Design of a Synthetic Collagen-Binding Peptidoglycan that Modulates

Design of a Synthetic Collagen-Binding Peptidoglycan that Modulates Collagen Fibrillogenesis. John E. Paderi ... Publication Date (Web): August 5, 200...
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Design of a Synthetic Collagen-Binding Peptidoglycan that Modulates Collagen Fibrillogenesis John E. Paderi and Alyssa Panitch* Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47907 Received June 24, 2008; Revised Manuscript Received July 2, 2008

The ubiquity of collagen in mammalian tissues, with its host of structural and chemical functions, has motivated its research in many fields, including tissue engineering. The organization of collagen is known to affect cell behavior and the resulting structural integrity of tissues or tissue engineered scaffolds. Of particular interest are proteoglycan (PG) interactions with collagen and their influence on collagen assembly. These natural molecules provide unique chemical and mechanical cues and are known to modulate collagen fibrillogenesis. Research has been limited to PGs extracted and purified from animal sources and has the drawbacks of limited design control and costly purification. Consequently, we have designed a synthetic peptidoglycan based on decorin, a collagenbinding PG. The synthetic peptidoglycan containing a collagen-binding peptide with a single dermatan sulfate side chain specifically binds to collagen, delays fibrillogenesis, and increases collagen gel stiffness as decorin does. This design can be tailored with respect to the peptide sequence and attached glycosaminoglycan chain, offering unique control with relative ease of manufacturing.

Introduction The ubiquity of type I collagen and its ability to self-assemble in vitro provides great potential for uses in tissue engineering. Collagen gels, which can be formed under physiological conditions, have been widely used as cell scaffolds for a variety of target tissues.1 The intricate organization of collagen molecules forming periodic D-banding structures of networked collagen fibrils provides a three-dimensional environment that contains a host of biological and mechanical signals required for cellular activity. Cellular responses can be altered by incorporating other extracellular matrix (ECM) molecules, such as glycosaminoglycans (GAGs) and proteoglycans (PGs), which not only themselves present unique chemical and mechanical cues, but also alter the organization of the collagen network.2,3 Collagen assembly is widely studied, and particularly interesting are the effects of GAGs and PGs on collagen organization and fibrillogenesis.2-6 Decorin is a small leucine-rich PG (SLRP) composed of a protein core and a dermatan sulfate (DS) side chain. The decorin core binds collagen with high affinity in the nanomolar range, and while collagen self-assembles in vitro, the decorin-collagen interactions delay fibrillogenesis presumably by inhibiting lateral aggregation of collagen monomer.7,8 TEM images of collagenous tissues stained with cupromeronic blue show PGs located on the surface of fibrils, and PGs are believed to play a role in modulating fibril diameter.2,9,10 Bound to the surface of collagen fibrils, decorin is believed to enhance the mechanical integrity of collagen structures as in the sliding filament model proposed by Scott.11,12 The charge distribution on adjacent DS side chains allows for transient DS-DS interactions providing additional mechanical support. The polysaccharide chains of PGs may also bridge collagen fibrils resulting in fibril bundles. Using decorin as a model, we designed a peptidoglycan containing a collagen-binding peptide sequence with a single * To whom correspondence should be addressed. E-mail: apanitch@ purdue.edu.

covalently attached DS side chain. We hypothesized that the peptidoglycan would affect collagen fibrillogenesis and the viscoelastic properties of collagen gels. Our peptidoglycan design mimics naturally occurring SLRPs but can be tailored with respect to the peptide sequence as well as the GAG identity; this biomimetic molecule modulates collagen organization and incorporates the chemical and mechanical cues of the GAG into collagen-based scaffolds.

Materials and Methods Materials. Pepsin-treated Nutragen type I collagen from bovine hide was purchased from Inamed Biomaterials (Freemont, CA) at a stock concentration of 6.4 mg/mL in 10 mM HCl. Dermatan sulfate (DS; 41 kDa and 6.85% sulfur) and its oxidized form containing an average of 1.1 aldehydes per polymer chain were purchased from Celsus Laboratories (Cincinnati, OH). Decorin from bovine tendon (100 kDa, 50 kDa of which is DS) was purchased from Sigma-Aldrich (St. Louis, MO). Amino acids were purchased from Anaspec (San Jose, CA), Knorr resin was purchased from SynBioSci Corporation (Livermore, CA), and all other supplies were purchased from VWR (West Chester, PA) or Sigma-Aldrich unless otherwise noted. The peptide sequences RRANAALKAGELYKSILYGC (SILY) and ZRRANAALKAGELYKSILYGC (ZSILY), where Z designates dansylglycine, were synthesized on a solid phase Symphony Peptide Synthesizer (Protein Technologies) with Knorr resin. Peptides were cleaved from the resin using 92.5% TFA, 2.5% MilliQ water, 2.5% triisopropylsilane, and 2.5% ethanedithiol. Peptides were purified by ¨ KTA Explorer FPLC (GE reverse phase chromatography on an A Healthcare) using a C-18 column (Grace-Vydac) with acetonitrile and water, and purity was confirmed by mass spectroscopy as described.13 Conjugation of SILY to Dermatan Sulfate. Oxidized DS was conjugated to the aldehyde-reactive end of the heterobifunctional crosslinker PDPH (Pierce, Rockford, IL) following manufacturer’s protocol and is illustrated in Scheme 1. Oxidized DS was dissolved in coupling buffer (0.1 M sodium phosphate, 0.25 M sodium chloride, pH 7.2) to a concentration of 1.2 mM. PDPH was added in 10-fold molar excess, and the reaction proceeded at room temperature for 2 h. Excess PDPH ¨ KTA (229 Da) was removed by gel filtration chromatography with an A

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Scheme 1. Conjugation of Oxidized DS (oxDS) to SILY

Purifier FPLC (GE Healthcare) using a column packed with Sephadex G-25 and equilibrated with MilliQ water. Pure DS-PDPH was lyophilized and stored at -80 °C until conjugation with SILY. The number of PDPH molecules attached to each DS molecule was determined by a protocol provided by Pierce, in which the sulfhydryl-reactive end of PDPH was reduced with DTT and the production of pyridine-2-thione, a byproduct of the disulfide exchange, was measured by absorbance at 343 nm. For conjugation with SILY, the free sulfhydryl-reactive end of PDPH was reacted with 5-fold molar excess SILY by dissolving DS-PDPH in coupling buffer to a final concentration of 5 mM. The reaction was monitored by the production of pyridine-2-thione, as described above. Excess SILY was removed by gel filtration and pure DS-SILY was lyophilized and stored at -20 °C until further testing. The same protocol was repeated for fluorescently-labeled ZSILY producing DS-ZSILY. SILY and DS-SILY Binding Assays. The binding activity of SILY to collagen was tested by two separate solid phase assays. For microplate assays, collagen or BSA was immobilized on a high-bind 96-well black/clear-bottom plate (Costar) by incubating each at 2 mg/ mL for 1 h at 37 °C in 10 mM HCl or 1×PBS, respectively. Wells were then washed three times with 1×PBS. Fluorescently-labeled ZSILY was dissolved in 1×PBS at varying concentrations from 100 µM to 10 nM and incubated in each treatment for 30 min at 37 °C. Unbound ZSILY was removed by washing three times with 1×PBS and fluorescence (ex: 335 nm em: 490 nm) was measured on a SpectraMax M-5 spectrophotometer (Molecular Devices, Sunnyvale, CA). The logistic saturation binding curve was fitted using MatLab software to determine the equilibrium binding constant KD. A similar protocol was followed to determine binding activity of DS-ZSILY to collagen with an additional step of blocking DS interactions by preincubating each well with DS followed by three rinses with 1×PBS. Surface plasmon resonance assays were performed on a Biacore 2000, as described previously.14 Collagen at 1 mg/mL in sodium acetate buffer, pH 4, was immobilized at a flow rate of 5 µL/min on flow cell 2 (FC-2) of a CM-3 chip using EDC/NHS chemistry. Unreacted esters were capped with ethanolamine. FC-1 was treated similarly without immobilizing collagen. SILY was dissolved in 1×HBS-EP buffer (Biacore) at varying concentrations from 100-1.5 µM and was flowed over FC-1 and 2 at 90 µL/min for 90 s with a dissociation time of 90 s. The chip was regenerated with 50 µL injections of 0.5 M NaOH. Concentrations were randomized and performed in triplicate and binding curves were evaluated using BIAevaluation software after subtracting the reference FC-1 from FC-2. Collagen Gel Preparation and Fibrillogenesis Assays. Samples compared in this study include collagen alone or collagen mixed with DS, DS-SILY, intact decorin, or free SILY peptide. Unless noted, treatment concentrations were added at a 10:1 molar ratio of collagen/ additive. Molar ratios were calculated by an estimate of molecular weights: collagen, 285 kDa; DS, 41 kDa; DS-SILY, 43.2 kDa; and Decorin, 100 kDa. Gels were formed by mixing stock collagen solution with 10×PBS, 1 M NaOH, and 1×PBS on ice to a final concentration of 4 mg/mL, pH of 7.4, and ionic strength of 164 mM. Additives were

Figure 1. DS-SILY conjugation characterization. After 2 h, a final ∆A343nm corresponded to 1.06 SILY molecules added to each DS molecule. Note that t ) 0 is not truly a zero time point due to the slight delay between the addition of SILY to the DS-PDPH and measurement of the solution at 343 nm.

added as a part of the final 1×PBS addition and incubated on ice for 30 min before initializing fibrillogenesis by warming to 37 °C. Fibrillogenesis was monitored turbidimetrically by absorbance at 313 nm as described.15-17 Samples of 50 µL were added at 4 °C to each well of a 384-well plate and measurements were taken at 1 min intervals for up to 3 h in a SpectraMax M-5 spectrophotometer set at 37 °C. Rheological Mechanical Testing. Collagen gel solutions were prepared as described, and 200 µL samples were pipetted onto a 20 mm diameter wettable surface of hydrophobic printed slides (Tekdon, Myakka City, FL). Gels were formed by incubating overnight at 37 °C in a humidified incubator prior to mechanical testing. Rheological tests were performed on a stress-controlled AR G-2 rheometer (TA Instruments, New Castle, DE) with a 20 mm diameter stainless steel parallel plate geometry head. Slides containing gels were clamped to the peltier plate heated to 37 °C, and normal force control of 0.25 N was used to lower the geometry to a gap height of 600 µM onto the gels. Stress and frequency sweeps were performed to determine the linear range, and all gels were tested at a constant stress of 1.0 Pa over a frequency range of 0.1 to 2.0 Hz. Statistics. All experiments were performed in triplicate and are presented as average ( SE. Significance was determined using Design Expert (StatEase, Minneapolis, MN) software with R ) 0.05.

Results Characterization of SILY and DS-SILY Conjugation. The reaction scheme for the conjugation of dermatan sulfate to SILY is shown in Scheme 1. The heterobifunctional cross-linker PDPH was first reacted with the available aldehyde groups on oxidized DS forming DS-PDPH. The number of PDPH molecules attached was determined by reacting DS-PDPH with DTT, producing free pyridine-2-thiol. This procedure provided by Pierce yielded 1.1 PDPH molecules per DS molecule (see Supporting Information), which agrees with the number of aldehydes present on oxidized DS, as provided by Celsus Laboratories. The free sulfhydryl on the terminal cysteine residue of SILY was then used to conjugate SILY to DS-PDPH, forming DS-SILY. The reaction was again monitored by the production of pyridine-2-thione, as measured by an increase in absorbance at 343 nm. SILY and DS-PDPH did not absorb significantly at 343 nm over the coupling buffer alone, and absorbance differences were calculated with coupling buffer as a reference. Shown in Figure 1, after reacting SILY with DSPDPH for 2 h, an average of 1.06 SILY peptides were conjugated to each DS molecule. The extinction coefficient of pyridine-2-thione at 343 nm (8080 M-1 cm-1) was used to calculate the moles of SILY reacted in the disulfide exchange

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Figure 2. Binding Affinity of SILY and ZSILY to Collagen. (A) Microplate assay and (B) representative Biacore curves SILY binding to immobilized collagen. Equilibrium binding yields a KD ) 0.86 µM, while KD calculated from on-off rates gives 1.2 µM with chi2 < 1, indicating a good fit.

Figure 3. Binding of DS-ZSILY to collagen. Microplate assay of fluorescently-labeled DS-ZSILY conjugate to immobilized collagen. DS-ZSILY binds specifically to the collagen treated surface. NT ) untreated wells.

because there is a 1:1 molar ratio of free pyridine-2-thione to added SILY as in Scheme 1. Binding of SILY and DS-SILY to Collagen. Fluorescentlylabeled SILY was found to bind specifically to the collagen surface of a 96-well plate in a dose-dependent manner. The saturation binding curve shown in Figure 2 indicates an equilibrium binding KD of 0.86 µM, as determined from the inflection point of the fitted logistic curve. Binding of SILY without the dansylglycine fluorescent tag was determined by Biacore assays. SILY bound specifically to the collagen surface, and KD ) 1.2 µM was calculated from the on-off binding kinetics determined by BIA evaluation software with a chi2 < 1, indicating a good fit. Fluorescently-labeled DS-ZSILY was found to bind specifically to the collagen surface of a 96-well plate in a dosedependent manner shown in Figure 3, demonstrating that conjugation to DS does not eliminate peptide-collagen binding. Turbidity Measurements. Collagen fibrillogenesis in the presence of DS, decorin, DS-SILY, and free SILY peptide are shown in Figure 4. Concentrations of these additives were tested at 10:1 and 1:1 molar ratios of collagen/additive. DS-SILY at a 10:1 molar ratio delays fibrillogenesis and decreases the optical density at 313 nm to a greater degree than decorin at the same ratio. At a 1:1 ratio of collagen to DS-SILY, absorbance values

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reached approximately half of the 10:1 ratio treatment. Free SILY peptide at a 1:1 molar ratio had little effect on fibrillogenesis relative to collagen, whereas DS increased the rate of fibrillogenesis both at 10:1 and 1:1 ratios. Rheological Mechanical Tests. The complex moduli (G*) of collagen gels formed in the presence of DS, decorin, DSSILY, or free SILY peptide are shown in Figure 5, and a summary of storage moduli (G′) over all experimental conditions are presented in Table 1 and reported as the average G′. Frequency sweeps from 0.1 to 2.0 Hz were tested with a constant stress of 1.0 Pa; however, data is only shown or averages calculated up to 1.0 Hz because the gels broke at higher values. At all frequencies, the storage modulus (G′) was greater than the loss modulus (G′′), indicating a gel-like material. At a 10:1 molar ratio of collagen/treatment, DS, decorin, and DS-SILY increased G* over collagen alone, whereas free SILY peptide showed no statistical differences compared to collagen. Decorin and DS-SILY significantly increased G* approximately 2.5 times collagen alone, whereas DS increased G* approximately 1.7 times that of collagen. DS-SILY showed the same trend of increased G* at a lower concentration of 30:1; however, at 5:1 molar ratio, G* of DS-SILY was not statistically different from DS.

Discussion The aim of this study was to create a synthetic collagenbinding peptidoglycan inspired by the native SLRP decorin and to compare its influence on collagen fibrillogenesis and the rheological mechanical properties of collagen gels to that of decorin. SILY and DS-SILY Bind to Collagen. The peptide sequence RRANAALKAGELYKCILY, derived from the platelet receptor to collagen I, was modified to use a c-terminal cystine sulfhydryl group for conjugate chemistry. The internal cysteine was exchanged for a serine, thereby substituting the -OH group of the serine for the -SH group of the cysteine, and a glycine spacer followed by a terminal cysteine was added to the c-terminus of original sequence yielding RRANAALKAGELYKSILYGC, abbreviated here as SILY. The binding activity of SILY to collagen was confirmed by microplate and Biacore assays in which KD values of 0.86 µM and 1.2 µM were determined, respectively. These KD values are 2 orders of magnitude higher than the original sequence;18 however, specific binding to collagen with relatively high affinity is observed and the sequence contains only one free sulfhydryl group, making it a suitable candidate for directed conjugation to DS. The observed decrease in binding affinity may be due to the modification of the peptide sequence. While the -OH group of serine is chemically similar to the -SH group of cysteine, the higher nucleophilicity may play an important role in the binding affinity of the original sequence. The glycine and terminal cysteine residues that were added to the original sequence may also be responsible in part for the decreased binding affinity due to charge interactions or steric hindrance of the active binding sequence to collagen. Finally, the original work by Chiang and Kang found the KD ) 10 nm; the assay was performed with human placental type I collagen, while this work used type I collagen from bovine hide. Any of these factors may be contributing to the decrease in KD observed in the current system. The conjugation of oxidized DS to SILY was achieved using the heterobifunctional cross-linker PDPH, which is reactive to aldehyde and sulfhydryl groups. Conjugation of the PDPH

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Figure 4. Measurements of turbidity changes during fibrillogenesis at (A) 10:1 and (B) 1:1 molar ratios of collagen:additive. DS-SILY at 1:1 nearly inhibits fibrillogenesis, while free SILY peptide has little delaying effect. Table 1. Average G′ (Pa) over All Experimental Conditions G′avg (Pa) over frequencies 0.1-1.0 Hz molar ratio collagen DS decorin DS-SILY SILY

Figure 5. Rheological mechanical properties of collagen gels reported as G/ (complex modulus) formed at (A) 10:1, (B) 30:1, and (C) 5:1 molar concentrations of collagen/additive. Data is presented as average ( SE. Differences between treatments are significant (R ) 0.05) other than (A), (, collagen; ×, SILY; (C) 9, DS; and, b, DSSILY.

hydrazide to oxidized DS resulted in 1.1 PDPH molecules per DS (see Supporting Information). SILY was then added by disulfide exchange to the PDPH-DS conjugate and the ratio of SILY/DS in the final DS-SILY bioconjugate was 1.06. The same procedure was repeated for fluorescently-labeled ZSILY, yielding DS-ZSILY, which was used in a microplate assay to determine the binding activity of the conjugate to collagen.

30:1 44 59

10:1 24 40 56 61 19

5:1 59 60

DS-ZSILY bound specifically to the collagen-coated surface in a dose-dependent manner, although a saturation point was not achieved at concentrations up to 100 µM. Relative to the amount of free ZSILY bound on the collagen surface in a similar assay, DS-ZSILY bound to a much higher degree. The greater than expected binding of DS-ZSILY at the collagen surface may be due to interactions between the negatively-charged DS and the positively-charged ZSILY peptide, whereby specifically bound DS-ZSILY sequesters additional DS-ZSILY molecules through electrostatic interactions between the DS chains and ZSILY peptides of neighboring molecules. While altering the ionic strength of the buffer could reduce unwanted DS interactions, it would also alter the KD for the DS-ZSILY to collagen interaction. For this reason, the actual KD was not determined, but definitive binding alone was demonstrated. DS-SILY Delays Fibrillogenesis. Turbidity has been widely used to measure the rate of collagen fibrillogenesis.15-17 Decorin is known to increase gelling time and decrease optical density and is generally agreed to decrease fibril diameter.4,8,15,19 Turbidity curves in Figure 4 show delayed fibrillogenesis and decreased optical density in the presence of decorin, and a greater delay and decrease in optical density in the presence of DS-SILY. The higher ratio of 1:1 collagen to DS-SILY was also tested, resulting in marked inhibition of fibrillogenesis. The lower absorbance values of approximately half that of the collagen control could be indicative of smaller diameter fibrils, and while fibrillogenesis is delayed, it is not completely inhibited. It is also possible that a further increase in absorbance would be observed over a longer time span and that the 3 h turbidity assay does not capture the complete fibrillogenesis in the case of added DS-SILY. DS by contrast increased the rate of fibrillogenesis over collagen alone, while peptide alone had little effect on fibrillogenesis. These results suggest that DS-SILY may operate by a similar mechanism to decorin, where the peptidoglycan, like the decorin core protein, binds along specific regions of the collagen molecule inhibiting collagen self-association by lateral aggregation, thus resulting in delayed fibrillogenesis. At a 1:1 ratio, where theoretically every collagen monomer contains a bound DS-SILY molecule, fibrillogenesis is nearly inhibited. The selfassociation of collagen may, however, compete for DS-SILY binding and allow for some degree of collagen aggregation and

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fibrillogenesis. Decorin in high concentrations has been shown to completely prevent fibril formation;15 however, the decorin core protein affinity for collagen is in the nanomolar range,7 whereas the KD of SILY is approximately 1 µM and, thus, DSSILY-collagen interactions are more susceptible to replacement by collagen-collagen interactions than are decorin-collagen interactions when decorin and DS-SILY are present at equal concentration. The free SILY peptide at a high 1:1 peptide/collagen ratio minimally delayed collagen fibrillogenesis. The decorin core protein has been shown to delay fibrillogenesis similarly to intact decorin;16,20however, in comparing the two molecules, there is a significant size difference. The core protein of decorin is about 40 kDa, whereas SILY is only 1.3 kDa, so it is likely that when the decorin core protein binds to the collagen molecule, self-association of collagen is prevented by steric hindrance, unlike SILY, which may be too small to have the same effect. DS alone was found to increase both the rate of fibrillogenesis and the overall turbidity at the 10:1 ratio and to a greater degree at the 1:1 ratio. DS has been shown to interact with collagen at physiologic conditions,21 so it is possible that DS sequesters collagen monomers, allowing for collagen aggregation and fibrillogenesis without inhibiting fibrillogenesis. Whereas DSSILY or decorin remain bound on the surface of the collagen fibril, inhibiting further aggregation of collagen, DS may act more by bringing together collagen molecules without interfering with collagen-collagen interactions, thus accelerating collagen aggregation and fibrillogenesis. Decorin and DS-SILY Increase Collagen Gel Stiffness. The viscoelastic properties of gels were tested by rheological studies. Figure 5 depicts the complex modulus (G*), which is a measure of gel stiffness derived from the storage and loss moduli. In the presence of DS, gel stiffness increased approximately 1.7 times over that of collagen alone. The addition of decorin and DS-SILY further increased gel stiffness about 2.5 times that of collagen gels. The same trends were observed in storage modulus values (G′), which are summarized by average G′ in Table 1. Averages are reported in Table 1 for quick reference, though G′ values increase with frequency, which was consistent for all experimental conditions. Differences in gel stiffness may be attributed to a number of factors including the degree of fibril branching, fibril size, length, and physical interactions including DS-DS, DS-collagen, or peptide/protein core-collagen.21-23 Effects of a collagen-binding proteoglycan and peptidoglycan, in this case, decorin and DS-SILY, are more complex than just DS-collagen interactions and seem to play a more important role in modulating fibrillogenesis, as is illustrated by the delay and decrease in optical density observed in turbidity curves. At a low ratio of 30:1 collagen/additive, DS and DS-SILY followed the same trends, both increasing gel stiffness over that of collagen alone, and DS-SILY producing stiffer gels than DS. At higher 5:1 molar ratios, however, DS-SILY was equal to DS stiffness, indicating that at higher DS-SILY concentrations, the gel stiffness begins to decrease. This result follows the observations with turbidity, in which at a 1:1 ratio fibrillogenesis shows marked inhibition, which suggests that, as the concentration of DS-SILY increases, a critical concentration may be reached where fibril formation is completely inhibited and, thus, the viscoelastic properties of collagen gels will decrease or gels will not form at all.

Conclusions We describe here a novel synthetic collagen-binding peptidoglycan based on the collagen-binding peptide sequence

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RRANAALKAGELYKSILYGC, abbreviated here as SILY, which is conjugated to a DS chain. This design is based on the small-leucine rich PG decorin, a well-characterized collagenbinding PG known to delay or inhibit collagen fibrillogenesis in vitro. DS-SILY was found to delay collagen fibrillogenesis and lower overall turbidity in a dose-dependent manner similarly to decorin. Gels formed in the presence of decorin and DSSILY resulted in stiffer gels than collagen alone or collagen with DS, although at higher concentrations of DS-SILY, gel stiffness began to show a decrease in stiffness relative to DS. The synthetic collagen-binding peptidoglycan described here can be synthesized with relative ease and can be tailored with respect to the peptide sequence as well as the GAG chain and thus provides a biomimetic approach of modulating collagen fibrillogenesis that may have potential application for tissue engineered collagen scaffolds. We are further exploring the mechanism by which DS-SILY and other synthetic collagen-binding peptidoglycans have on collagen fibrillogenesis. We are also interested in the effects of these molecules and the altered collagen gel network on cells and the potential impact for tissue engineering applications. Acknowledgment. This work was funded in part by the National Science Foundation (CAREER: CBET 0651643). Supporting Information Available. Characterization of DSPDPH conjugation. This information is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) Freed, L. E.; Guilak, F.; Guo, X. E.; Gray, M. L.; Tranquillo, R.; Holmes, J. W.; Radisic, M.; Sefton, M. V.; Kaplan, D.; VunjakNovakovic, G. Tissue Eng. 2006, 12 (12), 3285–3305. (2) Scott, J. E.; Haigh, M. Biochem. J. 1988, 253 (2), 607–610. (3) Stuart, K. A.; Panitch, A. Biopolymers 2008, in press. (4) Bierbaum, S.; Douglas, T.; Hanke, T.; Scharnweber, D.; Tippelt, S.; Monsees, T. K.; Funk, R. H. W.; Worch, H. J. Biomed. Mater. Res. 2006, 77A (3), 551–562. (5) Douglas, T.; Heinemann, S.; Mietrach, C.; Hempel, U.; Bierbaum, S.; Scharnweber, D.; Worch, H. Biomacromolecules 2007, 8 (4), 1085– 1092. (6) Lelu, S. P.; Pluen, A. Macromol. Symp. 2007, 256 (1), 175–188. (7) Nareyeck, G.; Seidler, D. G.; Troyer, D.; Rauterberg, J.; Kresse, H.; Schonherr, E. Eur. J. Biochem. 2004, 271 (16), 3389–3398. (8) Vogel, K. G.; Trotter, J. A. Collagen Relat. Res. 1987, 7 (2), 105– 114. (9) Reed, C. C.; Iozzo, R. V. Glycoconjugate J. 2003, 19 (4-5), 249– 255. (10) Svensson, L.; Oldberg, A.; Heinegard, D. Osteoarthritis Cartilage 2001, 9, S23-S28. (11) Scott, J. E. J. Physiol. (Cambridge, U.K.) 2003, 553 (2), 335–343. (12) Scott, J. E.; Orford, C. R. Biochem. J. 1981, 197 (1), 213–&. (13) Seal, B. L.; Panitch, A. Acta Biomaterialia 2006, 2 (3), 241–251. (14) Lee, J. Y.; Choo, J. E.; Choi, Y. S.; Park, J. B.; Min, D. S.; Lee, S. J.; Rhyu, H. K.; Jo, I. H.; Chung, C. P.; Park, Y. J. Biomaterials 2007, 28 (29), 4257–4267. (15) Douglas, T.; Heinemann, S.; Bierbaum, S.; Scharnweber, D.; Worch, H. Biomacromolecules 2006, 7 (8), 2388–2393. (16) Milan, A. M.; Sugars, R. V.; Embery, G.; Waddington, R. J. Calcif. Tissue Int. 2005, 76 (2), 127–135. (17) Williams, B. R.; Gelman, R. A.; Poppke, D. C.; Piez, K. A. J. Biol. Chem. 1978, 253, 6578–6585. (18) Chiang, T. M.; Kang, A. H. J. Clin. InVest. 1997, 100 (8), 2079– 2084. (19) Vogel, K. G.; Paulsson, M.; Heinegard, D. Biochem. J. 1984, 223 (3), 587–597. (20) Kuc, I. M.; Scott, P. G. Connect. Tissue Res. 1997, 36 (4), 287–296. (21) Obrink, B. Eur. J. Biochem. 1973, 34 (1), 129–137. (22) Hsu, S.; Jamieson, A. M.; Blackwell, J. Biorheology 1994, 31 (1), 21–36. (23) Scott, J. E. FASEB J. 1992, 6 (9), 2639–2645.

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