Biometric surfactant polymers designed for shear-stable endothelialization on biomaterials

We have developed a series of extracellular matrix (ECM)-like biomimetic surfactant polymers to improve endothelial cell adhesion and growth on vascular biomaterials. These polymers provide a single-step procedure for modifying the surface of existing biomaterials and consist of a poly(vinyl amine) (PVAm) backbone with varying ratios of cell-binding peptide (RGD) to carbohydrate (maltose), ranging from 100% RGD:0% maltose to 50% RGD:50% maltose. Three biomimetic surfaces, as well as a fibronectin (FN)-coated glass surface were seeded at confluence with human pulmonary artery endothelial cells (HPAECs) and exposed to shear stresses ranging from 0-40.6 dyn/cm2 for periods of 2 h and 6 h. Surfaces were examined for HPAEC coverage and cytoskeletal arrangement as a function of time and shear stress. In general, after 6 h of shear exposure, EC retention on 100% RGD > FN > 75% RGD > 50% RGD. The 100% RGD surface maintained more than 50% of its initial EC monolayer at low to moderate shear stresses whereas all other surfaces dropped to approximately 40% or less in the same shear stress range. The most stable surface, 100% RGD, showed a significant increase in cytoskeletal organization at all shear stresses greater than 2.5 dyn/cm2. In contrast, there was no real change in cytoskeletal organization on the FN surface, and there was a decrease on the 75% RGD surface over time. These results indicate that increasing surface peptide density can control EC shear stability. Furthermore, improved shear stability increases with increasing peptide density and is related to the EC's ability to reorganize its cytoskeleton.     

The selective modulation of endothelial cell mobility on RGD peptide containing surfaces by YIGSR peptides

The ability of the biomimetic peptides YIGSR, PHSRN and RGD to selectively affect adhesion and migration of human microvascular endothelial cells (MVEC) and vascular smooth muscle cells (HVSMC) was evaluated. Cell mobility was quantified by time-lapse video microscopy of single cells migrating on peptide modified surfaces. Polyethylene glycol (PEG) hydrogels modified with YIGSR or PHSRN allowed only limited adhesion and no spreading of MVEC and HVSMC. However, when these peptides were individually combined with the strong cell binding peptide RGD in PEG hydrogels, the YIGSR peptide was found to selectively enhance the migration of MVEC by 25% over that of MVEC on RGD alone (p<0.05). No corresponding effect was observed for HVSMC. This suggests that the desired response of specific cell types to tissue engineering scaffolds could be optimized through a combinatory approach to the use of biomimetic peptides.     

RGD modified polymers: biomaterials for stimulated cell adhesion and beyond

Since RGD peptides (R: arginine; G: glycine; D: aspartic acid) have been found to promote cell adhesion in 1984 (Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule, Nature 309 (1984) 30), numerous materials have been RGD functionalized for academic studies or medical applications. This review gives an overview of RGD modified polymers, that have been used for cell adhesion, and provides information about technical aspects of RGD immobilization on polymers. The impacts of RGD peptide surface density, spatial arrangement as well as integrin affinity and selectivity on cell responses like adhesion and migration are discussed.     

Surface modification for controlled studies of cell-ligand interactions

This work describes a method for coupling cell adhesion peptides to hydrophobic materials for the purpose of controlling surface peptide density while simultaneously preventing nonspecific protein adsorption. PEO/PPO/PEO triblock copolymers (Pluronic F108) were equipped with terminal pyridyl disulfide functionalities and used to tether RGD containing peptides to polystyrene (PS). The density of F108 on PS was 1.4 E5 +/- 2.12 E1 molecules/microm2. XPS and ToF SIMS indicated that the F108 coating was homogeneous and that the unmodified and activated F108 distributed evenly on PS. By mixing unmodified F108 with PDS-activated F108 prior to adsorption, it was possible to vary peptide density between 0 and 8.7 E4 +/- 2.66 E3 peptides/microm2, while otherwise, maintaining consistent surface properties. GRGDSY grafted PS supported cell attachment, spreading, and development of cytoskeletal structure, all of which were found to increase with increasing peptide density. Cell proliferation followed this same trend, however, maximal growth occurred at a submaximal peptide density. Cell aspect ratio varied in a biphasic manner with GRGDSY density. F108 coated PS and GRGESY grafted PS were inert to cell adhesion. Cells released from GRGDSY grafted PS upon addition of either a reducing agent or free GRGDSY, which indicates that cell-substrate interactions were mediated solely by the tethered peptides.     

Surface-immobilization of adhesion peptides on substrate for ex vivo expansion of cryopreserved umbilical cord blood CD34+ cells

The interaction between integrins and extracellular matrix proteins play an important role in the regulation of hematopoiesis. Human hematopoietic progenitor cells express very late antigen-4 (VLA-4) and VLA-5, which mediate their interaction with fibronectin by recognizing the connecting segment-1 (CS-1 and RGD motifs, respectively. In this study, we investigated the ex vivo expansion of human umbilical cord blood (UCB) CD34+ cells on synthetic substrates surface-immobilized with peptides containing the CS-1 binding motif (EILDVPST) and the RGD motif (GRGDSPC). These peptides were covalently conjugated to poly(ethylene terephthalate) (PET) film at a surface density of 2.0-2.3 nmol/cm2. UCB CD34+ cells were cultured for 10 days in serum-free medium supplemented with recombinant human thrombopoietin, stem cell factor, flt3-ligand and interleukin 3. The highest cell expansion fold was observed on the CS-1 peptide-modified surface, where total nucleated cells, total colony forming unit, and long-term culture initiating cells were expanded by 589.6+/-58.6 (mean+/-s.d.), 76.5+/-8.8, and 3.2+/-0.9-fold, respectively, compared to unexpanded cells. All substrates surface-immobilized with peptides, including the control peptides, were more efficient in supporting the expansion of CD34+, CFU-GEMM and LTC-ICs than tissue culture polystyrene surface. Nevertheless, after 10-days of ex vivo expansion from 600 CD34+ cells, only cells cultured on CS-1-immobilized surface yielded positive engraftment, even though the frequency was low. PET surface immobilized with RGD peptide was less efficient than that with CS-1 peptide. Our results suggest that covalently immobilized adhesion peptides can significantly influence the proliferation characteristics of cultured UCB CD34+ cells.     

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.     

Arg-Gly-Asp (RGD) peptides alter hepatic killing of Candida albicans in the isolated perfused mouse liver model.

The isolated perfused mouse liver model was used to study the effect of Arg-Gly-Asp (RGD)-containing peptides on hepatic trapping and killing of Candida albicans. After extensive washing, 10(6) C. albicans CFU were infused into mouse livers. At the time of recovery, 63% +/- 2% (mean +/- standard error of the mean) of the infused C. albicans CFU were recovered from the liver and 14% +/- 1% were recovered from the effluent for a total recovery of 77% +/- 2%. This indicates that 86% +/- 9% of the original inoculum was trapped by the liver and that 23% +/- 2% was killed within the liver. Prior to their infusion into livers, 10(7) CFU of C. albicans were incubated at 37 degrees C for 30 min in the presence of various RGD peptides (0.1 mg/ml). Repeatedly, more than 90% of the infused RGD-treated C. albicans was trapped by the perfused liver. In comparison with the 23% killing rate observed in control livers, perfused livers killed approximately 40 to 50% of the infused C. albicans treated either with fibronectin, PepTite 2000, RGD, or RGDS. Hepatic killing of C. albicans treated with PepTite 2000 or fibronectin was dose dependent. Treatment of C. albicans with GRGDTP, GRGDSP, GRADSP, or GRGESP did not alter the ability of the perfused liver to kill C. albicans, suggesting that a degree of specificity for RGD peptides is associated with an increased ability of liver to kill RGD-treated C. albicans. Together, the data suggest that RGD peptides bind to a receptor on the surface of C. albicans, thereby increasing hepatic, and presumably Kupffer cell, killing of C. albicans. Natural or synthetic RGD peptides may serve as opsonins promoting C. albicans killing by Kupffer cells.     

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.     

Human endothelial cell interaction with biomimetic surfactant polymers containing Peptide ligands from the heparin binding domain of fibronectin

Biomimetic materials that mimic the extracellular matrix (ECM) provide a means to control cellular functions such as adhesion and growth, which are vital to successful engineering of tissue-incorporated biomaterials. Novel "ECM-like" biomimetic surfactant polymers consisting of a poly(vinyl amine) backbone with pendant cell-adhesive peptides derived from one of the heparin-binding domains of fibronectin were developed to improve endothelial cell adhesion and growth on vascular biomaterials. Heparin-binding peptide (HBP) sequences, alone and in combination with RGD peptides, were examined for their ability to promote human pulmonary artery endothelial cell (HPAEC) adhesion and growth (HBP1, WQPPRARI; HBP2, SPPRRARVT; HBP1:RGD; and HBP2:RGD) and compared with cell adhesion and growth on fibronectin and on negative control polymer surfaces in which alanines were substituted for the positively charged arginine residues in the two peptides. The results showed that HPAECs adhered and spread equally well on all HBP-containing polymers and the positive fibronectin control, showing similar stress fiber and focal adhesion formation. However, the HBP alone was unable to support long-term HPAEC growth and survival, showing a loss of focal adhesions and cytoskeletal disorganization by 24 h after seeding. With the addition of RGD, the surfaces behaved similarly or better than fibronectin. The negative control polymers showed little to no initial cell attachment, and the addition of soluble heparin to the medium reduced initial cell adhesion on both the HBP2 and HBP2:RGD surfaces. These results indicate that the HBP surfaces promote initial HPAEC adhesion and spreading, but not long-term survival.     

Maleimide-thiol coupling of a bioactive peptide to an elastin-like protein polymer

Recombinant elastin-like protein (ELP) polymers display several favorable characteristics for tissue repair and replacement as well as drug delivery applications. However, these materials are derived from peptide sequences that do not lend themselves to cell adhesion, migration, or proliferation. This report describes the chemoselective ligation of peptide linkers bearing the bioactive RGD sequence to the surface of ELP hydrogels. Initially, cystamine is conjugated to ELP, followed by the temperature-driven formation of elastomeric ELP hydrogels. Cystamine reduction produces reactive thiols that are coupled to the RGD peptide linker via a terminal maleimide group. Investigations into the behavior of endothelial cells and mesenchymal stem cells on the RGD-modified ELP hydrogel surface reveal significantly enhanced attachment, spreading, migration and proliferation. Attached endothelial cells display a quiescent phenotype.     

Generation of cell adhesive substrates using peptide fluoralkyl surface modifiers

Previous studies reported on the delivery of vitamin E to the surface of a polycarbonate polyurethane (PCNU) to produce antioxidant surfaces, using a bioactive fluorinated surface modifer (BFSM). In the current report, a cell adhesive peptide sequence was coupled to the BFSM, and when blended into PCNU, generated a cell adhesive substrate. An NH2-GK*GRGD-CONH2 peptide sequence (referred to as RGD) with a dansyl label (*) on the lysine residue was coupled via the N-terminal to a BFSM precursor molecule. The resulting RGD BFSM was purified and the pmol peptide/mg BFSM value was assayed by amino acid quantification. The migration of the RGD BFSM in a PCNU blend was confirmed by X-ray photoelectron spectroscopy analysis. U937 macrophage-like cells and human monocytes were seeded onto the PCNU and blends of PCNU with non-bioactive fluorinated surface modifier or the RGD BFSM, in order to study the cell response. Both U937 cells and human monocytes adhered in greater numbers to the RGD BFSM substrate when compared to unmodified PCNU or the blend of PCNU with the non-bioactive fluorinated surface modifying macromolecule substrate. The study demonstrated a novel approach for the introduction of peptides onto the surface of polymers by modifying the surface from within the polymer as opposed to the use of cumbersome post-surface modification techniques. The generation of a peptide substrate points to the possibility of producing complex bioactive surfaces using various peptide BFSMs or pharmaceuticals simultaneously to manipulate cell functions.     

Cyclo-(DfKRG) peptide grafting onto Ti-6Al-4V: physical characterization and interest towards human osteoprogenitor cells adhesion

In the present paper, specific interest has been devoted to the design of new hybrid materials associating Ti-6Al-4V alloy and osteoprogenitor cells through the grafting of two RGD containing peptides displaying a different conformation (linear RGD and cyclo-DfKRG) onto titanium surface. Biomimetic modification was performed by means of a three-step reaction procedure: silanization with APTES, cross-linking with SMP and finally immobilization of peptides thanks to thiol bonding. The whole process was performed in anhydrous conditions to ensure homogeneous biomolecules layout as well as to guarantee a sufficient amount of biomolecules grafted onto surfaces. The efficiency of this new route for biomimetic modification of titanium surface was demonstrated by measuring the adhesion between 1 and 24 h of osteoprogenitor cells isolated from HBMSC. Benefits of the as-proposed method were related to the high concentration of peptides grafted onto the surface (around 20 pmol/mm(2)) as well as to the capacity of cyclo-DfKRG peptide to interact with integrin receptors. Moreover, High Resolution beta-imager (using [(35)S]-Cys) has exhibited the stability of peptides grafted onto the surface when treated in harsh conditions.     

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.     

The effect of RGD density on osteoblast and endothelial cell behavior on RGD-grafted polyethylene terephthalate surfaces

Hybrid materials combining polyethylene terephthalate and different types of cells (endothelial and osteoblastic cells) have been developed thanks to the covalent grafting of different densities of RGD containing peptides onto the polymer surface. Biomimetic modifications were performed by means of a three-step reaction procedure: creation of COOH functions, coupling agent grafting and the immobilization of the RGDC peptides. High resolution μ-imager was used to evaluate RGD densities (varying between 0.6 and 2.4 pmol/mm²) and has exhibited the stability of the surface grafted peptides when treated in harsh conditions. The efficiency of this route for biomimetic modification of a PET surface was demonstrated by measuring the adhesion of MC3T3 and HSVEC cells and by focal adhesion observation. Results obtained prove that a minimal RGDC density of 1 pmol/mm² is required to improve MC3T3 and HSVEC cells responses. Indeed, cells seeded onto a RGDC-modified PET with a density higher than 1 pmol/mm² were able to establish focal adhesion as visualized by fluorescence microscope compared to cells immobilized onto unmodified PET and RGDC-modified PET with densities lower than 1 pmol/mm². Moreover, the number of focal contacts was enhanced by the increase of RGDC peptide densities grafted onto the material surface. With this study we proved that the density of peptides immobilized on the surface is a very important parameter influencing osteoblast or endothelial cell adhesion and focal contact formation.     

RGD peptide-conjugated poly(dimethylsiloxane) promotes adhesion, proliferation, and collagen secretion of human fibroblasts

A novel technique for conjugating Arg-Gly-Asp (RGD) peptides to poly(dimethylsiloxane) (PDMS) surfaces as well as its application to cell culture is presented in this paper. This technique performs RGD conjugation to PDMS through photochemical immobilization of functional NHS groups to PDMS surface followed with linking RGD peptide to the surface via coupling reaction with NHS. A bifunctional photolinker, N-sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-SANPAH), was used to conjugate RGD peptide to the surface. Compared to existing methods for peptide conjugation to PDMS, this technique is convenient, efficient, and free of organic contamination to PDMS surfaces. It can also be used to conjugate other peptides or proteins to most polymeric materials. In addition, cell culture studies showed that the RGD-conjugated PDMS surfaces promoted the adhesion, proliferation, and collagen production of human skin fibroblasts (HSFs). Finally, the RGD-conjugated PDMS surfaces are resistant to autoclaving and UV irradiation, which enables them to be repeatedly used in cell culture studies.     

Adhesion of MC3T3-E1 cells to RGD peptides of different flanking residues: detachment strength and correlation with long-term cellular function

We synthesized a series of RGD peptides and immobilized them to an amine-functional self-assembled monolayer using a modified maleimide-based conjugate technique that minimizes nonspecific interactions. Using a spinning disc apparatus, a trend in the detachment strength (tau(50)) of RGD peptides of different flanking residues was found: RGDSPK > RGDSVVYGLR approximately RGDS > RGES. Using blocking monoclonal antibodies, cellular adhesion to the peptides was shown to be primarily alpha(v)-integrin-mediated. In contrast, the tau(50) value of the cells on fibronectin (Fn)-coated substrates of similar surface density was 6-7 times higher and involved both alpha(5)beta(1) and alpha(v)beta(3) integrins. Cellular spreading was enhanced on RGD peptides after 1 h when compared to RGE and unmodified substrates. However, no significant differences were observed between the different RGD peptides. Long-term function of MC3T3-E1 cells was also evaluated by measuring alkaline phosphatase (ALP) activity and mineral deposition. Among the four peptides, RGDSPK exhibited the highest level of ALP activity after 11 days and mineralization after 15 days and reached comparable levels as Fn substrates after 15 and 24 days, respectively. These findings collectively illustrate both the advantages and limitations of enhancing cellular adhesion and function by the design of RGD peptides.     

Modulation of differentiation and mineralization of marrow stromal cells cultured on biomimetic hydrogels modified with Arg-Gly-Asp containing peptides

We synthesized biomimetic hydrogels modified with an osteopontin-derived peptide (ODP) and used them as a substrate for in vitro culture of marrow stromal cells (MSCs) to investigate the effect of the biomimetic surface on differentiation of MSCs into osteoblasts. Proliferation and biological assays for 16 days proved that MSCs became differentiated into osteoblasts secreting osteogenic phenotypic markers such as alkaline phosphatase (ALP), osteopontin, and mineralized calcium. In addition, there was an additive effect of the cell-binding peptide on differentiation and mineralization of MSCs cultured in the presence of soluble osteogenic supplements in cell culture media. For example, calcium content at day 16 on peptide-modified hydrogels was significantly higher than on tissue culture polystyrene. Two general trends were observed: (1) proliferation of MSCs decreased as the amount of differentiation markers increased, and (2) higher peptide concentrations accelerated the differentiation of MSCs. On the hydrogel modified with ODP, ALP activity exhibited a maximum value of 36.7 +/- 4.2 pmol/cell/h at day 10 for the concentration of 2 micromol/g while the culture time needed for maximum ALP activity occurred on day 13 for the lower concentrations. On the same hydrogel, the calcium content at day 10 was 21.4 +/- 2.3 ng/cell for the peptide concentration of 2 micromol/g and 1.0 +/- 0.3 ng/cell for 1.0 micromol/g. We used Gly-Arg-Gly-Asp-Ser (GRGDS) for modification of the hydrogel as a comparison to the results with ODP. However, osteoblast development was not significantly affected by the nature of the binding peptide sequences. These results suggest that MSC function can be modulated by variation of the peptide concentration in biomimetic hydrogels used for scaffold-based bone tissue engineering.     

Alginate type and RGD density control myoblast phenotype

Alginates are being increasingly used for cell encapsulation and tissue engineering applications; however, these materials cannot specifically interact with mammalian cells. We have covalently modified alginates of varying monomeric ratio with RGD-containing cell adhesion ligands using carbodiimide chemistry to initiate cell adhesion to these polymers. We hypothesized that we could control the function of cells adherent to RGD-modified alginate hydrogels by varying alginate polymer type and cell adhesion ligand density, and we have addressed this possibility by studying the proliferation and differentiation of C2C12 skeletal myoblasts adherent to these materials. RGD density on alginates of varying monomeric ratio could be controlled over several orders of magnitude, creating a range of surface densities from 1-100 fmol/cm(2). Myoblast adhesion to these materials was specific to the RGD ligand, because adhesion could be competed away with soluble RGD in a dose-dependent manner. Myoblast proliferation and differentiation could be regulated by varying the alginate monomeric ratio and the density of RGD ligands at the substrate surface, and specific combinations of alginate type and RGD density were required to obtain efficient myoblast differentiation on these materials.     

Protein and surface effects on monocyte and macrophage adhesion, maturation, and survival

Cell adhesion and maturation can be affected by the protein adsorption profile on the surface of an implanted biomaterial. In this study we have investigated how surface chemistry and adsorbed proteins can modulate monocyte and macrophage adhesion, IL-13-induced foreign-body giant cell formation, and apoptosis in vitro. Compared to a dimethylsilane-modified surface (DM), a surface modified with RGD peptides had no effect on adhesion density, foreign-body giant cell (FBGC) formation, or apoptosis in nondepleted serum conditions. The depletion of specific adhesive proteins affected adhesion, FBGC formation, and apo- ptosis. While the depletion of fibronectin and vitronectin had no overall effect compared to nondepleted serum conditions, the depletion of IgG from serum caused a significant decrease in initial adherent cell density [1000 +/- 200 compared to 2460 +/- 590 (p = 0.02)], a significant decrease in FBGC formation [2% compared to 17% (p = 0.02)], and a significant increase in the level of apoptosis [57% compared to 32% (p = 0.01)] on DM. The lowered initial adherent cell density on DM was not observed on the RGD surface, indicating that the RGD surface promotes increased initial adhesion. However, the RGD surface does not affect FBGC formation (i.e., macrophage fusion) or levels of apoptosis, which remained comparable to those on the DM surfaces at days 7 and 10.     

Three-dimensional culture of differentiating marrow stromal osteoblasts in biomimetic poly(propylene fumarate-co-ethylene glycol)-based macroporous hydrogels

This study assesses the ability of biomimetic poly(propylene fumarate-co-ethylene glycol)-based hydrogels to sustain the differentiation of marrow stromal cells (MSCs) to the osteoblastic phenotype and to produce a mineralized matrix in vitro. Macroporous hydrogels based on poly(propylene fumarate-co-ethylene glycol) with and without covalently linked RGD cell-adhesive peptide were synthesized and seeded with rat MSCs suspended in media or in a type I collagen solution. Cells suspended in media were found to adhere to RGD-modified but not to unmodified hydrogels. Cells suspended in a collagen solution were entrapped after collagen gelation and proliferated independent of the peptide modification of the hydrogel. Hydrogel modification with RGD peptide was sufficient to allow for the adhesion and differentiation of MSCs to the osteoblastic phenotype in the presence of osteogenic culture supplements. MSCs seeded with a collagen gel onto RGD-modified macroporous hydrogels after 28 days of culture showed a significant increase in cell numbers, from 15,200 +/- 2,000 to 208,600 +/- 69,700 cells (p < 0.05). Moreover, significant calcium deposition was apparent after 28 days of culture in RGD-modified hydrogels for cells suspended in a collagen gel in comparison to cells suspended in media, 3.47 +/- 0.26 compared to 0.82 +/- 0.20 mg Ca(2+) per scaffold (p < 0.05). Confocal microscopy revealed that MSCs suspended in a collagen gel and cultured on RGD-modified hydrogels for 28 days were adhered to the surface of the hydrogel while MSCs suspended in a collagen gel and cultured on unmodified hydrogels were located within the pores of and not in direct contact with the hydrogel surface. The results demonstrate that these biomimetic hydrogels facilitate the adhesion and support the differentiation of MSCs to the osteoblastic phenotype in the presence of osteogenic culture media.