Patent Application
20110269231
A paper copy of the Sequence Listing and a computer readable form of the sequence listing provided herein, containing the file named “28243-141-P100257US02_ST25.txt”, which is 3,720 bytes in size (measured in MS-DOS), and are herein incorporated by reference. This Sequence Listing consists of SEQ ID NOs: 1-12.
The present disclosure generally relates to biomaterials presenting synthetic peptides thereon and methods of preparing the biomaterials. More particularly, the present disclosure relates to biomaterials having a substrate comprising synthetic peptides thereon. The synthetic peptides may be proteoglycan-binding peptides and/or glycosaminoglycan-binding peptides. The present disclosure further relates to methods of using the biomaterials. Specifically, the present disclosure relates to methods of sequestering endogenous proteoglycans and endogenous glycosaminoglycans, methods of increasing stem cell proliferation, methods of reducing spontaneous stem cell differentiation, and methods of enhancing induced osteogenic differentiation using the biomaterials.
Serum is a relatively inexpensive cell culture supplement that provides a source of biomolecules that adsorb to polymeric cell culture substrates and support cell growth. Biomolecule adsorption is random and non-specific, however, which introduces difficulties when trying to immobilize specific subsets of biomolecules onto a cell culture substrate. Moreover, supplementation of biomolecules in cell culture systems to elicit changes in cell behavior typically requires a supraphysiologic concentration of the biomolecules, which likely provides limited insight into biomolecule function within the in vivo context. One alternative to non-specific adsorption is to covalently immobilize synthetic analogs of serum-derived biomolecules onto culture substrates.
Proteoglycans (PGs) are macromolecular complexes having a core protein and covalently tethered glycosaminoglycan (GAG) chains. PGs are present on the cell surface or within the extracellular environment of most mammalian cell types. Within the pericellular environment, PGs organize the extracellular matrix (ECM) through non-covalent interactions with ECM proteins (e.g., fibronectin and collagen type-I), and regulate cell function through non-covalent interactions with growth factors (e.g., fibroblast growth factor-2 (FGF-2)) and cell surface receptors (e.g., L-selectin). Recently, endogenous PGs have emerged as key regulators of diverse tissue development processes. For example, the spatially- and temporally-regulated expression of cartilage-specific sulfated proteoglycan is integral to chick limb development, while proper capillary branch formation during lung branching morphogenesis requires the expression of vascular endothelial growth factor isoforms bearing a heparin-binding domain. Additionally, a deficiency in the expression of perlecan, a proteoglycan common to the basement membrane, results in lethal chondrodysplasia. In light of these observations, the incorporation of exogenous GAGs or PGs has emerged as a promising mechanism to impart PG-mediated regulation over cell function into biomaterials for tissue regeneration therapies. However, biomaterials that properly mimic native extracellular environments by sequestering endogenous, cell-secreted PGs remain poorly characterized.
A key mechanism of PG-mediated regulation of cell behavior is the specific, non-covalent binding of PGs to growth factors, a class of soluble signaling proteins that regulate diverse cell processes, such as proliferation and differentiation. For example, interactions between platelet-derived growth factor (PDGF) and chondroitin sulfate inhibit PDGF-mediated phosphorylation of PDGF-receptor-β and, in turn, inhibit PDGF-mediated fibroblast proliferation. Moreover, binding of bone morphogenetic proteins (BMPs) to cell surface heparin sulfates down-regulates BMP-mediated osteogenesis in C2C12 cells. Additionally, a well-characterized example of PG-mediated up-regulation of growth factor signaling involves the fibroblast growth factor (FGF) family. In particular, the GAGs heparin and heparin sulfate mediate the dimerization of FGFs, such as FGF-1,-2, and -4, that is required for FGF-mediated activation of cell surface FGF receptors. These results demonstrate that PG-growth factor interactions are important up- and down-regulators of growth factor activity and, in turn, growth factor-mediated modulation of cell behavior.
For the foregoing reasons, there is a need for biomaterials having molecules that sequester proteoglycans and/or glycosaminoglycans and regulate stem cell behavior. Specifically, it would be advantageous if the biomaterials were capable of non-covalently sequestering endogenous, cell-secreted and/or serum-derived PGs so as to introduce PG-mediated regulation over cell function for regenerative medicine and tissue engineering applications.
The present disclosure is generally directed to biomaterials and to methods of preparing the biomaterials. The biomaterials may be used in methods for sequestering endogenous proteoglycans and/or endogenous glycoaminoglycans, and methods for regulating stem cell behavior. More particularly, in one aspect, the present disclosure is directed to a biomaterial comprising a synthetic peptide thereon. The synthetic peptide is selected from the group consisting of a synthetic proteoglycan-binding peptide, a synthetic glycosaminoglycan-binding peptide, and combinations thereof In one particular aspect, the biomaterial is a self assembled monolayer (SAM).
In some embodiments, the synthetic proteoglycan-binding peptide is a heparin-binding peptide. In other embodiments, the synthetic proteoglycan-binding peptide is a chondroitin sulfate-binding peptide. In other embodiments, the synthetic glycosaminoglycan-binding peptide is a hyaluronic acid glycosaminoglycan-binding peptide.
In other embodiments, the biomaterials may further include a cell-adhesion peptide.
Another aspect of the present disclosure includes methods of preparing the biomaterials including attaching a synthetic peptide to a substrate. In one embodiment, the synthetic peptide may be attached to the substrate by incubating the substrate in a solution comprising the synthetic peptide.
Another aspect of the present disclosure includes methods of sequestering endogenous proteoglycans and/or endogenous glycosaminoglycans by exposing the endogenous proteoglycans and/or endogenous glycosaminoglycans to a biomaterial having a synthetic peptide thereon.
Another aspect of the present disclosure includes methods of reducing spontaneous stem cell differentiation by culturing stem cells in the presence of a biomaterial having a synthetic peptide thereon.
Another aspect of the present disclosure includes methods of enhancing induced osteogenic differentiation by culturing stem cells in the presence of a biomaterial having a synthetic peptide thereon.
Another aspect of the present disclosure includes methods of increasing stem cell proliferation by culturing stem cells in the presence of a biomaterial having a synthetic peptide thereon.
The disclosure will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:
While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described below in detail. It should be understood, however, that the description of specific embodiments is not intended to limit the disclosure to cover all modifications, equivalents and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present disclosure, suitable methods and materials are described below.
The present disclosure is generally directed to biomaterials. As used herein, “biomaterial” refers to any material, natural or man-made, that comprises whole or part of a living structure or biomedical device which performs, augments, or replaces a natural function.
In one suitable embodiment, the present disclosure is directed to a biomaterial, wherein the biomaterial is a self-assembled monolayer (SAM) including a substrate having a synthetic peptide thereon. While described herein as being a SAM, it should be understood by one skilled in the art that any biomaterial known in the art can be used in place of the described SAM without departing from the scope of the present disclosure.
Suitable substrates for use in the biomaterials may be, for example, a metal-containing substrate. Suitable metals for use in the metal-containing substrates may include, for example, gold, silver, titanium, glass, diamond, and glassy carbon. In one embodiment, the metal-containing substrate may further include a polyethylene glycol-containing molecule.
Other suitable biomaterial substrates include synthetic hydrogels, such as hydrogels prepared from a synthetic polymer. Suitable synthetic polymers may include, for example, polyethylene-glycol, polyacrylic acid, poly-N-isopropylacrylamide, poly(propylene fumarate-co-ethylene glycol), and self-assembling peptides.
In another aspect, the substrate may be a polymer derived from a natural source. Suitable polymers derived from a natural source may include, for example, an alginate and/or a chitosan.
In another aspect, the substrate may be, for example, a mineralized material. Suitable mineralized materials may be, for example, hydroxyapatite.
As described above, the substrate of the biomaterials (e.g., SAMs) of the present disclosure include at least one synthetic peptide thereon. The synthetic peptide may be selected from a synthetic proteoglycan-binding peptide, a synthetic glycosaminoglycan-binding peptide, and combinations thereof. Suitable synthetic proteoglycan-binding peptides may be, for example, heparin-binding peptides and chondroitin sulfate-binding peptides.
Suitable synthetic heparin-binding peptides may be derived from fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), heparin binding epidermal growth factor (heparin binding EGF), platelet-derived growth factor (PDGF), and bone morphogenic protein (BMP). In one particular embodiment, the heparin-binding peptide may be derived from FGF-2 and BMP-2, for example.
Suitable synthetic chondroitin sulfate-binding peptides may be derived from a protein, such as midkine.
By way of example, suitable synthetic peptides may be, for example, SEQ ID NO: 1 (KRTGQYKL); SEQ ID NO: 2 (TYRSRKY); SEQ ID NO: 3 (KRTGQYKLGSKTGPGQK); SEQ ID NO: 4 (QAKHKQRKRLKSSC); SEQ ID NO: 5 (SPKHHSQRARKKNKNC); SEQ ID NO: 6 (XBBXBX; where B=basic residue and X=hydropathic residue); and SEQ ID NO: 7 (XBBBXXBX; where B=basic residue and X=hydropathic residue).
Suitable synthetic glycosaminoglycan-binding peptides may be hyaluronic acid-binding peptides.
Suitably, the biomaterial substrate includes a surface density of the synthetic peptide of less than about 2%. Particularly suitable surface densities of the synthetic peptide may be from about 0.1% to about 2%. As used herein, “surface density” refers to the mole fraction of the synthetic peptide (e.g., heparin-binding peptide, the chondroitin sulfate-binding peptide, and the hyaluronic acid glycosaminoglycan-binding peptide) attached to the substrate of the biomaterial (e.g., SAM substrate).
In another aspect, the substrate may further include a cell-adhesion peptide. As used herein, “cell-adhesion peptide” refers to a peptide that contributes to or enhances attachment of a cell to the substrate. Suitable cell-adhesion peptides may be, for example, an integrin-binding peptide. Suitable integrin-binding peptides may be, for example, SEQ ID NO: 8 (RGDSP).
In one aspect, the substrate includes an integrin-binding peptide at a surface density of from about 0.1% to about 5%.
Another aspect of the present disclosure includes methods of preparing biomolecules including a substrate having a synthetic peptide thereon. One embodiment relates to a method of preparing a biomaterial wherein the biomaterial is a SAM. The method includes preparing SAMs including a substrate having synthetic peptide thereon. The method includes attaching a synthetic peptide to a substrate. The synthetic peptide may be attached over the entire surface of a substrate or spatially patterned within a region of the substrate. In one embodiment, the synthetic peptide may be attached to the substrate by incubating the substrate in a solution including the synthetic peptide for a suitable period of time such that the synthetic peptide attaches to the substrate. A suitable period of time may be, for example, from about 30 minutes to about 80 minutes.
The methods may further include washing and drying the substrate having the synthetic peptide attached thereto. Suitable drying of the substrate may be by drying in a nitrogen-containing atmosphere. A suitable nitrogen-containing atmosphere for drying the substrate may be, for example, drying under a stream of nitrogen.
Another aspect of the present disclosure includes methods of sequestering endogenous proteoglycans and/or endogenous glycosaminoglycans using the biomaterials described above. The methods include exposing endogenous proteoglycans and/or endogenous glycosaminoglycans to the previously described biomaterials and SAMs. As used herein, “endogenous proteoglycans” and “endogenous glycosaminoglycans” are proteoglycans and glycosaminoglycans that are secreted into the culture medium by the cultured cells and/or in a mixture such as, for example, serum.
Another aspect of the present disclosure includes methods of increasing stem cell proliferation. The methods include culturing stem cells in the presence of the previously described biomaterials. As used herein, “increasing stem cell proliferation” refers to an increase in the number of stem cells as a function of time as compared to control cells cultured in the presence of biomaterials without a synthetic proteoglycan-binding peptide and/or a synthetic glycosaminoglycan-binding peptide attached.
Suitable stem cells may be, for example, mesenchymal stem cells. The mesenchymal stem cells may be, for example, human mesenchymal stem cells (hMSCs). hMSCs have the capacity to differentiate down multiple mesenchymal lineages, such as bone and cartilage. The stem cells may be, for example, H9 human embryonic stem cells. The umbilical vein endothelial cells may be, for example, human umbilical vein endothelial cells (HUVECs). Particularly suitable cells may be any adherent cell type. The term “adherent cell type” is used herein according to its ordinary meaning to refer to any cell that can attach to or interact with the substrate and/or molecules or coatings on a substrate. Thus, an adherent cell type may be, for example, embryonic stem cells, induced pluripotent stem cells, fibroblasts, and muscle cells. Examples of non-adherent cells that are not expected to attach to or interact with the substrate and/or molecules or coatings on a substrate may be, for example, blood cells.
Another aspect of the present disclosure includes methods of reducing spontaneous stem cell differentiation. The methods comprise culturing stem cells in the presence of the previously described biomaterials. As used herein, “reducing spontaneous stem cell differentiation” refers to the maintenance by the stem cells of multipotency upon multiple population doublings. Multipotent hMSCs, for example, are typically defined in the art as mononuclear cells positive for mesenchymal stromal cell markers including, for example, CD73, CD90, and CD105.
Another aspect of the present disclosure includes a tissue regeneration system including a substrate having a synthetic peptide thereon. The synthetic peptide may be, for example, a synthetic proteoglycan-binding peptide, a synthetic glycosaminoglycan-binding peptide, and combinations thereof
The disclosure will be more fully understood upon consideration of the following non-limiting Examples.
While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described below in detail. It should be understood, however, that the description of specific embodiments is not intended to limit the invention to cover all modifications, equivalents and alternatives falling within the spirit and scope of the invention as defined by the appended claims.
The following materials and reagents were used for Examples 1-13.
Gold substrates (5 nm Cr, 100 nm Au and 2 nm Ti, 10 nm Au) were obtained from Evaporated Metal Films (Ithaca, N.Y.). 11-tri(ethylene glycol)-undecane-1-thiol (HS - - - EG3) was synthesized. 11-carboxyhexa(ethylene glycol)-undecane-1-thiol (HS - - - EG6 - - - COOH) was obtained from Prochimia (sopot, Poland). All peptides were synthesized using a standard solid phase peptide synthesis protocol for Fmoc-protected amino acids activated by hydroxybenzotriazole (HOBt) and Diisopropylcarbodiimide (DIC). Piperidine, dimethylformamide (DMF), triisoproylsilane (TIPS) and PD 173074 were obtained from Sigma-Aldrich (St. Louis, Mo.). Fmoc-protected amino acids and Rink amide MBHA peptide synthesis resin were obtained from NovaBiochem (San Diego, Calif.). Hydroxybenzotriazole (HOBt) was obtained from Advanced Chemtech (Louisville, Ky.). Diisopropylcarbodiimide (DIC) was obtained from Anaspec (San Jose, Calif.). Trifluroacetic acid (TFA) and diethyl ether were obtained from Fisher Scientific (Fairlawn, N.J.). Absolute ethanol was obtained from AAPER Alcohol and Chemical Co. (Shelbyville, Ky.). Human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) were obtained from Cambrex (North Brunswick, N.J.). The EGM-2 bullet kit for endothelial cell culture was obtained from Lonza (Walkersville, Mass.). 1× minimum essential medium, alpha (αMEM) and Medium 199 were obtained from CellGro (Mannassas, Va.). MSC-qualified fetal bovine serum was obtained from Hyclone (Logan, Utah). Goat anti-human DC105 antibody was obtained from BD Transductions (San Jose, Calif.). Rabbit anti-human CD90 antibody was obtained from Abgent (San Diego, Calif.). AlexaFluor-488-tagged donkey anti-goat and AlexaFluor-647-tagged goat anti-rabbit secondary antibodies were obtained from Invitrogen (Carlsbad, Calif.).
In this Example, SAMs including gold substrates were prepared. Specifically, gold substrates were cut, sonicated in ethanol for 3 minutes, washed with ethanol, and dried under a stream of nitrogen prior to monolayer formation. Monolayers were formed by immersing clean gold substrates in an ethanolic solution of 99% HS - - - EG3:1% HS - - - EG6 - - - COOH for typical protein binding experiments, or in an ethanolic solution containing 96% HS - - - EG6 - - - COOH for typical cell culture experiments. After monolayer formulation, the gold substrates were removed from the ethanolic solution, washed with ethanol, and dried under a stream of nitrogen.
Peptide conjugation to monolayers was achieved using one of two strategies: (1) carboxylate groups were “activated” by incubating SAMS in an aqueous solution containing 100 mM N-Hydroxysuccinimide (NHS) and 250 mM 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) for 10 minutes, followed by washing with DI H2O and ethanol, and drying under a stream of nitrogen; and (2) NHS-activated SAMs were incubated in a 1× PBS solution containing 250 μM peptide for 60 minutes, followed by washing with DI H2O, 0.1% sodium dodecyl sulfate (SDS), DI H2O and ethanol, and drying under a stream of nitrogen. In the second strategy, 1) CuBr, Na-Asc, and TBTA were dissolved in DMSO at a concentration of 2 mM, 2) an acetylene-bearing peptide was dissolved in HEPES (0.1 M, pH 8.5) at a concentration of 2 mM, 3) the DMSO solution containing CuBr, Na-Asc, and TBTA and the HEPES solution containing peptide were then mixed at a 1:1 ratio by vortexing, followed by sonication for 10 minutes, 4) gold substrates bearing azide-termini were immersed in this solution and allowed to incubate at room temperature for 60 minutes, and 5) at the reaction endpoint, gold substrates were washed sequentially with DI H2O, 0.1% sodium dodecyl sulfate (SDS) in water, DI H2O, and ethanol, followed by drying under a stream of nitrogen.
Polarization-modulated infrared reflectance-absorbance spectroscopy (PM-IRRAS) was used to characterize the binding of heparin PGs from fetal bovine serum onto SAMs presenting a synthetic peptide derived from the heparin-binding domain of FGF-2, KRTGQYKL (SEQ ID NO: 1). Control SAMs were prepared using a scrambled, non-functional peptide, TYRKKGLQ (SEQ ID NO: 9).
Specifically, a Nicolet Magna-IR 860 FT-IR spectrometer with photoelastic modulator (available as PEM-90 from Hinds Instruments (Hillsboro, Oreg.), synchronous sampling demodulator (available as SSD-100 from GWC Technologies, (Madison, Wis.), and a liquid nitrogen-cooled mercury cadmium telluride detector were used to record infrared spectra of SAMs on gold films. Spectra of each sample were obtained at an incident angle of 83° with modulation centered at 1500 cm−1. For each sample, 500 scans were taken at a resolution of 4 cm−1 per modulation center. Data collected as differential reflectance vs. wave number were converted to absorbance units vs. wave number by the method outlined by Frey and co-workers (Frey, B. L.; Corn, R. M.; Weibel, S. C., ed.; Wiley & Sons: New York, 2002; p 1042.).
IR spectra collected from 1% KRTGQYKL-SAMs demonstrated a significant increase in IR absorbance at wavenumbers corresponding to amide I, amide II, and sulfonate functionalities when compared to 1% TYRKKGLQ-SAMs (
To further characterize the specificity of heparin PG binding onto KRTGQYKL-SAMs, the dependence of heparin on PG-KRTGQYKL (SEQ ID NO: 1) binding was assessed. 1% KRTGQYKL-SAMs were incubated for 20 minutes in FBS or FBS treated with heparin lyase I (FBS and 10 units heparin lyase I), an enzyme that cleaves highly sulfated domains of heparin, but does not efficiently cleave heparan sulfate or other GAGs. KRTGQYKL-SAMs were compared to SAMs prepared using a scrambled, non-functional peptide, TYRKKGLQ (SEQ ID NO: 9). IR spectra collected from 1% KRTGQYKL-SAMs after incubation in serum treated with heparin lyase I demonstrated IR absorbance over the entire spectral range that was similar to the baseline IR spectrum collected from a 1% KRTGQYKL-SAM immediately after peptide immobilization (
PM-IRRAS was used to characterize FGF-2 binding onto KRTGQYKL-SAMs. 1% KRTGQYKL SAMs were incubated in a lx PBS solution (pH 7.4) containing 10 μg/mL recombinant human FGF-2 for 20 minutes and analyzed using PM-IRRAS. IR spectra collected from 1% KRTGQYKL-SAMs after exposure to FGF-2 demonstrated no significant change in IR absorbance over the entire spectral range when compared to baseline IR spectra collected from 1% KRTGQYKL-SAMs immediately after peptide immobilization (
It is now believed that heparin PG binding to KRTGQYKL SAMs is mediated by reversible, non-covalent interactions. A surface plasmon resonance detector was used to characterize the reversible nature of heparin PG binding onto KRTGQYKL SAMs. 1% KRTGQYKL-SAMs were exposed to various volume fractions of serum to determine the maximal change in surface refractive index at the end of the binding phase, as well as the rate of release of bound molecules from the substrate after the binding phase.
Results demonstrated that exposure to 10% serum resulted in a significant increase in the surface refractive index on 1% KRTGQYKL-SAMs, while a low level of non-specific binding was observed on SAMs presenting 1% TYRKKGLQ (SEQ ID NO: 9). These results validated PM-IRRAS data demonstrating that 1% KRTGQYKL-SAMs specifically bind to heparin PGs present in FBS (
The above-results demonstrated that SAMs presenting a peptide derived from the heparin-binding domain of FGF-2 specifically sequestered heparin-bearing PGs from serum via reversible, non-covalent interactions. Moreover, the observed reversibility of heparin PG binding on these surfaces indicated that the density of heparin PG bound to the substrate will directly depend on the concentration of heparin PGs present in the cell culture medium. These results suggested the applicability of SAMs presenting KRTGQYKL to elucidate the influence of sequestered heparin PGs on stem cell behavior.
In this Example, the influence of endogenous heparin sequestered by SAMs having a synthetic PG-binding peptide on human mesenchymal stem cell (hMSC) behavior was characterized. hMSCs were cultured on SAMs presenting 2% RGDSP (SEQ ID NO: 8) and either 2% KRTGQYKL (SEQ ID NO: 1) or 2% TYRKKGLQ (SEQ ID NO: 9) for 72 h in medium supplemented with 10% FBS.
It was found that hMSC number as a function of time was significantly greater on RGDSP/KRTGQYKL-SAMs when compared to RGDSP/TYRKKGLQ-SAMs after 24, 48, and 72 h of culture in medium supplemented with 10% FBS (
The adhesion of hMSCs on SAMs presenting RGDSP (SEQ ID NO: 8) and KRTGQYKL (SEQ ID NO: 1) at co-varying surface densities was investigated to determine if interactions between cell surface PGs and KRTGQYKL mediate the enhanced proliferation of hMSCs observed on RGDSP/KRTGQYKL SAMs. hMSCs were cultured on SAMs presenting surface densities at 1% RGDSP (SEQ ID NO: 8):1% KRTGQYL (SEQ ID NO: 1); 1% RGDSP:0.5% KRTGQYL; 1% RGDSP:0.1% KRTGQYL; 0.1% RGDSP:1% KRTGQYL; 0.1% RGDSP:0.5% KRTGQYL; and 0.1% RGDSP:0.1% KRTGQYL. Culture media was serum-free or contained 10% FBS.
Results demonstrated that the extent of hMSC spreading was independent of RGDSP (SEQ ID NO: 8) or KRTGQYKL (SEQ ID NO: 1) density in the presence or absence of serum, and indicated that hMSC adhesion was not influenced by interactions between the substrate and cell surface heparan sulfate PGs, or cell-surface receptors and heparin PGs bound to the substrate (
Proliferation of hMSCs cultured on KRTGQYKL-SAMs in medium supplemented with FBS and PD 173074 was determined PD 173074 is a soluble inhibitor of FGF-2 and FGF-4 cognate receptors FGF receptor-1 and -3. hMSCs were cultured on SAMs presenting 2% RGDSP (SEQ ID NO: 8) and either 2% KRTGQYL (SEQ ID NO: 1) or TYRKKGLQ (SEQ ID NO: 9) in medium supplemented with 200 nM PD 173074 and 10% FBS.
Results demonstrated that the increased proliferation of hMSCs during culture in serum-supplemented medium on KRTGQYKL SAMs was completely abolished in the presence of PD 173074, which indicates that an FGF family member is involved in the increased hMSC proliferation observed on KRTGQYKL SAMs (
Human umbilical vein endothelial cells (HUVECs) as a model cell type were used to investigate the correlation between the surface density of bound heparin PGs, the concentration of soluble FGF-2, and the extent of up-regulation of FGF-mediated cell proliferation.
HUVECs were cultured according to the protocol supplied by the manufacturer. Sub-confluent cells were harvested from the plate, suspended in M199 supplemented with 10% FBS, and counted using a hemacytometer. Cells were collected as a pellet by centrifugation at 1100 rpm for 5 minutes, the media was decanted off of the pellet, and the cells were suspended in fresh serum-supplemented M199 at a density of 20,000 cells/250 μL. SAMs presenting 2% RGDSP (SEQ ID NO: 8) and various densities of KRTGQYKL (SEQ ID NO: 1) were prepared by incubating NHS-activated SAMs in 1× PBS solutions containing 125 μM RGDSP (SEQ ID NO: 8), and various molar ratios of KRTGQYKL (SEQ ID NO: 1) and TYRKKGLQ (SEQ ID NO: 9) at a total concentration of 125 μM Immediately after peptide conjugation, HUVECs were seeded on the SAM substrates at a density of 10,000 cells/cm2 in M199 supplemented with 10% FBS. After allowing the HUVECs to attach to the SAMs overnight, the substrates were washed with 1× PBS to remove any loosely bound cells and placed in M199 supplemented with 10% FBS and 1 or 5 ng/mL rhFGF-2. T=0 hr for each experiment was designated as the point immediately after placing the SAMs in fresh culture medium supplemented with FGF-2. Brightfield photomicrographs (40× mag) of each substrate were collected on an Olympus IX51 inverted epifluorescent microscope at t=0, 24, 48, and 72 hrs, and the total number of cells per viewing area was counted manually.
Results demonstrated that HUVEC number as a function of time was dependent on both KRTGQYKL (SEQ ID NO: 1) surface density and concentration of exogenous FGF-2 supplement present in the cell culture media at t=0 hours (
The proliferation of hMSCs on RGDSP/KRTGQYKL-SAMs in medium supplemented with 10% serum compared to proliferation on RGDSP/TYRKKGLQ-SAMs in medium supplemented with 10% serum and various concentrations of FGF-2 was investigated.
hMSCs were expanded at low density on tissue culture treated polystyrene plates using the method described previously by Sotiropoulou et al. to maintain pluripotency (Sotiropoulou et al., “Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells,” Stem Cells, 24:462 (2006)). At passage 6, cells were harvested from the plate, suspended in medium supplemented with 10% fetal bovine serum, and counted using a hemacytometer. Cells were collected as a pellet by centrifugation at 1100 rpm for 5 minutes, the media was decanted off of the pellet, and the cells were suspended in αMEM supplemented with 10% FBS at a density of 20,000 cells/250 μL. SAMs presenting 2% RGDSP (SEQ ID NO: 8) and various surface densities of KRTGQYKL (SEQ ID NO: 1) or TYRKKGLQ (SEQ ID NO: 9) were prepared using previously described methods Immediately after peptide conjugation, hMSCs were seeded on the SAM substrates at a density of 2,000 cells/cm2 in αMEM supplemented with 10% FBS and FGF-2 at concentrations of 0 ng/mL, 1 ng/mL, and 5 ng/mL. After allowing the hMSCs to attach to the SAMs overnight, the substrates were washed with 1× PBS to remove any loosely bound cells and placed in one of the following culture conditions: αMEM supplemented with 0.01, 0.1, 1, or 10% FBS, or αMEM supplemented with 10% FBS and 200 nM PD173054. T=0 hr for each experiment was designated as the point immediately after placing the SAMs in the appropriate culture medium. Brightfield photomicrographs (40× mag) of each substrate were collected on an Olympus IX51 inverted epifluorescent microscope at t=0, 24, 48, and 72 hrs. The total number of cells per viewing area was counted manually for 3 samples per condition.
Results demonstrated that a FGF-2 concentration of 5 ng/mL was required to stimulate hMSCs proliferation on RGDSP/TYRKKGLQ-SAMs at a rate similar to that observed on KRTGQYKL-SAMs during the first 24 hours of culture (
The influence of substrate-bound heparin PGs and, in turn, locally-enhanced endogenous FGF activity, on hMSC phenotype after multiple population doublings was investigated. Multipotent hMSCs are typically defined as mononuclear cells positive for mesenchymal stromal cell markers, including CD73, CD90, and CD105. hMSC phenotype was characterized using quantitative PCR (qPCR) and immunocytochemistry after 72 hours of culture on 2% RGDSP/2% KRTGQYKL-SAMs or 2% RGDSP/2% TYRKKGLQ-SAMs.
qPCR analysis demonstrated that the CD73, CD90, and CD105 mRNA expression levels of hMSCs decreased in all conditions during the 72 hour culture period (
The influence of spatially patterned RGDSP (SEQ ID NO: 8) and KRTGQYKL (SEQ ID NO: 1) presentation and, in turn, spatial localization of heparin PG sequestration on hMSC proliferation was investigated. hMSCs were cultured on 2% RGDSP/2% KRTGQYKL-SAMs or 2% RGDSP/2% TYRKKGLQ-SAMs during culture in medium supplemented with 10% FBS as described herein.
SAMs were prepared as previously described. Low passage number hMSCs were harvested from polystyrene plates and seeded on SAMs presenting RGDSP (SEQ ID NO: 8), RGDSP(SEQ ID NO: 8)/KRTGQYKL(SEQ ID NO: 1), or RGDSP(SEQ ID NO: 8)/TYRSRKY(SEQ ID NO: 2), at specified surface densities at a cell density of 2500 cells/cm2. Cells were cultured in αMEM basal medium supplemented with 10% FBS, 50 μg/mL 2-phosphate ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone for 7 days. Brightfield images of cells were collected to measure cell projected area and quantification of cell number as a function of time. At the end of the 7-day culture period, alkaline phosphatase activity was analyzed using the SensoLyte FDP alkaline phosphatase assay kid (Anaspec, Fremont, Calif.).
Results demonstrated that RGDSP (SEQ ID NO: 8) bound to alpha-5 beta-1 integrins and ligation of alpha-5 beta-1 integrins enhanced osteogenic differentiation of hMSCs (
The following materials and reagents were used for Examples 14-18.
Gold substrates (5 nm Cr, 100 nm Au or 2 nm Ti, 10 nm Au) were from Evaporated Metal Films (Ithaca, N.Y.). 11-tri(ethylene glycol)-undecane-1-thiol (HS - - - EG3), Piperidine, dimethylformamide (DMF), triisoproylsilane (TIPS), acetone, 99.999% cuprous bromide (CuBr), dimethylsulfoxide (DMSO), Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), and sodium ascorbate (Na-Asc) were from Sigma-Aldrich (St. Louis, Mo.). 11-carboxylic acid-hexa(ethylene glycol)-undecane-1-thiol (HS - - - EG6 - - - COOH) and 11-azidohexa(ethylene glycol)-undecane-1-thiol (HS - - - EG6 - - - N3) were purchased from Prochimia (Sopot, Poland). Fmoc-protected amino acids and Rink amide MBHA peptide synthesis resin were from NovaBiochem (San Diego, Calif.). Hydroxybenzotriazole (HOBt) was from Advanced Chemtech (Louisville, Ky.). Diisopropylcarbodiimide (DIC) and Fmoc-(R)-3-amino-5-hexynoic acid were from Anaspec (San Jose, Calif.). Trifluoroacetic acid (TFA) and diethyl ether were from Fisher Scientific (Fairlawn, N.J.). Absolute ethanol was from AAPER Alcohol and Chemical Co. (Shelbyville, Ky.). Human mesenchymal stem cells (hMSCs) were from Cambrex (North Brunswick, N.J.). 1× minimum essential medium, alpha was from CellGro (Mannassas, Va.). MSC-qualified fetal bovine serum was from Invitrogen (Carlsbad, Calif.). 0.05% Trypsin and penicillin/streptomycin were from Hyclone (Logan, Utah). Actin cytoskeleton staining kit and FITC-conjugated secondary antibody were from Chemicon (Billerica, Mass.).
Peptide synthesis: Peptides were synthesized using standard Fmoc solid phase peptide synthesis on a 316c automated peptide synthesizer (CSbio, Menlo Park, Calif.). Rink amide MBHA resin was used as the solid phase, and HOBt and DIC were used for amino acid activation and coupling. After coupling the final amino acid, incubation of resin in TFA, TIPS, and deionized (DI) water (95:2.5:2.5) for 4 hours released the peptide from the resin and removed protecting groups. The peptide was then extracted from the TFA/TIPS/H2O cocktail by precipitation with cold diethyl ether. Lyophilized peptides were analyzed on a Bruker Reflex II MALDI-TOF mass spectrometer (Billerica, Mass.) using dihydroxybenzoic acid (DHB) (10mg/mL) as matrix in acetonitrile:DI water (7:3).
In this Example, SAMs including gold substrates were prepared. Specifically, gold substrates were cut, sonicated in ethanol for 3 minutes, washed with ethanol, and dried under a stream of nitrogen prior to monolayer formation. Monolayers were formed by incubating clean gold substrates in an ethanolic solution of HS - - - EG3, HS - - - EG6 - - - N3 and HS - - - EG6 - - - COOH at various molar ratios (2 mM total thiol concentration) overnight. After monolayer formation, gold substrates were removed from the ethanolic solution, washed with ethanol, and dried under a stream of nitrogen.
In this Example, SAMs prepared as described above were immersed in solution with various proteoglycans to determine the ability of SAMs to sequester the proteoglycan.
Immediately after SAM formation as described in Example 15, SAM substrates were immersed in an aqueous solution containing 100 mM NHS and 250 mM EDC for 10 minutes to convert the surface carboxylate groups to amine-reactive NHS-esters. After 10 minutes, the substrates were washed briefly with DI H2O and ethanol and dried under a stream of nitrogen. NHS-ester-terminated SAMs were then incubated in a 1× PBS solution containing 500 mM amine-terminated RGESP (SEQ ID NO: 10) or TYRSRKY (SEQ ID NO: 2) (pH 7.5) for 60 minutes. After 60 minutes, gold substrates were washed sequentially with DI water, 0.1% sodium dodecyl sulfate in water, DI water, and ethanol, followed by drying under a stream of nitrogen. CuBr and Na-Asc were dissolved in DMSO at a concentration of 2 mM by sonicating for 10 minutes. TBTA was then dissolved in this solution at a concentration of 2 mM by sonicating for an additional 10 minutes. Lyophilized acetylene-bearing RGDSP (SEQ ID NO: 8) was dissolved in HEPES (0.1 M, pH 8.5) to achieve a peptide concentration of 2 mM. The DMSO solution containing CuBr, Na-Asc, and TBTA and the HEPES solution containing RGDSP (SEQ ID NO: 8) were then mixed at a 1:1 ratio by vortexing, followed by sonication for 10 minutes. Azide-terminated gold substrates were immersed in this solution and allowed to incubate at room temperature for 60 minutes. At the reaction endpoint, gold substrates were washed sequentially with DI water, 0.1% sodium dodecyl sulfate in water, DI water, and ethanol, followed by drying under a stream of nitrogen.
PM-IRRAS analysis of SAMs: Infrared spectra of the SAMs on gold films were recorded using a Nicolet Magna-IR 860 FT-IR spectrometer with photoelastic modulator (available as PEM-90 from Hinds Instruments (Hillsboro, Oreg.)), synchronous sampling demodulator (available as SSD-100 from GWC Technologies (Madison, Wis.)), and a liquid nitrogen-cooled mercury cadmium telluride detector. All spectra were obtained at an incident angle of 83° with modulation centered at 1500 cm−1 and 2500 cm−1. For each sample, 500 scans were taken at a resolution of 4 cm−1 per modulation center. Data was collected as differential reflectance vs. wave number.
The results are shown in
Correlation Between Reactive Moiety Density and Peptide Density: A plot of the COOH mole fraction in ethanol during SAM formation versus the amide I peak area after conjugation of RGESP (SEQ ID NO: 10) to COOH demonstrated a linear correlation (
In this Example, the binding of serum-derived heparin proteoglycans on SAMs were evaluated.
SAMs presenting 1% TYRSRKY (SEQ ID NO: 2) or 1% scrambled, non-functional peptide SKTYYRR (SEQ ID NO: 11) were prepared using previously described methods. Briefly, SAMs comprised of 1% HS - - - EG6 - - - COOH and 99% HS - - - EG3 were immersed in an aqueous solution of 100 mM NHS/250 mM EDC for 10 minutes, followed by incubation in a 1× PBS (pH 7.4) solution containing 500 mM TYRSRKY (SEQ ID NO: 2) or SKTYYRR (SEQ ID NO: 11) Immediately following the peptide immobilization steps, SAMs were incubated in a 50:50 (v/v) solution of 1× PBS (pH 7.4) and FBS for 20 minutes. After the serum incubation step, SAMs were rinsed briefly with DI H2O and were dried under a stream of nitrogen. The molecular composition of biomolecules bound on the SAM was then analyzed using PM-IRRAS.
IR spectra collected from 1% TYRSRKY SAMs after incubation in a 50% FBS solution demonstrated a significant increase in amide I (λ=1666 cm−1), amide II (λ=1550 cm−1), methylene (λ=1460, 1400 cm−1), sulfate (λ=1260, 1080 cm−1), and carbohydrate (λ=1100 cm−1) absorbance when compared to 1% SKTYYRR SAMs after incubation in a 50% FBS solution (
In this Example, the behavior of hMSCs on RGESP- and RGDSP-presenting SAMs was explored to demonstrate the applicability of orthogonally-reactive SAMs as cell culture substrates in a well-defined model system. To maintain multipotency, hMSCs were expanded at low density on tissue culture treated polystyrene plates. At passage 6, cells were harvested from the plate, suspended in medium supplemented with 10% fetal bovine serum, and counted using a hemacytometer. Cells were collected as a pellet by centrifugation at 1100 rpm for 5 minutes, the media was decanted off of the pellet, and the cells were suspended in fresh αMEM at a density of 20,000 cells/250 μL. SAM preparation and peptide conjugation were performed using the protocols described above Immediately after peptide conjugation, SAMs were placed into 1 mL of 1× PBS (pH 7.4) in a 12-well tissue culture plate to prevent degradation of the monolayer due to air oxidation. PBS was aspirated from the wells and replaced with 1.25 mL of αMEM, followed by addition of 250 μL of the cell suspension directly over the SAM substrate in each well. Plates were then gently rocked for 10 seconds to evenly distribute cells over the substrate surface. Substrates were then incubated for a specified time frame (12 hrs for RGESP/RGDSP SAMs, 4 hrs for RGDSP/TYRSRKY SAM in a humid environment at 37° C., 5% CO2 to allow hMSC attachment. At the end of the attachment period, the hMSC growth media was aspirated from the well and the substrates were gently washed with sterile 1× PBS to remove any loosely bound cells. The 1× PBS solution was then replaced with fresh medium. Brightfield photomicrographs of cells were then collected using an Olympus IX51 inverted microscope.
The results demonstrated that a significant number of hMSCs were present on all SAMs presenting RGDSP (SEQ ID NO: 8) (i.e. RGDSP=0.0001, 0.001, or 0.01), but were absent on the SAM presenting RGESP (SEQ ID NO: 10) alone (i.e. RGDSP=0) (
Analysis of projected cell area on SAMs presenting different RGDSP (SEQ ID NO: 8) and RGESP (SEQ ID NO: 10) surface densities demonstrated that the extent of hMSC spreading is dependent on RGDSP (SEQ ID NO: 8) surface density (
In this Example, the influence of an integrin-binding peptide, RGDSP (SEQ ID NO: 8) and a proteoglycan-binding peptide TYRSRKY (SEQ ID NO: 2) on hMSC adhesion was evaluated.
hMSCs were seeded on the SAMs as described previously. After washing away loosely bound cells using 1× PBS, cytoskeletal immunostaining of hMSCs was performed by following the protocol supplied by the manufacturer. Briefly, a 4% paraformaldehyde solution in 1× PBS was added to the wells for 15 minutes to fix the cells, followed by a 5 minute incubation in a 1× PBS solution containing 0.05% Tween-20 to permeabilize the cells. Wells were subsequently blocked to prevent non-specific antibody adsorption using a 1× PBS solution containing 0.1 wt % bovine serum albumin. After blocking, a 1× PBS solution containing an anti-Vinculin primary antibody was added to each well and allowed to incubate at room temperature for 60 minutes. The wells were then washed gently three times using a 1× PBS solution containing 0.1 wt % bovine serum albumin. Immediately after washing, a 1× PBS solution containing a fluorescein-tagged mouse anti-human IgG secondary antibody and a TRITC-tagged anti-Phalloidin antibody was added to each well and allowed to incubate at room temperature for 45 minutes. Substrates were then washed using the method described previously. Cytoskeletal staining was analyzed using an Olympus IX51 inverted epifluorescent microscope equipped with FITC and TRITC filter cube sets.
Analysis of projected cell area of hMSCs on SAMs presenting 0.1-1.0% TYRSRKY (SEQ ID NO: 2) (
Interestingly, analysis of projected cell area on SAMs presenting 0.1% RGDSP (SEQ ID NO: 8) and 0.1-1% TYRSRKY (SEQ ID NO: 2) in the presence of 10% FBS (
These results demonstrated that an integrin binding moiety and a proteoglycan binding moiety work in concert to influence hMSC adhesion on 2-D substrates. Additionally, these results demonstrated that soluble biomolecules present during cell culture can compete with cell surface biomolecules for material binding sites and, in turn, directly influence specific cell-material interactions. Although SAM instability can limit the long-term efficacy of these materials when characterizing the influence of immobilized biomolecules on cell function over the course of weeks, SAMs presenting peptides are commonly used to characterize cell-material interactions over a relatively short-term (e.g. hours to days). The results herein demonstrated that SAMs presenting orthogonally-reactive moieties are useful base materials to characterize the concerted influence of two biochemically-distinct peptides on stem cell adhesion, and suggest widespread applicability of these materials to characterize additional stem cell material interactions mediated by immobilized biomolecules.
In this Example, the binding of a synthetic KRT peptide to hydroxyapatite was evaluated.
A 50 μM peptide solution containing the synthetic KRT peptide, KRTGQYKLGGGAAAA(Gla)PRR(Gla)VA(Gla)L (SEQ ID NO: 12), was incubated with 1 mg of hydroxyapatite particles for 30 minutes at 37° C. After incubation, the concentration of peptide in solution was determined using a micro-BCA assay (Pierce) as a measure of the depletion of the peptide from solution due to hydroxyapatite binding.
Results demonstrated that the hydroxyapatite particles bound 66.08 nmol/m2 of peptide. These results demonstrate that binding of the KRT peptide to hydroxyapatite allows for the fabrication or coating of orthopedic implants with calcium phosphate to sequester endogenous growth factors such as FGF2 or BMP-2. In turn, this would promote bone regeneration.
H9 human embryonic stem cells (hESCs) were cultured on oligo(ethylene-glycol) functionalized alkanethiolate self-assembled monolayers (SAMs) presenting 7.5% RGDSP (SEQ ID NO: 8) and 2.5% KRTGQYKL (SEQ ID NO: 1) to determine if the surface could 1) support cell adhesion and proliferation and 2) maintain the undifferentiated state of the hESCs. hESCs are traditionally cultured on undefined surfaces such as a mouse embryonic feeder layer or MATRIGEL (BD Biosciences).
H9 hESCs were seeded in DMEM/F12 basal medium and allowed to attach for 2 hours. After attachment, media was changed to mTeSR1 supplemented with the ROCK inhibitor, Y27632. The media was replenished every 48 hours during time-lapse imaging. Cells were imaged over 120 hours and during that time, cell spreading and proliferation was observed. When the cells reached a near confluent monolayer, they were fixed and stained with Hoechst nuclear stain and an antibody against human Oct4 (a marker of hESC pluripotency). As shown in FIGS. 45(A)-(F) and 46(A)-(C). SAMs presenting 7.5% RGDSP (SEQ ID NO: 8) and 2.5% KRTGQYKL (SEQ ID NO: 1) promoted hESC attachment, proliferation and self-renewal.
This application claims the benefit of U.S. Provisional Application No. 61/326,945 filed Apr. 22, 2010, which disclosure is incorporated by reference in its entirety.
This invention was made with United States government support awarded by the National Institutes of Health under grant number R01HL093282. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 61326945 | Apr 2010 | US |
