Interactions of corneal epithelial cells and surfaces modified with cell adhesion peptide combinations

In order to facilitate the adhesion of corneal epithelial cells to a poly dimethyl siloxane (PDMS) substrate ultimately for the development of a synthetic keratoprosthesis, PDMS surfaces were modified by covalent attachment of combinations of cell adhesion and synergistic peptides derived from laminin and fibronectin. Peptides studied included YIGSR and its synergistic peptide PDSGR from laminin and the fibronectin derived RGDS and PHSRN. Surfaces were modified with combinations of peptides determined by an experimental design. Peptide surface densities, measured using 125-I labeled tyrosine containing analogs, were on the order of pmol/cm2. Surface density varied as a linear function of peptide concentration in the reaction solution, and was different for the different peptides examined. The lowest surface density at all solution fractions was obtained with GYRGDS, while the highest density was consistently obtained with GYPDSGR. These results provide evidence that the surfaces were modified with multiple peptides. Water contact angles and XPS results provided additional evidence for differences in the chemical composition of the various surfaces. Significant differences in the adhesion of human corneal epithelial cells to the modified surfaces were noted. Statistical analysis of the experimental adhesion results suggested that solution concentration YIGSR, RGDS, and PHSRN as well as the interaction effect of YIGSR and PDSGR had a significant effect on cell interactions. Modification with multiple peptides resulted in greater adhesion than modification with single peptides only. Surface modification with a control peptide PPSRN in place of PHSRN resulted in a decrease in cell adhesion in virtually all cases. These results suggest that surface modification with appropriate combinations of cell adhesion peptides and synergistic peptides may result in improved cell surface interactions.     

Effect of RGD secondary structure and the synergy site PHSRN on cell adhesion, spreading and specific integrin engagement

The relationship between the form of cell adhesion, ligand presentation, and cell receptor function was characterized using model Langmuir-Blodgett supported films, containing lipid-conjugated peptide ligands, in which isolated variables of the ligand presentation were systematically altered. First, the conformation of an adhesive Arginine-Glycine-Aspartic acid (RGD) peptide was varied by synthesizing linear and looped RGD peptide-containing amphiphiles and subsequently measuring the impact on the function of human umbilical vein endothelial cells. Secondly, the contribution of non-contiguous ligands to cellular engagement was assessed using multi-component biomimetic films. The peptide amphiphiles were composed of fibronectin-derived headgroups--GRGDSP, and its synergy site Pro-His-Ser-Arg-Asn (PHSRN)--attached to hydrocarbon tails. The peptide amphiphiles were diluted using polyethylene glycol (PEG) amphiphiles, where PEG inhibited non-specific cell adhesion. Cells adhered and spread on GRGDSP/PEG systems in a dose-dependent manner. The presentation of GRGDSP influenced integrin cell surface receptor specificity. Results demonstrated that beta1-containing integrins mediated adhesion to the linear GRGDSP presentation to a greater extent than did the alphavbeta3 integrin, and looped GRGDSP preferentially engaged alphavbeta3. GRGDSP/PHSRN/PEG mixtures that closely mimicked the RGD-PHSRN distance in fibronectin, enhanced cell spreading over their two-component analogues. This study demonstrated that controlling the microenvironment of the cell was essential for biomimetics to modulate specific binding and subsequent signaling events.     

Smooth muscle cell growth in photopolymerized hydrogels with cell adhesive and proteolytically degradable domains: synthetic ECM analogs for tissue engineering

Photopolymerizable polyethylene glycol (PEG) derivatives have been investigated as hydrogel tissue engineering scaffolds. These materials have been modified with bioactive peptides in order to create materials that mimic some of the properties of the natural extracellular matrix (ECM). The PEG derivatives with proteolytically degradable peptides in their backbone have been used to form hydrogels that are degraded by enzymes involved in cell migration, such as collagenase and elastase. Cell adhesive peptides, such as the peptide RGD, have been grafted into photopolymerized hydrogels to achieve biospecific cell adhesion. Cells seeded homogeneously in the hydrogels during photopolymerization remain viable, proliferate, and produce ECM proteins. Cells can also migrate through hydrogels that contain both proteolytically degradable and cell adhesive peptides. The biological activities of these materials can be tailored to meet the requirements of a given tissue engineering application by creating a mixture of various bioactive PEG derivatives prior to photopolymerization.     

Adhesion of alpha5beta1 receptors to biomimetic substrates constructed from peptide amphiphiles

Biomimetic membrane surfaces functionalized with fragments of the extracellular matrix protein, fibronectin, are constructed from mixtures of peptide and polyethylene glycol (PEG) amphiphiles. Peptides from the primary binding loop, GRGDSP, were used in conjunction with the synergy site peptide, PHSRN, in the III(9-10) sites of human fibronectin. These peptides were attached to dialkyl lipid tails to form peptide amphiphiles. PEG amphiphiles were mixed in the layer to minimize non-specific adhesion in the background. GRGDSP and PEG amphiphiles or GRGDSP, PHSRN, and PEG amphiphiles were mixed in various ratios and deposited on solid substrates from the air-water interface using Langmuir-Blodgett techniques. In this method, peptide composition, density, and presentation could be controlled accurately. The effectiveness of these substrates to mimic native fibronectin is evaluated by their ability to generate adhesive forces when they are in contact with purified activated alpha5beta1 integrin receptors that are immobilized on an opposing surface. Adhesion is measured using a contact mechanical approach (JKR experiment). The effects of membrane composition, density, temperature, and peptide conformation on adhesion to activated integrins in this simulated cell adhesion setup were determined. Addition of the synergy site, PHSRN, was found to increase adhesion of alpha5beta1, to biomimetic substrates markedly. Increased peptide mobility (due to increased experimental temperature) increased integrin adhesion markedly at low peptide concentrations. A balance between peptide density and steric accessibility of the receptor binding face to alpha5beta1 integrin was required for highest adhesion.     

Attachment and spreading of fibroblasts on an RGD peptide-modified injectable hyaluronan hydrogel

Hyaluronan (HA) hydrogels resist attachment and spreading of fibroblasts and most other mammalian cell types. A thiol-modified HA (3,3'-dithiobis(propanoic dihydrazide) [HA-DTPH]) was modified with peptides containing the Arg-Gly-Asp (RGD) sequence and then crosslinked with polyethylene glycol (PEG) diacrylate (PEGDA) to create a biomaterial that supported cell attachment, spreading, and proliferation. The hydrogels were evaluated in vitro and in vivo in three assay systems. First, the behavior of human and murine fibroblasts on the surface of the hydrogels was evaluated. The concentration and structure of the RGD peptides and the length of the PEG spacer influenced cell attachment and spreading. Second, murine fibroblasts were seeded into HA-DTPH solutions and encapsulated via in situ crosslinking with or without bound RGD peptides. Cells remained viable and proliferated within the hydrogel for 15 days in vitro. Although the RGD peptides significantly enhanced cell proliferation on the hydrogel surface, the cell proliferation inside the hydrogel in vitro was increased only modestly. Third, HA-DTPH/PEGDA/peptide hydrogels were evaluated as injectable tissue engineering materials in vivo. A suspension of murine fibroblasts in HA-DTPH was crosslinked using PEGDA plus PEGDA peptide, and the viscous, gelling mixture was injected subcutaneously into the flanks of nude mice; gels formed in vivo following injection. After 4 weeks, growth of new fibrous tissue had been accelerated by the sense RGD peptides. Thus, attachment, spreading, and proliferation of cells is dramatically enhanced on RGD-modified surfaces but only modestly accelerated in vivo tissue formation.     

Enhancing hepatocyte adhesion by pulsed plasma deposition and polyethylene glycol coupling

Decreased hepatocyte adhesion to polymeric constructs limits the function of tissue engineered hepatic assist devices. We grafted adhesion peptides (RGD and YIGSR) to polycaprolactone (PCL) and poly-L-lactic acid (PLLA) in order to mimic the in vivo extracellular matrix and thus enhance hepatocyte adhesion. Peptide grafting was done by a novel technique in which polyethylene glycol (PEG)-adhesion peptide was linked to allyl-amine coated on the surface of PCL and PLLA by pulsed plasma deposition (PPD). Peptide grafting density, quantified by radio-iodinated tyrosine in YIGSR, was 158 fmol/cm(2) on PLLA and 425 fmol/cm(2) on PCL surfaces. The adhesion of hepatocytes was determined by plating 250,000 hepatocytes/well (test substrates were coated on 12 well plates) and quantifying the percentage of adhered cells after 6 h by MTT assay. Adhesion on PCL surfaces was significantly enhanced (p < 0.05) by both YIGSR (percentage of adhered cells = 53 +/- 7%) and RGD (53 +/- 12%) when compared to control surfaces (31 +/- 8%). Hepatocyte adhesion on PLLA was significantly (p < 0.05) enhanced on PLLA-PEG-RGD surfaces (76 +/- 14%) compared to control surfaces (42 +/- 19%) and more (68 +/- 25%) but not statistically significant (p = 0.15) on PLLA-PEG-YIGSR surfaces compared to control surfaces. These results indicate that hepatocyte adhesion to PCL and PLLA based polymeric surfaces can be enhanced by a novel adhesion peptide grafting technique using pulsed plasma deposition and PEG cross-linking.     

Functionalized self-assembling peptide nanofiber hydrogels mimic stem cell niche to control human adipose stem cell behavior in vitro

A class of designer functionalized self-assembling peptide nanofiber scaffolds developed from self-assembling peptide RADA16-I (AcN-RADARADARADARADA-CONH₂) has become increasingly attractive not only for studying spatial behaviors of cells, but also for developing approaches for a wide range of medical applications including regenerative medicine, rapid hemostasis and cell therapy. In this study, we report three functionalized self-assembling peptide hydrogels that serve as a three-dimensional (3-D) artificial microenvironment to control human adipose stem cell (hASC) behavior in vitro. Short peptide motifs SKPPGTSS (bone marrow homing motif), FHRRIKA (heparin-binding motif) and PRGDSGYRGDS (two-unit RGD cell adhesion motif) were used to extend the C-terminus of RADA16-I to obtain functionalized peptides. Atomic force microscopy confirmed the formation of self-assembling nanofibers in the mixture of RADA16-I peptide and functionalized peptides. The behaviors of hASCs cultured in 3-D peptide hydrogels, including migration, proliferation and growth factor-secretion ability, were studied. Our results showed that the functionalized peptide hydrogels were suitable 3-D scaffolds for hASC growth with higher cell proliferation, migration and the secretion of angiogenic growth factors compared with tissue culture plates and pure RADA16-I scaffolds. The present study suggests that these functionalized designer peptide hydrogels not only have promising applications for diverse tissue engineering and regenerative medicine applications as stem cell delivery vehicles, but also could be a biomimetic 3-D system to study nanobiomaterial–stem cell interactions and to direct stem cell behaviors.     

Monocyte activation in response to polyethylene glycol hydrogels grafted with RGD and PHSRN separated by interpositional spacers of various lengths

Polyethylene glycol (PEG) is often cited as a "stealth" polymer, capable of resisting both protein adsorption and cell adhesion. By extension, PEG would then be expected to limit the host response. Monocyte-derived macrophages play an integral role in inflammation, and thus their response to a material can potentially dictate the overall host response to a biomaterial. In the present study, monocyte responses following interaction with a photopolymerized PEG hydrogel were compared with those from standard tissue culture polystyrene (TCPS). Additionally, the effect of the spacing between RGD and PHSRN, the corresponding synergy sequence on fibronectin (FN), was evaluated using peptides with differing spacer lengths grafted to the PEG hydrogel. Monocyte adherent density on the PEG-only hydrogel was comparable with that of TCPS; however, the secretion of the proinflammatory molecules interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF) increased dramatically following monocyte interaction with PEG-only hydrogels as compared with TCPS. The matrix metalloproteinase-2 (MMP-2) concentration was similar for all surfaces, while both the matrix metalloproteinase-9 (MMP-9) and FN concentrations were above the range of the assay for all substrates. Cell density was higher on the PHSRNG(13)RGD grafted substrate as compared with PHSRNG(6)RGD, but neither sequence increased cell density versus RGD alone. Although protein concentration did sometimes vary with different peptides, this variation was minimal in comparison with the surface effects between TCPS and the PEG-only hydrogel. This study explores the roles of PEG and FN-derived peptides on monocyte activation.     

Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering

Poly(ethylene glycol) (PEG) hydrogels were investigated as encapsulation matrices for osteoblasts to assess their applicability in promoting bone tissue engineering. Non-adhesive hydrogels were modified with adhesive Arg-Gly-Asp (RGD) peptide sequences to facilitate the adhesion, spreading, and, consequently, cytoskeletal organization of rat calvarial osteoblasts. When attached to hydrogel surfaces, the density and area of osteoblasts attached were dramatically different between modified and unmodified hydrogels. A concentration dependence of RGD groups was observed, with increased osteoblast attachment and spreading with higher RGD concentrations, and cytoskeleton organization was seen with only the highest peptide density. A majority of the osteoblasts survived the photoencapsulation process when gels were formed with 10% macromer, but a decrease in osteoblast viability of approximately 25% and 38% was seen after 1 day of in vitro culture when the macromer concentration was increased to 20 and 30wt%, respectively. There was no statistical difference in cell viability when peptides were added to the network. Finally, mineral deposits were seen in all hydrogels after 4 weeks of in vitro culture, but a significant increase in mineralization was observed upon introduction of adhesive peptides throughout the network.     

Biodegradable poly(ethylene glycol)–peptide hydrogels with well-defined structure and properties for cell delivery

In this study, biodegradable PEG–peptide hydrogels have been synthesized using Click chemistry. A series of Arg-Gly-Asp (RGD) containing peptides were prepared via a solid phase synthesis approach, which were further functionalized with azide to yield peptide azide or peptide diazide. A tetra-hydroxy terminated 4-arm PEG was functionalized with acetylene and was reacted with peptide azide/diazide and/or PEG diazide to produce hydrogels via a copper mediated 1,3-cycloaddition (Click chemistry) generating a triazole linkage as the networking forming reaction. The gelation time ranged from 2 to 30 min, depending on temperature, catalyst and precursor concentration, as well as peptide structure. The resulting hydrogels were characterized by swelling, viscoelastic properties and morphology as well as their ability for cell attachment and proliferation. Hydrogels cross-linked by peptide diazide yielded higher storage modulus (G′) with shorter spacers between azide groups. As expected, the swelling degree decreased while the G′ increased with increasing the concentration of the precursors as a result of increased cross-linking density. Primary human dermal fibroblasts were used as model cells to explore the possibility of using the RGD peptide hydrogels for cell-based wound healing. The attachment and proliferation of the cells on the hydrogels were evaluated. The RGD peptide hydrogels synthesized with a peptide concentration of 2.7–5.4 mm achieved significantly improved cell attachment and greater cell proliferation rate when compared to the hydrogels without RGD peptides. These hydrogels may provide a platform technology to deliver cells for tissue repair.     

Human microvascular endothelial cell growth and migration on biomimetic surfactant polymers

Successful engineering of a tissue-incorporated vascular prosthesis requires cells to proliferate and migrate on the scaffold. Here, we report on a series of "ECM-like" biomimetic surfactant polymers that exhibit quantitative control over the proliferation and migrational properties of human microvascular endothelial cells (HMVEC). The biomimetic polymers consist of a poly(vinyl amine) (PVAm) backbone with hexanal branches and varying ratios of cell binding peptide (RGD) to carbohydrate (maltose). Proliferation and migration behavior of HMVEC was investigated using polymers containing RGD: maltose ratios of 100:0, 75:25 and 50:50, and compared with fibronectin (FN) coated glass (1 microg/cm2). A radial Teflon fence migration assay was used to examine HMVEC migration at 12 h intervals over a 48 h period. Migration was quantified using an inverted optical microscope, and HMVEC were examined by confocal microscopy for actin and focal adhesion organization/ arrangement. Over the range of RGD ligand density studied (approximately 0.19-0.6 peptides/nm2), our results show HMVEC migration decreases with increasing RGD density in the polymer. HMVEC were least motile on the 100% RGD polymer (approximately 0.38-0.6 peptides/nm2) with an average migration of 0.20 mm2/h in area covered, whereas HMVEC showed the fastest migration of 0.48+/-0.06 mm2/h on the 50% RGD surface ( approximately 0.19-0.30 peptides/nm2). In contrast, cell proliferation increased with increasing surface peptide density; proliferation on the 50% RGD surface was 1.5%+/-0.06/h compared with 2.2%+/-0.07/h on the 100% RGD surface. Our results show that surface peptide density affects cellular functions such as growth and migration, with the highest peptide density supporting the most proliferation but the slowest migration.     

Adhesive and mechanical properties of hydrogels influence neurite extension

Photopolymerizable polyethylene glycol (PEG) hydrogels conjugated with bioactive ligands were examined for their use as scaffolds in peripheral nerve regeneration applications. The bioactivity and mechanical properties of PEG hydrogels can be tailored through the integration of bioactive factors (adhesion ligands, proteolytic sites, growth factors) and the alteration of PEG concentrations, respectively. For peripheral nerve regeneration, it will be important to determine the type and concentration of the bioactive molecules required to improve neurite extension. In this study, cell adhesion ligands (RGDS, IKVAV, and YIGSR) were covalently attached to PEG hydrogels. Both the type and concentration of cell adhesion ligand used affected neurite extension. Extension from PC12 cells was greater on hydrogels with RGDS incorporated than IKVAV, and the optimal concentration for each ligand was different. Cells adhered to but did not extend neurites on hydrogels with YIGSR. Cells did not adhere to hydrogels containing RGES. Furthermore, different combinations of these ligands affected neurite extension to different degrees. The mechanical properties of the hydrogels also significantly affected neurite extension. PC12 cells grown on more flexible hydrogels exhibited the greatest degree of neurite extension. PEG hydrogels have thus been developed with varying biochemical and mechanical properties that may enhance nerve regeneration.     

Corneal epithelial cell growth over tethered-protein/peptide surface-modified hydrogels

In this study, we investigated the corneal epithelial cell growth rate and adhesion to novel hydrogels with (1) extracellular matrix proteins [fibronectin, laminin, substance P, and insulin-like growth factor-1 (IGF-1)] and (2) peptide sequences [RGD and fibronectin adhesion-promoting peptide (FAP)] tethered to their surface on poly(ethylene glycol) (PEG) chains. The growth rate to confluence of primary rabbit cornea epithelial cells was compared for plain polymethacrylic acid-co-hydroxyethyl methacrylate (PHEMA/MAA) hydrogels, PHEMA/MAA hydrogels coated with extracellular matrix proteins or peptides, and PHEMA/MAA hydrogels with tethered extracellular matrix proteins or peptides on the surface. The development of focal adhesions by the epithelial cells grown on the surfaces was determined by F-actin staining. Little to no epithelial cell growth occurred on the plain hydrogel surfaces throughout the 15-day culture period. Of the coated hydrogels, only the fibronectin-coated surfaces showed a significant increase in cell growth compared to plain hydrogels (p < 0.009). However, even these surfaces reached a maximum of only 20% confluence. Laminin, fibronectin adhesion-promoting peptide (FAP), and fibronectin/laminin (1:1) tether-modified hydrogels all achieved 100% confluence by the end of the culture period, although the rates at which confluence was reached differed. F-actin staining showed that focal adhesions were formed for the laminin, FAP, and fibronectin/laminin tether-modified surfaces. The results support the hypothesis that tethering certain extracellular matrix proteins and/or peptides to the hydrogel surface enhances epithelial cell growth and adhesion, compared with that seen for protein-coated or plain hydrogel surfaces.     

Biofunctionalization of collagen for improved biological response: scaffolds for corneal tissue engineering

Residual dendrimer amine groups were modified with incorporate COOH group containing biomolecules such as cell adhesion peptides into collagen scaffolds. YIGSR, as a model cell adhesion peptide, was incorporated into both the bulk structure of the gels and onto the gel surface. The effects of the peptide modified collagen gels on corneal epithelial cell behavior were examined with an aim of improving the potential of these materials as tissue-engineering scaffolds. YIGSR was first chemically attached to dendrimers and the YIGSR attached dendrimers were then used as collagen crosslinkers, incorporating the peptide into the bulk structure of the collagen gels. YIGSR was also attached to the surface of dendrimer crosslinked collagen gels through reaction with excess amine groups. The YIGSR modified dendrimers were characterized by H-NMR and MALDI mass spectra. The amount of YIGSR incorporated into collagen gels was determined by (125)I radiolabelling at maximum to be 3.1-3.4 x 10(-2)mg/mg collagen when reacted with the bulk and 88.9-95.6 microg/cm(2) when attached to the surface. The amount of YIGSR could be tuned by varying the amount of peptide reacted with the dendrimer or the amount of modified dendrimer used in the crosslinking reaction. It was found that YIGSR incorporation into the bulk and YIGSR modification of surface promoted the adhesion and proliferation of human corneal epithelial cells as well as neurite extension from dorsal root ganglia.     

Fibronectin modulates macrophage adhesion and FBGC formation: the role of RGD, PHSRN, and PRRARV domains.

To probe the role of human plasma fibronectin in modulating human blood-derived macrophage adhesion and fusion to form multinucleated foreign-body giant cells (FBGC), a series of biomimetic oligopeptides based on the functional structure of fibronectin was designed and synthesized. Peptides incorporated the RGD and PHSRN integrin-binding sequences from FIII-10 and FIII-9 modules, respectively, and the PRRARV sequence from the C-terminal heparin-binding domain, either alone or in combination. Peptides were immobilized onto a polyethyleneglycol-based polymer substrate. The following conclusions were reached. Fibronectin modulated macrophage adhesion and the extent (i.e., size) of FBGC formation on control surfaces in the presence of serum proteins. Macrophages adhered to all substrates with relatively subtle differences between adhesion mediated by RGD, PHSRN, PRRARV, or combinations thereof. beta1-integrin subunit was essential in macrophage adhesion to peptide-grafted networks in a receptor-peptide specific manner, whereas beta3-integrin subunit was less important. Macrophage adhesion to PRRARV was mediated primarily by the direct interaction with integrins. RGD or PHSRN alone did not provide an adequate substrate for macrophage fusion to form FBGCs. However, the PHSRN synergistic site and the RGD site in a single oligopeptide provided a substrate for FBGC formation that was statistically comparable to that on the positive control material in the presence of serum proteins. This response was highly dependent upon the relative orientation between RGD and PHSRN. PRRARV did not support FBGC formation. These results demonstrate the importance of fibronectin and, specifically, the synergy between RGD and PHSRN domains, in supporting macrophage fusion to form FBGCs.     

Photo-crosslinkable, biomimetic, and thermo-sensitive pluronic grafted hyaluronic acid copolymers for injectable delivery of chondrocytes

Hyaluronic acid (HA) grafted with Pluronic F127 copolymer was used as biomimetic hydrogels for cell delivery. The graft copolymer was synthesized by conjugating amine end-capped Pluronic F127 to carboxylic groups of HA using coupling agents. The synthesized HA-g-Pluronic exhibited thermo-sensitive sol-gel transition behaviors over the temperature range of 20-40 degrees C. HA-g-Pluronic copolymers with vinyl groups were photo-crosslinked to prepare more robust hydrogels for cell cultivation. For improved cellular adhesion and proliferation, cell adhesive peptide (Arg-Gly-Asp (RGD)) was additionally conjugated to the HA backbone. The resultant thermo-sensitive, photocrosslinkable, and RGD modified HA-g-Pluronic copolymers were used to encapsulate and cultivate bovine chondrocytes in vitro. A tissue containing cartilage-like components such as GAG and type II collagen was successfully produced within the hydrogels, indicating that the synthesized HA-g-Pluronic copolymers can be potentially used as an injectable cell carrier.     

Study on the potential of RGD- and PHSRN-modified alginates as artificial extracellular matrices for engineering bone

Alginate is a polysaccharide that can be crosslinked by divalent cations, such as calcium ions, to form a gel. Chemical modification is typically used to improve its cell adhesive properties for tissue engineering applications. In this study, alginates were modified with peptides containing RGD (arginine–glycine–aspartic acid) or PHSRN (proline–histidine–serine–arginine–asparagine) sequences from fibronectin to study possible additive and synergistic effects on adherent cells. Alginates modified with each peptide were mixed at different ratios to form gels containing various concentrations and spacing between the RGD and PHSRN sequences. When normal human osteoblasts (NHOsts) were cultured on or in the gels, the ratio of RGD to PHSRN was found to influence cell behaviors, especially differentiation. NHOsts cultured on gels composed of RGD- and PHSRN-modified alginates showed enhanced differentiation when the gels contained >33 % RGD-alginate, suggesting the relative distribution of the peptides and the presentation to cells are important parameters in this regulation. NHOsts cultured in gels containing both RGD- and PHSRN-alginates also demonstrated a similar enhancement tendency of calcium deposition that was dependent on the peptide ratio in the gel. However, calcium deposition was greater when cells were cultured in the gels, as compared to on the gels. These results suggest that modifying this biomaterial to more closely mimic the chemistry of natural cell adhesive proteins, (e.g., fibronectin) may be useful in developing scaffolds for bone tissue engineering and provide three-dimensional cell culture systems which more closely mimic the environment of the human body.     

Selective adhesion of astrocytes to surfaces modified with immobilized peptides.

Under serum-free conditions, rat skin fibroblasts, but not cortical astrocytes, selectively adhered to glass surfaces modified with the integrin-ligand peptide RGDS. In contrast, astrocytes, but not fibroblasts, exhibited enhanced adhesion onto substrates modified with KHIFSDDSSE, a peptide that mimics a homophilic binding domain of neural cell adhesion molecule (NCAM). Astrocyte and fibroblast adhesion onto substrates modified with the integrin ligands IKVAV and YIGSR as well as the control peptides RDGS and SEDSDKFISH were similar to that observed on aminophase glass (reference substrate). This study is the first to demonstrate the use of immobilized KHIFSDDSSE in selectively modulating astrocyte and fibroblast adhesion on material surfaces, potentially leading to materials that promote specific functions of cells involved in the response(s) of central nervous system tissues to injury. This information could be incorporated into novel biomaterials designed to improve the long-term performance of the next generation of neural prostheses.     

Enzymatic conjugation of a bioactive peptide into an injectable hyaluronic acid–tyramine hydrogel system to promote the formation of functional vasculature

In this study, one-step enzyme-mediated preparation of a multi-functional injectable hyaluronic-acid-based hydrogel system is reported. Hydrogel was formed through the in situ coupling of phenol moieties by horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂), and bioactive peptides were simultaneously conjugated into the hydrogel during the gel formation process. The preparation of this multi-functional hydrogel was made possible by synthesizing peptides containing phenols which could couple with the phenol moieties of hyaluronic-acid–tyramine (HA–Tyr) during the HRP-mediated crosslinking reaction. Preliminary studies demonstrated that two phenol moieties per molecule resulted in a consistently high degree of conjugation into the HA–Tyr hydrogel network, unlike the one modified with one phenol moiety per molecule. Therefore, an Arg–Gly–Asp (RGD) peptide bearing two phenol moieties (phenol₂–poly(ethylene glycol)–RGD) was designed for conjugation to endow the HA–Tyr hydrogel with adhesion signals and enhance its bioactivities. Human umbilical vein endothelial cells (HUVECs) cultured on or within the RGD-modified hydrogels showed significantly different adhesion behavior, from non-adherence on the HA–Tyr hydrogel to strong adhesion on hydrogels modified with phenol₂–poly(ethylene glycol)–RGD. This altered cell adhesion behavior led to improved cell proliferation, migration and formation of capillary-like network in the hydrogel in vitro. More importantly, when HUVECs and human fibroblasts (HFF1) were encapsulated together in the RGD-modified HA–Tyr hydrogel, functional vasculature was observed inside the cell-laden gel after 2 weeks in the subcutaneous tissue. Taken together, the in situ conjugation of phenol₂–poly(ethylene glycol)–RGD into HA–Tyr hydrogel system, coupled with the ease of incorporating cells, offers a simple and effective means to introduce biological signals for preparation of multi-functional injectable hydrogels for tissue engineering application.     

Extracellular matrix-mimetic poly(ethylene glycol) hydrogels engineered to regulate smooth muscle cell proliferation in 3-D

The goal of this project is to engineer a defined, synthetic poly(ethylene glycol) (PEG) hydrogel as a model system to investigate smooth muscle cell (SMC) proliferation in three-dimensions (3-D). To mimic the properties of extracellular matrix, both cell-adhesive peptide (GRGDSP) and matrix metalloproteinase (MMP) sensitive peptide (VPMSMRGG or GPQGIAGQ) were incorporated into the PEG macromer chain. Copolymerization of the biomimetic macromers results in the formation of bioactive hydrogels with the dual properties of cell adhesion and proteolytic degradation. Using these biomimetic scaffolds, the authors studied the effect of scaffold properties, including RGD concentration, MMP sensitivity, and network crosslinking density, as well as heparin as an exogenous factor on 3-D SMC proliferation. The results indicated that the incorporation of cell-adhesive ligand significantly enhanced SMC spreading and proliferation, with cell-adhesive ligand concentration mediating 3-D SMC proliferation in a biphasic manner. The faster degrading hydrogels promoted SMC proliferation and spreading. In addition, 3-D SMC proliferation was inhibited by increasing network crosslinking density and exogenous heparin treatment. These constructs are a good model system for studying the effect of hydrogel properties on SMC functions and show promise as a tissue engineering platform for vascular in vivo applications.