of individual human cancer cells in a living organism without damage to the host.

β2-microglobulin is a signaling and growth-promoting factor for human prostate cancer bone metastasis

The protein factor β2-microglobulin (β2M), purified from the conditioned medium of human prostate cancer cell lines, stimulated growth and enhanced osteocalcin (OC) and bone sialoprotein (BSP) gene expression in human prostate cancer cells by activating a cyclic AMP (cAMP)–dependent protein kinase A signaling pathway. When β2M was overexpressed in prostate cancer cells, it induced explosive tumor growth in mouse bone through increased phosphorylated cAMP-responsive element binding protein (CREB) and activated CREB target gene expression, including OC, BSP, cyclin A, cyclin D1, and vascular endothelial growth factor. Interrupting the β2M downstream signaling pathway by injection of the β2M small interfering RNA liposome complex produced an effective regression of previously established prostate tumors in mouse bone through increased apoptosis as shown by immunohistochemistry and activation of caspase-9, caspase-3, and cleavage of poly(ADP-ribose) polymerase. These results suggest that β2M signaling is an attractive new therapeutic target for the treatment of lethal prostate cancer bone metastasis. (Cancer Res 2006; 66(18): 9108-16)

Biofunctionalization of electrospun PCL-based scaffolds with perlecan domain IV peptide to create a 3-D pharmacokinetic cancer model

Because prostate cancer cells metastasize to bone and exhibit osteoblastic features (osteomimicry), the interrelationships between bone-specific microenvironment and prostate cancer cells at sites of bone metastasis are critical to disease progression. In this work the bone marrow microenvironment in vitro was recreated both by tailoring scaffolds physical properties and by functionalizing electrospun polymer fibers with a bioactive peptide derived from domain IV of perlecan heparan sulfate proteoglycan. Electrospun poly (epsilon-caprolactone) (PCL) fibers and PCL/gelatin composite scaffolds were modified covalently with perlecan domain IV (PlnDIV) peptide. The expression of tight junction protein (E-cadherin) and focal adhesion kinase (FAK) phosphorylation on tyrosine 397 also were investigated. The described bioactive motif significantly enhanced adherence and infiltration of the metastatic prostate cancer cells on all modified electrospun substrates by day 5 post-seeding. Cells cultured on PlnDIV-modified matrices organized stress fibers and increased proliferation at statistically significant rates. Additional findings suggest that presence of PlnDIV peptide in the matrix reduced expression of tight junction protein and binding to PlnDIV peptide was accompanied by increased focal adhesion kinase (FAK) phosphorylation on tyrosine 397. We conclude that PlnDIV peptide supports key signaling events leading to proliferation, survival, and migration of C4-2B cancer cells; hence its incorporation into electrospun matrix is a key improvement to create a successful three-dimensional (3-D) pharmacokinetic cancer model.

Potential Role for Heparan Sulfate Proteoglycans in Regulation of Transforming Growth Factor-β (TGF-β) by Modulating Assembly of Latent TGF-β-binding Protein-1

Latent transforming growth factor-β-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in storage of latent TGF-β in the ECM and regulate its availability. We have previously identified fibronectin as a key molecule for incorporation of LTBP1 and TGF-β into the ECM of osteoblasts and fibroblasts. Here we provide evidence that heparan sulfate proteoglycans may mediate binding between LTBP1 and fibronectin. We have localized critical domains in the N terminus of LTBP1 that are required for co-localization with fibronectin in osteoblast cultures and have identified heparin binding sites in the N terminus of LTBP1 between residues 345 and 487. Solid-phase binding assays suggest that LTBP1 does not bind directly to fibronectin but that the binding is indirect. Heparin coupled to bovine serum albumin (heparin-BSA) was able to mediate binding between fibronectin and LTBP1. Treatment of primary osteoblast cultures with heparin or heparin-BSA but not with chondroitin sulfate impaired LTBP1 deposition onto fibronectin without inhibiting expression of LTBP1. Inhibition of LTBP1 incorporation was accompanied by reduced incorporation of latent TGF-β into the ECM, with increased amounts of soluble latent TGF-β. Inhibition of attachment of glycosaminoglycans to the core proteins of proteoglycans by β-d-xylosides also reduced incorporation of LTBP1 into the ECM. These studies suggest that heparan sulfate proteoglycans may play a critical role in regulating TGF-β availability by controlling the deposition of LTBP1 into the ECM in association with fibronectin.