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.     

Adhesion and migration of marrow-derived osteoblasts on injectable in situ crosslinkable poly(propylene fumarate-co-ethylene glycol)-based hydrogels with a covalently linked RGDS peptide

Marrow-derived osteoblasts were cultured on poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)) based hydrogels modified in bulk with a covalently linked RGDS model peptide. A poly(ethylene glycol) spacer arm was utilized to covalently link the peptide to the hydrogel. Three P(PF-co-EG) block copolymers were synthesized with varying poly(ethylene glycol) block lengths relative to poly(ethylene glycol) spacer arm. A poly(ethylene glycol) block length of nominal molecular weight 2000 and spacer arm of nominal molecular weight 3400 were found to reduce nonspecific cell adhesion and show RGDS concentration dependent marrow-derived osteoblast adhesion. A concentration of 100 nmol/mL RGDS was sufficient to promote adhesion of 84 +/- 17% of the initial seeded marrow-derived osteoblasts compared with 9 +/- 1% for the unmodified hydrogel after 12 h. Cell spreading was quantified as a method for evaluating adhesivity of cells to the hydrogel. A megacolony migration assay was utilized to assess the migration characteristics of the marrow-derived osteoblasts on RGDS modified hydrogels. Marrow-stromal osteoblasts migration was greater on hydrogels modified with 100 nmol/mL linked RGDS when compared with hydrogels modified with 1000 nmol/mL linked RGDS, while proliferation was not affected. These P(PF-co-EG) hydrogels modified in the bulk with RGDS peptide are potential candidates as in situ forming scaffolds for bone tissue engineering applications.     

Osteogenic differentiation of rat bone marrow stromal cells cultured on Arg-Gly-Asp modified hydrogels without dexamethasone and beta-glycerol phosphate

In this study, we investigated the effect of signaling peptides incorporated into oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels on in vitro differentiation and mineralization of marrow stromal cells (MSCs) cultured in media without soluble osteogenic supplements (dexamethasone and beta-glycerol phosphate). When MSCs were cultured for 16 days on OPF hydrogels modified with Arg-Gly-Asp (RGD) containing peptides, the normalized cell number was dependent on the peptide concentration between days 0 and 5 and reached comparable values at day 10 regardless of the concentration. The alkaline phosphatase (ALP) activity of MSCs on the peptide-modified OPF hydrogels was also concentration-dependent: ALP activity showed peaks on day 10 or day 13 on OPF hydrogels modified with 2.0 and 1.0 micromol peptide/g, which were significantly greater than those on the OPF hydrogels modified with 0.1 micromol peptides/g or no peptide. A characteristic marker of osteoblastic differentiation, osteopontin (OPN), was detected for all the test groups. However, OPN secretion between days 0 and 10 was significantly higher on the peptide modified hydrogels compared to that on tissue culture-treated polystyrene. Taken together, the results indicate that the presence of signaling peptide allows for a favorable microenvironment for MSCs to differentiate into osteoblasts and produce mineralized matrix, although the soluble factors may further enhance calcium deposition. These findings further support the usefulness of OPF hydrogels as scaffolds for guided bone regeneration, and represent an initial step in exploring the complex relationship between soluble and insoluble factors in osteogenic differentiation on biodegradable materials.     

Modulation of marrow stromal osteoblast adhesion on biomimetic oligo[poly(ethylene glycol) fumarate] hydrogels modified with Arg-Gly-Asp peptides and a poly(ethyleneglycol) spacer

Novel oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels functionalized with cell adhesion peptides were prepared, and the effects of incorporated peptide density and macromolecular structure of hydrogels on attachment and morphology of marrow stromal cells (MSCs) were evaluated. Poly(ethylene glycol) (PEG; number average molecular weight of 930, 2860, and 6090) was used to synthesize OPF. A model peptide, Gly-Arg-Gly-Asp (GRGD), was incorporated into OPF hydrogels after being coupled to acrylated PEG of molecular weight 3400. The increase of incorporated peptide concentration enhanced MSC attachment to OPF hydrogels of PEG of molecular weight of 930 and 2860. However, the number of attached MSCs to OPF hydrogels of PEG (molecular weight 6090) remained constant regardless of the peptide density. The length of PEG in OPF also influenced cell attachment. When 1 micromole peptide/g hydrogel was incorporated into the OPF hydrogels, the degree of cell attachment at 12 h relative to the initial seeding density was 93.9 +/- 5.9%, 64.7 +/- 8.2%, and 9.3 +/- 6.6% for OPF hydrogels prepared with PEG of molecular weights of 930, 2860, and 6090, respectively. However, the crosslinking density of hydrogels did not significantly affect cell attachment. The interaction was sequence specific, in that MSC attachment to GRGD-modified hydrogels was competitively inhibited when cells were incubated in the presence of 0.5 mM soluble GRGD before cell seeding. These results suggest that we can modulate MSC attachment to OPF hydrogels by altering the peptide density and the molecular structure of OPF hydrogels.     

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.     

Endothelialization of microporous YIGSR/PEG-modified polyurethaneurea

Bioactive polyurethaneurea modified with polyethylene glycol (PEG) and the endothelial cell-adhesive peptide YIGSR was synthesized and fabricated into microporous scaffolds. This material has shown appropriate mechanical properties for vascular graft applications, resists platelet adhesion, and promotes endothelialization. In the current study, microporous scaffolds were formed by a gasfoaming and salt-leaching method. The scaffolds showed highly interconnected open pores throughout the matrices, with porosity of approximately 78% and pore sizes of 20-200 microm. The peptide modified scaffolds showed superior mechanical properties over peptide-free scaffolds (tensile strength, 1.4 +/- 0.03 versus 0.19 +/- 0.01 MPa; p < 0.01). Bovine aortic endothelial cells were seeded on the scaffolds, and cell attachment, proliferation, extracellular matrix production, and migration were investigated. Histological and scanning electron microscopy analysis showed that few cells adhered on peptide-free scaffolds, whereas confluent endothelial cell monolayers formed along the pores in peptide-modified scaffolds. DNA content, hydroxyproline production, and cell migration were also significantly greater in peptide-modified scaffolds.     

Migration of human vascular endothelial and smooth muscle cells.

Migration of endothelial and smooth muscle cells was studied in vitro by measuring the increase in surface area at specific time intervals of confluent cell colonies advancing under agarose gels that contained both Morgan's medium 199 and variuos types of sera. First passage cultures of endothelial cells or 3 to 6 passage smooth muscle cells were plated into wells punched in agarose gels, at a seeding density of 50,000 cells per well. At zero time the size of the cell colonies was 35.4 sq. mm. +/- standard error 0.1. Irradiation (1500 rads) did not affect the expansion of the cell colonies although 3H-thymidine uptake was inhibited. Endothelial cells migrated under the agarose gels concentrically as contiguous sheets. When exposed to either 20 per cent platelet-poor plasma serum, platelet-rich plasma serum, or whole blood serum, the average increase in surface area was approximately 9 sq. mm. per day. In contrast, arterial smooth muscle cell colonies expanded with an increment of approximately 9 sq. mm. per day when exposed to 10 per cent platelet-poor plasma serum but 12 sq. mm. per day when exposed to 10 per cent platelet-rich plasma serum (p less than 0.001). Platelet factors also had stimulatory effects on the migration of venous smooth muscle cells. Cytochalasin B, dibutyryl cyclic AMP, and theophylline inhibited the migration of both endothelial and smooth muscle cells, but the latter responded more to the inhibitory effects of all three agents. It is concluded that in contrast to vascular smooth muscle, endothelial cells do not require platelet factors for migration and are less responsive to specific inhibitors affecting cell movement.     

Cathepsin K-sensitive poly(ethylene glycol) hydrogels for degradation in response to bone resorption

We propose a new strategy of biomaterial design to achieve selective cellular degradation by the incorporation of cathepsin K-degradable peptide sequences into a scaffold structure so that scaffold biodegradation can be induced at the end of the bone formation process. Poly(ethylene glycol) diacrylate (PEGDA) hydrogels were used as a model biomaterial system in this study. A cathepsin K-sensitive peptide, GGGMGPSGPWGGK (GPSG), was synthesized and modified with acryloyl-PEG-succinimidyl carbonate to produce a cross-linkable cathepsin K-sensitive polymer that can be used to form a hydrogel. Specificity of degradation of the GPSG hydrogels was tested with cathepsin K and proteinase K as a positive control, with both resulting in significant degradation compared to incubation with nonspecific collagenases over a 24-h time period. No degradation was observed when the hydrogels were incubated with plasmin or control buffers. Cell-induced degradation was evaluated by seeding differentiated MC3T3-E1 osteoblasts and RAW264.7 osteoclasts on GPSG hydrogels that were also modified with the cell adhesion peptide RGDS. Resulting surface features and resorption pits were analyzed by differential interference contrast (DIC) and fluorescent images obtained with confocal microscopy. Results from both analyses demonstrated that GPSG hydrogels can be degraded specifically in response to osteoclast attachment but not in response to osteoblasts. In summary, we have demonstrated that by incorporating a cathepsin K-sensitive peptide into a synthetic polymer structure, we can generate biomaterials that specifically respond to cues from the natural process of bone remodeling.     

Synthesis of cell-adhesive dextran hydrogels and macroporous scaffolds

Dextran hydrogels have been previously investigated as drug delivery vehicles and more recently as macroporous scaffolds; however, the non-cell-adhesive nature of dextran has limited its utility for tissue engineering. To overcome this limitation, macroporous scaffolds of methacrylated dextran (Dex-MA) copolymerized with aminoethyl methacrylate (AEMA) were synthesized, thereby introducing primary amine groups for covalent immobilization of extracellular-matrix-derived peptides. The amino group density for hydrogels copolymerized with 0.5 wt% AEMA was found to be 36.1+/-0.4 micromol/cm(3) by elemental analysis. To further enhance cellular interaction, poly(Dex-MA-co-AEMA) hydrogels were modified with either CRGDS or a mixture of CDPGYIGSR and CQAASIKVAV (1:1, v/v) using sulfo-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). The immobilized peptide concentration was determined using amino acid analysis at: 2.6+/-0.9 micromol/cm(3) for CRGDS-derived hydrogels and 2.2+/-0.3 micromol/cm(3) plus 1.9+/-0.2 micromol/cm(3) for CDPGYIGSR plus CQAASIKVAV-derived hydrogels, respectively. Cellular interactions of primary embryonic chick dorsal root ganglia (DRGs) were compared on the hydrogels. Cell adhesion and neurite outgrowth on poly(Dex-MA) increased with copolymerization of AEMA and further improved with peptide modification and significantly for CDPGYIGSR/CQAASIKVAV-derived poly(Dex-MA-co-AEMA) hydrogels. Moreover, DRGs penetrated within the first 600 microm of the scaffolds, thereby demonstrating the potential of this scaffold for guided cell and axonal regeneration in vivo.     

Val-ala-pro-gly, an elastin-derived non-integrin ligand: smooth muscle cell adhesion and specificity

The elastin-derived peptide val-ala-pro-gly (VAPG) may be useful as a biospecific cell adhesion ligand for smooth muscle cells. By grafting the peptide sequence into a hydrogel material, we were able to assess its effects on smooth muscle cell adhesion and spreading. These materials are photopolymerizable hydrogels based on acrylate-terminated derivatives of polyethylene glycol (PEG). Because of their high PEG content, these materials are highly resistant to protein adsorption and cell adhesion. However, PEG diacrylate derivatives can be mixed with adhesive peptide-modified PEG monoacrylate derivatives to facilitate cell adhesion. Following photopolymerization, PEG monoacrylate derivatives are grafted into the hydrogel network formed by the PEG diacrylate. This results in covalent immobilization of adhesive peptides to the hydrogel via a flexible linker chain. The resistance of PEG to protein adsorption makes it an ideal material for this model system since cell-material interactions are limited to biomolecules that are covalently incorporated into the material. In this case we were able to demonstrate that VAPG is specific for adhesion of smooth muscle cells. It also was shown that fibroblasts, endothelial cells, and platelets cannot adhere to VAPG. In addition, not only was smooth muscle cell adhesion dependent on ligand concentration, but also cell spreading increased with increasing ligand concentration.     

Micropatterned surfaces modified with select peptides promote exclusive interactions with osteoblasts

Microcontact printing techniques were used to pattern circles (diameters 10. 50, 100, and 200 microm) of N1[3-(trimethoxysilyl)-propyl]diethylenetriamine (DETA) surrounded by octadecyltrichlorosilane (OTS) borders on borosilicate glass, a model substrate. The DETA regions were further modified by immobilization of either the cell-adhesive peptides Arginine-Glycine-Aspartic Acid-Serine (RGDS) and Lysine-Arginine-Serine-Arginine (KRSR) or the non-adhesive peptides Arginine-Aspartic Acid-Glycine-Serine (RDGS) and Lysine-Serine-Serine-Arginine (KSSR). After four hours under standard cell culture conditions but in the absence of serum, adhesion of either osteoblasts or fibroblasts on surfaces patterned with the non-adhesive peptides RDGS and KSSR was random and low. In contrast, both osteoblasts and fibroblasts adhered and formed clusters onto circles modified with the adhesive peptide RGDS, whereas only osteoblasts adhered and formed clusters onto the circles modified with KRSR, a peptide that selectively promotes adhesion of osteoblasts. These results provide evidence that patterning of select peptides can direct adhesion of specific cell lines exclusively to predetermined regions on material surfaces.     

Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration

Basic fibroblast growth factor (bFGF) was immobilized to hydrogel scaffolds with retention of mitogenic and chemotactic activity. The bFGF was functionalized in order to incorporate it covalently within polyethylene glycol (PEG) hydrogel scaffolds by reaction with acryloyl-PEG-NHS. Hydrogels were formed by exposing aqueous solutions of PEG diacrylate, acryloyl-PEG-RGDS, and acryloyl-PEG-bFGF to long-wavelength ultraviolet light in the presence of a photoinitiator. These bFGF-modified hydrogels with RGD adhesion sites were evaluated for their effect on vascular smooth muscle cell (SMC) behavior, increasing SMC proliferation by approximately 41% and migration by approximately 15%. A covalently immobilized bFGF gradient was formed using a gradient maker to pour the hydrogel precursor solutions and then photopolymerizing to lock in the concentration gradient. Silver staining was used to detect the bFGF gradient, which increased linearly along the hydrogel's length. Cells were observed to align on hydrogels modified with a bFGF gradient in the direction of increasing tethered bFGF concentration as early as 24 h after seeding. SMCs also migrated differentially, up the concentration gradient, on bFGF-gradient hydrogels compared to control hydrogels with and without a constant bFGF concentration. These hydrogel scaffolds may be useful for studying protein gradient effects on cell behavior and for directing cell migration in tissue-engineering applications.     

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.     

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.     

The effect of hydrogel charge density on cell attachment

The competitive growth patterns of osteoblasts and fibroblasts can determine if healthy bone or pathologic scar tissue is formed at a wound site. Cell interactions with various alloplastic biomaterials used for tissue-engineering applications is complex. Defined synthetic mediums are valuable for studying ionic and cell receptor-specific interactions. The objectives of this study were to determine if fibroblasts and osteoblasts differentially attached to HEMA and PEG hydrogels copolymerized with positive, negative, or neutral charge densities, or when grafted with specific integrin receptor RGD adhesion ligand. Cytoskeletal phenotypes were assessed with immunofluorescent microscopy and cell attachment assays. Osteoblast cell attachment to both HEMA and PEG hydrogels was significantly higher (P<0.01) as compared to fibroblast cells. Positively charged HEMA and PEG hydrogels supported the greatest cell attachment, followed by RGD grafted, negative, and neutral charge densities, respectively. Each of these conditions elicited nearly a two-fold increase in osteoblast cell attachment, as compared to fibroblasts. Cell attachment to serum-coated coverslips was used as the control. Immunofluorescent analysis showed that both cell types attached and spread better on the positively charged hydrogels. However, fibroblasts demonstrated less spreading as compared to osteoblasts. In conclusion, differences in hydrophilic properties differentially affect osteoblast and fibroblast cell attachment and spreading.     

In vitro osteogenic differentiation of marrow stromal cells encapsulated in biodegradable hydrogels

Novel hydrogel materials based on oligo(poly(ethylene glycol) fumarate) (OPF) crosslinked with a redox radical initiation system were recently developed in our laboratory as injectable cell carriers for orthopedic tissue engineering applications. The effect of OPF hydrogel material properties on in vitro osteogenic differentiation of encapsulated rat marrow stromal cells (MSCs) with and without the presence of osteogenic supplements (dexamethasone) was investigated. Two OPF formulations that resulted in hydrogels with different swelling properties were used to encapsulate rat MSCs (seeding density approximately 13 million cells/mL, samples 6 mm diameter x 0.5 mm thick before swelling) and osteogenic differentiation in these constructs over 28 days in vitro was determined via histology and biochemical assays for alkaline phosphatase, osteopontin and calcium. Evidence of MSC differentiation was apparent over the culture period for samples without dexamethasone, but there was large variability in calcium production between constructs using cells of the same source. Differentiation was also seen in samples cultured with osteogenic supplements, but calcium deposition varied depending on the source pool of MSCs. By day 28, osteopontin and calcium results suggested that, in the presence of dexamethasone, OPF hydrogels with greater swelling promoted embedded MSC differentiation over those that swelled less (43.7 +/- 16.5 microg calcium/sample and 16.4 +/- 2.8 microg calcium/sample, respectively). In histological sections, mineralized areas were apparent in all sample types many microns away from the cells. These experiments indicate that OPF hydrogels are promising materials for use as injectable MSC carriers and that hydrogel swelling properties can influence osteogenic differentiation of encapsulated progenitor cells.     

In vivo bone and soft tissue response to injectable,biodegradable oligo(poly(ethylene glycol) fumarate) hydrogels

This study was designed to assess in vivo bone and soft tissue behavior of novel oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels using a rabbit model. In vitro degradation of the OPF hydrogels was also investigated in order to compare with in vivo characteristics. Four groups of OPF hydrogel implants were synthesized by alternation of crosslinking density, poly(ethylene glycol) (PEG) block length of OPF, and cell-binding peptide content. The in vitro degradation rate of OPF hydrogels increased with decreasing crosslinking density of hydrogels, which was characterized by measuring weight loss and swelling ratio of hydrogels and medium pH change. Examination of histological sections of the subcutaneous and cranial implants showed that an uniform thin circumferential fibrous capsule was formed around the OPF hydrogel implants. Quantitative evaluation of the tissue response revealed that no statistical difference existed in capsule quality or thickness between implant groups, implantation sites or implantation times. At 4 weeks, there was a very limited number of inflammatory and multinuclear cells at the implant-fibrous capsule interface for all implants. However, at 12 weeks, OPF hydrogels with PEG block length of number average molecular weight 6090+/-90 showed extensive surface erosion and superficial fragmentation that was surrounded by a number of inflammatory cells, while OPF hydrogels with PEG block length of number average molecular weight 930+/-10 elicited minimal degradation. Constant fibrous capsule layers and number of inflammatory cells were observed regardless of the incorporation of cell-binding peptide and crosslinking density of OPF hydrogels with PEG block length of number average molecular weight 930+/-90. These results confirm that the degradation of implants can be controlled by tailoring the macromolecular structure of OPF hydrogels. Additionally, histological evaluation of implants proved that the OPF hydrogel is a promising material for biodegradable scaffolds in tissue engineering.     

Biomimetic modification of titanium dental implant model surfaces using the RGDSP-peptide sequence: a cell morphology study

Surface topography and (bio)chemistry are key factors in determining cell response to an implant. We investigated cell adhesion and spreading patterns of epithelial cells, fibroblasts and osteoblasts on biomimetically modified, smooth and rough titanium surfaces. The RGD bioactive peptide sequence was immobilized via a non-fouling poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) molecular assembly system, which allowed exploitation of specific cell-peptide interactions even in the presence of serum. As control surfaces, bare titanium and bio-inactive surfaces (scrambled RDG and unfunctionalized PLL-g-PEG) were used. Our findings demonstrated that surface topography and chemistry directly influenced the attachment and morphology of all cell types tested. In general, an increase in cell number and more spread cells were observed on bioactive substrates (containing RGD) compared to bio-inactive surfaces. More fibroblasts were present on smooth than on rough topographies, whereas for osteoblasts the opposite tendency was observed. Epithelial cell attachment did not follow any regular pattern. Footprint areas for all cell types were significantly reduced on rough compared to smooth surfaces. Osteoblast attachment and footprint areas increased with increasing RGD-peptide surface density. However, no synergy (interaction) between RGD-peptide surface density and surface topography was observed for osteoblasts neither in terms of attachment nor footprint area.     

Minute changes in composition of polymer substrates produce amplified differences in cell adhesion and motility via optimal ligand conditioning

We explored the interplay between substratum chemistry of polymeric materials and surface-adsorbed ligand concentration (human plasma fibronectin) in the control of cell adhesion and cell motility. We found that small changes in the chemical composition of a polymeric substratum had different effects on cellular motility--depending on the concentration of preadsorbed fibronectin. We used two tyrosine-derived polyarylates, poly(DTD diglycolate) and poly(DTD glutarate), as substrata for the seeding of NIH-3T3 fibroblasts. The only compositional difference between the two test polymers was that one single oxygen atom in the polymer backbone of poly(DTD diglycolate) had been substituted by a methylene group in the backbone of poly(DTD glutarate), The two polymers had closely matched hydrophobicity and physical properties. Flat, spin-coated surfaces of these polymers were pretreated with different concentrations of human plasma fibronectin (0-20 microg/ml). After seeding with NIH-3T3 fibroblasts, we examined the adhesion and motility behavior of these cells. We found that NIH-3T3 fibroblasts migrated significantly faster on poly(DTD diglycolate), but only when the polymer surfaces were pretreated with intermediate concentrations of fibronectin. Only at these intermediate levels of ligand conditioning, did the presence of an extra oxygen atom in the backbone of poly(DTD diglycolate) relative to poly(DTD glutarate) (i) alter the overall organization/concentration of the fibronectin; (ii) weaken cell attachment strength and inhibited excessive cell spreading; and (iii) promote cell motility kinetics. These findings indicate that the biological effect of minute changes in substratum chemistry is critically dependent on the level of surface-adsorbed cell-binding ligands.