âSmartâ Hydrogels and Genetically Engineered Stem Cells for Skeletal

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Biomacromolecules 2010, 11, 1516–1526

Peptide-Modified “Smart” Hydrogels and Genetically Engineered Stem Cells for Skeletal Tissue Engineering Shai Garty,*,†,‡,§ Nadav Kimelman-Bleich,†,| Zvi Hayouka,⊥ Daniel Cohn,§ Assaf Friedler,⊥ Gadi Pelled,|,# and Dan Gazit|,# Casali Institute of Applied Chemistry, Institute of Chemistry, Edmond J. Safra, The Hebrew University of Jerusalem, Givaat Ram Campus, Jerusalem, Israel, 91904, Skeletal Biotech Lab, Faculty of Dental Medicine, Hadassah Medical Campus, Ein Kerem, The Hebrew University of Jerusalem, Jerusalem, Israel, 91120, Department of Organic Chemistry, Institute of Chemistry, Edmond J. Safra, The Hebrew University of Jerusalem, Givaat Ram Campus, Jerusalem, Israel, 91904, and Department of Surgery and Cedars-Sinai Regenerative Medicine Institute (CS-RMI), Cedars Sinai Medical Center, Los Angeles, California 90048 Received February 9, 2010; Revised Manuscript Received April 15, 2010

Stimuli responsive or “smart” hydrogels are of interest for tissue-engineering applications, featuring the advantages of minimally invasive application. Currently, these materials have yet to be used as a biological replacement in restoring the function of damaged tissues or organs. The aim of this study was to demonstrate the advantages of thermoresponsive, peptide-containing hydrogels as a supportive matrix for genetically engineered stem cells. We used injectable hydrogels, enabling cell delivery to the desired site and providing adequate scaffolding postimplantation. Thermoresponsive hydrogels were developed based on amphiphilic block copolymers of polyethylene-oxide and polypropylene-oxide end-capped with methacrylate or maleimide entities and further reacted with RGD-containing peptides. Cell metabolic activity and survival within those hydrogels was studied, illustrating that the stable peptide-polymer conjugate is required for prolonged cell support. The unique polymer characteristics, combined with its enhanced cell interactions, suggest the potential use of these biomaterials in various tissue engineering applications.

1. Introduction Tissue engineering’s primary goal is the development of a biological replacement for damaged tissues and functionality restoration. Currently, the majority of tissue engineering strategies rely on the development of both cells and biomaterials. New scaffolds are being developed, mimicking the extracellular matrix (ECM), to support and organize cells into a threedimensional architecture and to present stimuli that direct cells to the growth and formation of a desired tissue.1 Mesenchymal stem cells (MSCs) can differentiate into osteogenic, adipogenic, chondrogenic,2 and tenogenic3 lineages. These cells are isolated from a variety of tissues4 and may be genetically engineered to overexpress osteogenic genes, thus, promoting differentiation of both the engineered MSCs and host stem cells.5 Genetically engineered MSCs expressing osteogenic genes promote bone formation in ectopic and orthotropic sites.5-7 Tet-off BMP-2 MSCs, derived from a C3H10t1/2-based cell line that was genetically engineered to express the osteogenic human BMP-2 gene under tetracycline-off regulation, have * To whom correspondence should be addressed. Phone: 1-206-543-3793. E-mail: [email protected]. † Garty Shai and Kimelman-Bleich Nadav contributed equally to this work. ‡ Currently at the Department of Ophthalmology, University of Washington Medical Center and Department of Bioengineering, College of Engineering and School of Medicine, University of Washington, Seattle, WA 98195. § Casali Institute of Applied Chemistry. | Skeletal Biotech Lab. ⊥ Department of Organic Chemistry. # Cedars Sinai Medical Center.

been shown to promote bone formation in several animal models, including bone defect regeneration and spinal fusion models.8,9 Combining genetically engineered stem cells and specialized biomaterials can lead to the development of biological treatments for pathological conditions currently receiving suboptimal treatment. One prominent pathway to accomplish that is by using hydrogels in tissue engineering applications that can be delivered in a minimally invasive manner10 and, once injected, have mechanical and structural properties similar to the desired tissue or ECM. Reverse thermoresponsive polymers, usually known as gels displaying reverse thermal gelation (RTG) constitute one of the most promising strategies for the development of injectable systems for minimally invasive biomedical applications. Water solutions of these materials display low viscosity at ambient temperature and exhibit a sharp viscosity increase following a small temperature rise, producing a semisolid gel at body temperature.11 There are several RTG displaying polymers. the most significant among them are poly(N-isopropylacrylamide) (pNIPAAm),12-15 block copolymers of poly(ethylene oxide)poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO),16-18 ethyl (hydroxyethyl) cellulose (EHEC) formulated with ionic surfactants that can be enzymatically degraded,19-21 and poly(ethylene glycol)-poly(lactic acid)-poly(ethylene glycol) triblocks (PEG-PLA-PEG) that degrade hydrolytically.22 Aqueous solutions of these systems have a lower critical solution temperature (LCST), resulting in a viscosity increase upon heating above this temperature. These thermoresponsive hydrogels are utilized in minimally invasive surgery23-25 and as scaffolds for tissue-engineering applications.26,27

10.1021/bm100157s  2010 American Chemical Society Published on Web 05/12/2010

Skeletal Tissue Engineering Scheme 1. Molecular Structures of PEO-PPO Block Copolymers: (A) Linear Bifunctional Poloxamer Pluronic F127 (MW 12.6 kDa) and (B) Branched Four-Arm Poloxamine Tetronic 1307 (MW 18 kDa); Both Polymers Contain 70 wt % PEO

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would lead to in situ scaffolds formation and improved cell viability for skeletal tissue engineering. Combining the advantages of phase change from liquid solution in delivering cells, to sturdy cross-linked hydrogel displaying enhanced biomechanical properties, supports the feasibility of bone substitute formation that can significantly promote future clinical applications. The applicability of these implants for a minimally invasive intervention has significant advantages as a first stratum for in situ generated skeletal tissue engineering.

2. Materials and Methods

One of the most important RTG-displaying materials is the family of amphiphilic block copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) linear triblock copolymers commercially available as Poloxamers or Pluronics16-18 and a four-arm block copolymer available as Tetronic (Scheme 1). Various types of Pluronic polymers have been largely investigated as drug carriers in controlled release applications,28-31 in the prevention of postoperative tissue adhesions,32 for cell encapsulation applications,33 as wound dressings,34 and as macroscopic engineered structures for tissue engineering.35 Tetronic polymers have been investigated mainly in drug delivery systems.36,37 In our previous report,35 we have demonstated the potential of water solutions of methacrylate modified amphiphilic polymers displaying reverse thermal gelation (RTG) to be injected into the target site and perform rapid cross-linking at body temperature, resulting in in situ scaffold formation.35,38-40 However, polymeric hydrogels containing a large percentage of polyethylene oxide (PEO) and water (>10%) are widely used as antiadhesion materials.41 To overcome this characteristic of PEO-rich thermoresponsive hydrogels, there is a need to design biomimetic materials that include proteins of the native extracellular matrix (ECM) or synthetic analog peptides.42 These moieties promote cell adhesion providing cellular responses toward tissue regenaration.43 Peptide-modified biomaterials enhance cell attachment in vitro44 and promote bone formation in vivo, resulting in an improved implant integration in various animal models.45-47 A stable bond between the peptide and the thermoresponsive polymer is essential to promote strong cell adhesion, because focal adhesion formation occurs only if the ligands withstand the cells’ contractile forces.48-50 The objective of the present study was the development of in situ generated implants for skeletal tissue engineering that are based on thermosensitive polymers. Two commercially available polymers were used: linear PEO99-PPO67-PEO99 triblock (Pluronic127) and the four-arm Tetronic T1307, comprising four block copolymers of PPO23-PEO72. These polymers’ hydroxyl end groups were modified with methacrylate further enabling cross-linking and with maleimide group to allow stable peptide conjunction. The peptides used in this work were RGD-based, with specific spacers and functional end groups. All peptides selected were cysteine-terminated to induce the addition reaction of its thiol group to the polymer’s unsaturated end groups. We hypothesized that thermosensitive peptide-containing hydrogels, preseeded with genetically engineered stem cells

2.1. Polymer Synthesis. Analytical grade solvents were dried using type 4A molecular sieves (BDH Chemicals Ltd., Poole, U.K.). Pluronic F127 NF (national formulary grade) and Tetronic T1107, T1307, and T908 polyethylene oxide (PEO) and polypropylene oxide (PPO) block copolymers were generously donated by BASF (Ludwigshafen, Germany). Methacryloyl chloride was obtained from Aldrich (SigmaAldrich Israel Ltd., Rehovot, Israel) and distilled before use. Triethylamine (TEA) was supplied by Riedel de-Haen (Sigma-Aldrich), p-maleimidophenyl-isocyanate (PMPI) and stannous 2-ethyl-hexanoate (Sn[Oct]2) were supplied by Sigma-Aldrich and used as received. A methacrylation reaction for cross-linkable F127-dimethacrylate (F127-dMA) preparation was performed as previously described.51 Briefly, 37.8 g (3.0 mmol) F127 (MW 12600) was poured into a 250 mL three-neck flask and dried for 3 h in a 120 °C oil bath under magnetic stirring and vacuum. The polymer was then cooled and dissolved in 75 mL of dry chloroform. Further cooling of the solution to 4 °C was performed in an ice bath. A total of 1.2 g triethyle amine (TEA; 12.0 mmol) were added to the solution. A freshly distilled methacryloyl chloride (2.5 g [24.0 mmol]) was diluted in 20 mL of chloroform and added dropwise to the cooled solution over a 2 h period while stirring. The reaction was then allowed to proceed for 24 h at room temperature. The crude product was resuspended in boiling toluene (100 mL) at 110 °C. The hot mixture was filtered several times to remove the byproduct of triethylammonium hydrochloride. The toluene solution was poured into 400 mL of petroleum ether (40-60 °C). The ensuing white solid product, F127-dMA, was filtered, washed with several portions of petroleum ether, and dried under vacuum at room temperature. All of the F127-dMA hydrogels were prepared in a manner previously described.35 In brief, 1.0 g F127-dMA was dissolved in 5 mL of HEPES/bicarbonate 25 mM buffer. Next, ammonium persulfate (APS, 20 mg predissolved in 100 µL water) and 20 µL of tetramethylene ethylenediamine (TEMED, predissolved in 100 µL water) were added. The 20% (wt/vol) hydrogel was incubated at 37 °C for 24 h. For all cell culture experiments, the polymers were predissolved under sterile conditions in Dulbecco’s modified eagle medium (DMEM, GIBCO) containing D-glucose, L-glutamine, and 25 mM HEPES buffer, without sodium pyruvate. The polymers were cross-linked within these samples using the same APS/TEMED catalytic pair. Multifunctional thermosensitive polymers preparation using Tetronic 1307 that was modified with heterobifunctional 4-(maleinimido)phenylisocyanate (PMPI) was performed as previously described.52 In brief, 3.6 g (0.2 mmol) Tetronic 1307 (MW 18000) were poured into a 250 mL three-neck flask and dried at 120 °C under vacuum. PMPI (18.0 mg [0.84 mmol]) and the Sn[Oct]2, 8.0 mg [0.02 mmol]), catalyst were added to the reaction mixture. The reaction proceeded at 80 °C for 60 min under nitrogen atmosphere with continuous magnetic stirring. The reacted polymer (T1307-PMPI) was then dissolved in chloroform (100 mL) precipitated at 600 mL petroleum-ether, 40-60 °C, washed repeatedly with additional portions of petroleum-ether (3 × 100 mL), and dried under vacuum at room temperature. Peptides were synthesized on an Applied Biosystems (ABI) 433A peptide synthesizer.53 The amino acids were purchased from Novabiochem (Calbiochem-Novabiochem Corp., San Diego, CA). Rink amide-MBHA resin was purchased from Merck (Darmstadt, Germany).

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All solvents and reagents used for peptide synthesis were HPLC grade. The peptides were purified on a Gilson HPLC using a reverse-phase C8 semipreparative column (ACE) with a gradient from 5 to 60% acetonitrile in water (both containing 0.001% (v/v) trifluoroacetic acid). Peptide sequences were CGRGDSY and GCGRGDSPG. Michael-type addition between the peptide and the polymer was conducted by reacting the cysteine (C) containing peptide-thiol side group with the carbon-carbon double bond end-capped polymer.54 Conjugation reaction between T1307-t-PMPI with the peptide CGRGDSY was done as follows: initially, the polymer was dissolved in bicarbonate buffer (0.25 M) and oxygen was removed by nitrogen bubbling for 30 min. Then, 0.94 g (5 × 10-5 mol) T1307-t-PMPI was added to the reaction vessel. After the polymer was dissolved, 1.9 mg (2.5 × 10-5 mol, 757 g/mol) peptide was added, and the reaction vessel was sealed and wrapped with aluminum foil. Following the reaction, the resulting peptide-modified polymer was extracted from the water solution to chloroform solution, following by product precipitation in cold ether. After fully drying in vacuum at room temperature, the addition reaction was analyzed using 1H NMR. The precise peptide concentration of the final water solution was determined as the ratio between the T1307t-PMPI-co-(peptide)n and F127-dMA in the final solution. 2.2. Polymer Characterization. 1H NMR spectra were recorded using a high-resolution Bruker AMX 300-MHz NMR imaging system for proton measurement (Bruker BioSpin GmbH, Rheinstetten, Germany). Peptide spectra were obtained at room temperature from 1% (wt/vol) D2O solutions. Polymer spectra were obtained from a 1% (wt/ vol) CDCl3 solution. The functional groups were characterized by performing an infrared spectroscopy (FT-IR) analysis using a Nicolet Avatar 360 FT-IR spectrometer (Thermo Scientific, Waltham, MA). The samples were prepared by solvent casting chloroform solutions directly on sodium chloride crystals (Sigma-Aldrich). The average molecular weights and polydispersity values were determined by using a gel permeation chromatography system (GPC, Waters Corp., Milford, MA) equipped with a differential separation module (Waters 2690) and a refractometer detector (Waters 410). The data were analyzed by a computer running Millenium chromatography manager software (Waters). The calibration standards consisted of polystyrenes (MW 472-360000 Da).55 Preparative high-performance liquid chromatography (HPLC, Gilson Inc., Middleton, WI) was used for peptide purification and an Applied Biosystems Voyager DE PRO MALDI-TOF mass spectrometer (Applied Biosystem, CA) was used for the peptide molecular weight analysis.53 The storage modulus (G′) was determined using a parallel-plates HAAKE RheoScope-1 rheometer (Thermo Haake, Langenselbold, Germany). Water solutions 30% (w/v) were investigated over the temperature range of 5 to 55 °C, at a 1 °C/min heating rate, with a constant frequency of 10 Hz and a shear stress of 5 Pa.56 2.3. Cell Culture. Tet-off BMP-2 MSCs were generated and cultured in a manner previously described.9 Doxycycline (DOX, 1 µg/ mL, Sigma-Aldrich, St. Louis, MO) was added to the cell medium to prevent hBMP-2 expression and cell differentiation during the culture period. When the cells reach near confluence, they were trypsinized and counted using the trypan blue exclusion method. Aliquots of 50000 cells were either resuspended in or seeded on top of 60 µL of each of the following hydrogels: F127-dMA + T-1307-t-PMPI-CGRGDSY, 0.3 mmol (denoted as PMPI-CGRGDSY 0.3 mmol); F127-dMA + T-1307-dMA-d-GCGRGDSPG, 0.3 or 0.6 mmol (denoted as GCGRGDSPG 0.3 mmol or GCGRGDSPG 0.6 mmol, accordingly); F127-dMA + T-1307-dMA-d-CGRGDSY, 0.6 mmol (denoted as CGRGDSY 0.6 mmol); and F127-dMA, 15% (w/v; no peptide). All hydrogels were cross-linked using the APS/TEMED system, as noted before. We also analyzed another control group of cells that were seeded onto the culture plate directly with no hydrogel. Seeded hydrogels were cultured in 96well plates for 3, 7, or 30 days in a humidified incubator containing 5% CO2 at 37 °C; the media were changed twice a week. To evaluate

Garty et al. the various hydrogels as scaffolds for skeletal tissue engineering, eight hydrogels were prepared for each group and analyzed as follows: five hydrogels were analyzed for cell viability and for DNA amount and three hydrogels were morphologically analyzed using electron microscopy. 2.4. High Resolution Scanning Electron Microscopy (HR-SEM). Sample preparation for the SEM included cell fixation, dehydration, and freeze-drying. The fixation procedure included removal of excess medium, washing twice using dimethylarsinic acid buffer (0.1 M, cacodylic acid, Sigma-Aldrich), and fixation using Karnovsky’s Fixative57 using 2.5% (v/v) glutaraldehyde (Sigma-Aldrich) in cacodylic acid buffer for 30 min at 37 °C. The samples were then dehydrated using graded ethanol concentrations from 50 to 100%, snap-frozen in liquid nitrogen, and lyophilized while kept at -50 °C. Electron micrographs were obtained using a Sirion high resolution scanning electron microscope (HR-SEM, FEI, Eindhoven, The Netherlands) with voltage set at 3-5 kV. Samples were sputter coated for 60 s until 10 nm palladium/gold (Pd/Au) coating formed. Ultrahigh spatial resolution was used for both structural studies and high-resolution analysis by performing energy-dispersive X-ray spectroscopy (EDS) with the aid of an EDS spectrometer equipped with a sapphire-Si (Li) detector. X-ray microanalysis enables the chemical analysis of a definitive section and, therefore, distinguishes between different entities (in this case hydrogels and cells) not only by their morphological characteristics but also by their chemical compositions. 2.5. DNA Content and Cell Viability. On days 3, 7, and 30 after seeding, the hydrogels were enzymatically digested as previously described.58 The digested sample materials were analyzed for DNA content by performing a PicoGreen assay (Molecular Probes, Eugene, OR) according to the manufacturer’s protocol.59 Briefly, 60 µL cell containing hydrogel samples were homogenized and enzymatically digested. The samples were then diluted into 1 mL buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and dye was added. Following a 5 min incubation, the samples were analyzed. The data represent the average ( standard deviation of five samples. Cell viability was determined on days 7 and 30 by performing AlamarBlue assays. The medium was replaced with medium containing 10% AlamarBlue dye (Serotec Ltd., Oxford, U.K.) and incubated for 5 h in a humidified incubator containing 5% CO2 at 37 °C. The extent of AlamarBlue reduction was determined according to the manufacturer’s protocol.60 Data is presented as average ( standard deviation of five samples. 2.6. Statistical Analysis. Two-tailed Student’s t test or ANOVA was performed to determine a significant difference between the experimental and control groups, with significance set at p < 0.05. Results are presented as means ( standard error.

3. Results 3.1. Characterization of Four-Arm Thermosensitive Polymers. Pluronic poloxamer and Tetronic poloxamine polymers were examined for both covalent cross-linking and peptide conjugation. The molecular structure of these amphiphilic block copolymers is based on polypropylene oxide central blocks and side blocks of polyethylene oxide. The Pluronics are linear triblock copolymers, whereas Tetronic are four-arm block copolymers. Water solutions of these polymers were prepared to characterize the reverse thermal gelation (RTG) phenomena. The unique performance of these block copolymers is well controlled by the molecular weight of the polymer, its hydrophilic/hydrophobic ratio, and the polymer concentration in the water solution. Initially, the reverse thermal gelation of Pluronic F127 (12.6 kDa), a well-investigated linear amphiphilic block copolymer, was investigated and compared to Tetronic polymers at different molecular weights: Tetronic T1107 (15.0 kDa), 1307 (18.0 kDa), and T908 (25.0 kDa). These polymers differ not only by their total molecular weight but also with their hydrophilic/hydrophobic ratio of 70% (mol) polyethylene oxide

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Figure 1. Elasticity moduli (G′) of the following 30% (w/v) polymer-water solutions at various temperatures: Tetronic 1307 (solid rhombus), Tetronic 1107 (empty squares), Tetronic 908 (triangle), and Pluronic F127 (circle). Note that at 37 °C the elasticity moduli of T1307 and T1107 are 34000 and 38000 Pa, respectively, whereas the elasticity modulus of F127 is 19,000 Pa and that of T908 is only 160 Pa. The initial gelation temperatures for T1307 and T1107 are 20.1 and 22.2 °C, respectively, and those for F127 and T908 are 13.6 and 36.3 °C, respectively. Scheme 2. Modification of the Poloxamine Tetronic 1307 to T1307-tetra-methacrylate

in T1107 and T1307 and 80% in T908. A rheological analysis was performed to determine the storage modulus (G′) at a temperature range from 5 to 55 °C. The sharp increase in G′ above the lower critical solution temperature (LCST) clearly reveals the phase change of the various systems, from liquid polymer solution to a sturdy gel. Figure 1 displays the results of the rheological analysis of different polymer-water solutions and demonstrates that at 37 °C, 30% (wt/vol) polymer-water solutions of tetronic 1307 and tetronic 1107 have G′ values of 34000 and 38000 Pa, respectively. The initial gelation temperature (Ti) was found to range between 20 and 24 °C, respectively. In addition, tetronic 908 at the same concentration displayed the RTG transition at a temperature higher than 37 °C. This rheological behavior could be related to the higher hydrophilic segment, affecting its enhanced solubility over a wider temperature range. As a comparison, F127 at the same concentration displays lower elasticity modulus value (19000 Pa) as well as lower gelation temperature (13.6 °C). 3.2. Cross-Linked Polymers. The enhanced rheological properties of these thermoresponsive polymers were still not sufficient as a sturdy cell substrate; therefore, much-improved mechanical properties were further achieved by covalently cross-

linking the hydrogel. The synthetic route taken in this study yielded reverse thermoresponsive gels that allow simultaneous cross-linking as well as peptide anchoring sites for further cell attachment. To achieve these requirements, multiblock tetronic copolymers were end-capped with a carbon-carbon double bond enabling rapid cross-linking by free radical polymerization in water solution while warming to body temperature and forming the added feature of a physical gel. In addition, peptide conjugation to the polymer can be achieved by Michael-type addition of cysteine containing peptides to the unsaturated double bond. Two pathways were chosen for the polymer modification. The first, a hydrolytically sensitive conjugated using methacrylic double bond attached to ester bond, and the second is a hydrolytically stable maleimide group, conjugated using an urethane bond. Tetronic-tetra-methacrylate derivatives were obtained by the reaction of the hydroxyl end groups with methacryloyl chloride. Scheme 2 shows the derivation of Tetronic 1307 using methacryloyl chloride in an organic solution, making a methacrylate end-capped four-arm polymer. Documentation of the reaction was obtained using 1H NMR analysis and FT-IR spectroscopy (data not shown).

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Figure 2. Elasticity moduli (G′) of cross-linked 30% (w/w) hydrogels containing T1307-tMA and F127-dMA. The G′ for the F127-dMA and T1307-tMA were 391500 and 302800 Pa, respectively. The G′ for the sample polymers were 19000 and 34000 Pa, respectively. The measurement was conducted for five different samples of each composition and each sample was measured over 36 sampling points. The statistical differences between the groups was determined by ANOVA, p < 0.01. Scheme 3. Conjunction of 4-(Maleinimido)phenyl-isocyanate (PMPI) to the Hydroxyl End-Group of the Polymer

Rheological data demonstrated that the presence of the four methacrylate terminal groups only slightly affected the Tetronic rheological performance. The native Tetronic 1307 exhibited a very similar elastic modulus temperature curve to that of the noncross-linked Tetronic 1307-tetramethacrylate (T1307-tMA). The T1307-tMA was then cross-linked in an aqueous solution by using the APS/TEMED redox catalytic pair, following the methods proposed by Sawheny et al.61 The modified Tetronic T1307-tMA water solutions were cross-linked and the elasticity modulus (G′) was compared to the cross-linked F127-dMA hydrogels. Figure 2 shows the elasticity modulus of a 30% (w/v) polymer at 37 °C, as measured in oscillation test. The G′ of the cross-linked F127-dMA hydrogel was 391500 Pa, whereas the four function Tetronic polymer displayed a slightly lower G′ of 302800 Pa. This might be explained by a well-packed structure of the F127-dMA polymer, forming a stiffer hydrogel. These findings led us to compose the final hydrogel as a mixture of F127-dMA and peptide-modified T1307-dMA-d-peptide, providing the means of fine-tuning the peptide concentration while gaining the desired mechanical properties. After demonstrating the peptide addition to the polymer, a more hydrolytic stable peptide conjugation group was developed using the hetero-bifunctional p-maleimidophenyl-isocyanate (PMPI). The reaction between the polymer’s hydroxyl end-group and the isocyanate forms a hydrolytically stable urethane bond. The reaction (Scheme 3) was performed in a polymer melt (80 °C) using Sn[Oct]2 catalyst. The yielded Tetronic-tetra-maleinimido-phenyl-isocyanate (T307-t-PMPI) was purified by recrystallization twice. Documentation of the reaction was verified by performing a 1H NMR analysis (data not shown). The PMPI maleimide group allows stable peptide conjugation. Additional

designing flexibility can be obtained by allowing the nonpeptide modified maleimide group to further cross-link with the hydrogel via radical polymerization. 3.3. Peptide Anchoring. Different peptide sequences were considered for conjugation to the polymers. Kessler et al.42 showed that a variety of RGD-containing peptides with different spacers and end-groups contribute to successful cell attachment. The RGD-based peptide sequences that were synthesized, CGRGDSY and GCGRGDSPG, contain features such as spacers and end-group amino acids. Another important feature is that these sequences contain cysteine (C), such as its thiol side group, that may react with the polymer’s double bond by a Michael addition to produce the peptide conjugation. The Michael-type addition reaction was performed in 0.1 M bicarbonate buffer, pH 8.5, following nitrogen bubbling that discards oxygen traces and prevents sulfur S-S bond formation.54 The reaction was performed successfully both for the methacryl bonds and for the maleimide ring. The conjugated peptide-polymers were further analyzed by performing H1 NMR (data not shown). The rheological performance of the polymer water solutions before and after peptide conjugation was determined using the rheometer by oscillation test over the temperature range 5 to 50 °C (Figure 3). This analysis revealed that the reverse thermoresponsiveness of the polymers was not altered. Only minor changes occurred in the initial gelation temperature, which decreased from 20.5 °C for F127-dMA (not cross-linked) to 15.7 °C for the peptide-modified F127. The elastic modulus at 37 °C decreased slightly to 11000 and 9700 Pa for the uncrosslinked F127-dMA and the peptide-modified F127-dpeptide, respectively. The cysteine containing peptide addition to PMPI-modified polymer T1307-t-PMPI was controlled by the relative peptide



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Figure 3. Elastic modulus (G′) of 20% (w/w) water solutions containing F127-dMA and F127-d-CGRGDSY. Scheme 4. Michael Addition of One of the Peptide Sequenced CGRGDSY to the Modified Polymer Previously Performed with (Maleinimido)phenyl-isocyanate (PMPI)

concentrations with respect to the polymer reactive groups in the reaction vessel. Scheme 4 depicts the peptide-modified polymer structure, which is T1307-t-PMPI-n-peptide (n ) 0-4). The ability to cross-link was inhibited under radical polymerization, creating a stable hydrogel at all temperatures. Water solutions of peptide modified hydrogels at different concentrations were prepared. The peptide concentration was manipulated both by the polymer conversion and by the peptide-modified polymer ratio to the nonpeptide carrying polymer in the solution. 3.4. High-Resolution Scanning Electron Microscopy (HR-SEM). HR-SEM was used to determine the semi-threedimensional (semi-3D) structure of the cells on top of and within the hydrogel. Prior to the analysis, the samples were fixed, lyophilized, and sputter-coated for 60 s using Pd/Au. Initially, the optimal polymer concentration was determined by varying the polymer concentration from 10 through 15, 20, and 25% (w/v), while keeping constant peptide concentration of 0.2 mM (Figure 4). The range of polymer concentration affected the rheological properties and the hydrogel porosity. We found that 15% (w/v) polymer enabled the optimal supportive environment for cell growth, providing a structure that is sturdy enough for cell attachment, while maintaining sufficient porosity and allowing cell growth and proliferation. As negative control, similar hydrogel compositions, without the peptides, were used.

Tet-off BMP2 cells were cultured for 1 week in the aforementioned 15% (wt/vol) hydrogels, with a peptide concentration of 0.2-0.6 mM. At a concentration of 0.2 mM, cell-to-matrix attachment was detected; this could be interpreted as a filopodial extension to the polymer (Figure 5A1,A2). A slight increase in peptide concentration to 0.3 mM made a bundle of cells interact with themselves and the polymer matrix (Figure 5B1,B2). At a peptide concentration of 0.5 mM, samples formed multiple interactions with the hydrogel that could suggest a good cell-matrix interaction, toward a beginning of ECM formation (Figure 5C1,B2). 3.5. Energy Dispersive X-ray Spectroscopy (EDS). Energy dispersive X-ray spectroscopy (EDS) was performed to distinguish between different entities of the cell-containing hydrogel samples. The beam spot was focused and obtained data on the bare hydrogel or the center of the uncoated cell. The normalized average values (n ) 3) of carbon (C), oxygen (O), and amine (N) for the cell compositions were 71.6 ( 3.6% (C), 23.0 ( 1.2% (O), and 5.4 ( 0.3% (N), while the hydrogel EDS values were 76.0 ( 3.7% (C), 21.0 ( 1.0% (O), and 3.0 ( 0.2% (N). The entities composition differed mostly by a higher amine/ carbon ratio of the cells with higher relative to carbon oxygen (32.1%) and amine (7.5%), as compared to the hydrogel composition of oxygen (27.6%) and amine (3.9%).

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Figure 4. Electron micrographs showing Tet-off BMP2MSCs in hydrogels. The total polymer concentrations are 10, 15, 20, and 25%. The polymers depicted consist of F127-dMA only in samples that did not contain peptide and F127-dMA + T1307-dMA-d-CGRGDSY in peptidemodified samples. All peptide-modified samples contain 0.2 mM peptide. The samples were fixed and lyophilized after 1 week. (A1) F127-dMA 10% (w/w; 10000×), (A2) F127-dMA 10% (w/w), CGRGDSY (0.2 mM; 10000×), (B1) F127-dMA 15% (w/w; 10000×), (B2) F127-dMA 15% (w/w), CGRGDSY (0.2 mM; 10000×), (C1) F127-dMA 20% (w/w; 10000×), (C2) F127-dMA 20% w/w, CGRGDSY (0.2 mM; 10000×), (D1) F127-dMA 25% (w/w; 10000×), (D2) F127-dMA 25% (w/w), CGRGDSY (0.2 mM; 10000×).

3.6. Cell Proliferation. A PicoGreen assay was performed to quantify dsDNA. The DNA content increased over time in the PMPI-CGRGDSY 0.3 mM group, showing a significant elevation between days 7 and 30. From this we inferred cell proliferation within the scaffold across time. In most other groups, the DNA content slightly decreased between day 3 and day 7 after seeding and then maintained the same level up to day 30. This dynamics suggests that some cell death occurred following seeding, but a steady state was reached at day 7 (Figure 6). On day 7, the DNA content in the F127-dMA groups was significantly higher than that in all other groups, with a 3.3- to 6.3-fold increase in DNA content in the F127-dMA 15%

group compared with all other groups, and a 1.7- to 3.2-fold increase in DNA content in the F127-dMA 15% on-top group compared with all other groups. On day 30, however, the DNA content in the PMPI-CGRGDSY 0.3 mM group was higher than that in all other groups and significantly higher than the DNA content in all but two groups, the CGRDSY 0.6 mM on-top group and the F127-dMA 15% on-top group. The DNA content in the PMPI-CGRGDSY 0.3 mM group was 4.9-fold higher than that in the control group (tet-off BMP2 cells cultured in a regular culture plate). The control groups formed a confluent monolayer by day 7, so that the analysis presents a DNA content comparison between the two-dimensional culture and the 3D



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Figure 5. Electron micrograph showing Tet-off BMP2MSCs and the peptide-modified hydrogel F127-dMA 15% + T1307-dMA-dCGRGDSY. The peptide concentrations are (A) 0.2 mM, A1 (5000×), A2 (10000×); (B) 0.3 mM, B1 (5000×), B2 (10000×); (C) 0.5 mM, C1 (5000×), C2 (10000×). Note that panels A1 and A2 show filopodial extensions, panels B1 and B2 show, in addition to filopodial extensions, an extracellular matrix (ECM), and panels C1 and C2 show multiple filopodial extensions (arrows), good cell-matrix interaction (arrows), and formation of ECM.

Figure 6. PicoGreen analysis of dsDNA in Tet-off BMP2MSCs in the peptide-modified hydrogels at 3 days, 1 week, and 1 month in culture. Changes in DNA content in the various groups over time. The data represent the average ( standard of five independent samples at each time point.

scaffolds. When we compared all groups, we noticed a 1.6- to 9-fold increase in DNA content in the PMPI-CGRGDSY 0.3

mM group on day 30. These data suggest that cell survival was elevated in this hydrogel.

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Figure 7. Metabolic activity measured by performing an AlamarBlue assay of peptide-modified hydrogels at 3 days, 1 week, and 1 month. Changes in metabolic activity in various groups over time. The data represent the average ( standard deviation of five independent samples for each time point.

3.7. Cell Viability. The metabolic activity of the cells was analyzed by performing an AlamarBlue assay. After three days in culture, the metabolic activity of the cells in all peptidemodified hydrogels was higher than that measured in unmodified hydrogels (in which no reduction of AlamarBlue was noted). The metabolic activity was lower in these groups than in the positive control samples (cells seeded without any hydrogel; Figure 7). After one week in culture, a decrease in the metabolic activity of most samples was noticed. This dynamics suggests that some cell death occurred following seeding but a steady state was reached at one week. After one month in culture, most hydrogel samples displayed a significant increase in AlamarBlue reduction, suggesting an elevation in cell metabolic activity over time. This increase was not noted in the positive control samples, probably due to limited growth area of the 2D culture control in comparison with the 3D experiential groups. A total of 30 days after seeding, a significant elevation (p < 0.01) in metabolic activity was noted in the PMPI-CGRGDSY 0.3 mM group compared with all other groups, with a 2.5- to 3.8-fold increase in AlamarBlue reduction in this group compared with the other groups. These data indicate that, over time, cell viability in the PMPI-CGRGDSY 0.3 mM group was better than in all other experimental groups, including the positive control samples.

4. Discussion In this study, we explored the feasibility of using several peptide-modified thermosensitive hydrogels as in situ forming scaffolds for skeletal tissue engineering. Evidently, the covalent bonds present in the cross-linked matrix prevent it from reverting to its soluble state once the temperature is reduced below the solution-gel transition. One should notice, however, that the thermoresponsiveness of these materials is important only at insertion time, when their enhanced injectability and full conformability play a key role. Once the solution is deployed at the site of performance and has gelled (as it adapts to body temperature), the RTG completes its initial clinical function. After this initial stage, loss of the reverse thermoresponsiveness of these matrices is immaterial. Having said that, the relatively high molecular weight between cross-link junctions allows the hydrogel to retain, although to a very limited extent, a residue of its RTG response, as discussed in our previous study.35

The synthesized peptides used in this study are RGD based, functioning as minimal peptide sequences necessary to promote cell attachment. Two different RGD peptide sequences were used: CGRGDSY and GCGRGDSPG. The peptide designs included cysteine (C) for thiol-mediated immobilization, allowing the thiol group to react with the unsaturated functional group of the polymer. Both peptides contain the GRGDS motif of fibronectin. While the first peptide contains tyrosine (Y) used for radioiodination, the latter peptide contains probably more receptor affine GRGDSP motif of fibronectin.42 Other amino acids were used as additional functional groups for cell attachment and as spacers enabling stereospecific cell attraction to the peptides. Improved peptide conjugation and rheological properties of the hydrogels were achieved by using Tetronic tetra-functional polymers. These polymer-water solutions displayed reverse thermoresponsiveness due to their unique structure of block copolymers of PEO and PPO at a specific ratio. The reaction design is based on a modified polymer that displays a stable covalent conjugation of peptides at high concentrations as well as cross-linking within the polymeric matrix. This concept was further investigated by using methacrylate end-groups incorporated within the polymer. A more stable peptide conjugation was accomplished using a heterobifunctional end-group of p-maleimidophenyl-isocyanate (PMPI). The PMPI reacted with the hydroxyl group of the polymer, forming a stable urethane bond and a maleimide group allowing both peptide conjugation and radical cross-linking. The PMPI molecule allowed for very stable peptide conjugation with enhanced efficiency. Sample preparation included combining different peptide concentrations with a constant polymer concentration in hydrogels. In this way, the rheological properties of the hydrogel were constrained and the peptide functionality varied. Various analytical tools were used to analyze cell-hydrogel interactions, including electron microscopy with X-ray spectroscopy, as well as cell activity and DNA content. Our data indicate that improved cell attachment and proliferation inside the 3D hydrogel matrix was first present at a peptide concentration of 0.2 mM. The polymer concentration that provided optimal rheological properties and cell attachment was determined to be 15% (w/v), which provided a supportive environment for


cell growth. The sturdy structure of the hydrogel provided sufficient mechanical properties needed for cell attachment and its bulk matrix facilitated cell proliferation. When the 15% (w/ v) polymer hydrogel containing 0.3 mM peptide was analyzed and compared with a standard culture plate used as a positive control, we noticed comparable ratios of cell survival throughout the entire experiment; the shape of the cell was typically spherical and quite a few cell-cell and cell-matrix interactions were evident after 1 week in culture. As the peptide concentration was raised to 0.5 mM and 0.6 mM, cell aggregates formed, displaying excessive cell-cell and cell-matrix interactions. It is important to note, however, that cell-matrix interactions were not ideal, probably due to the abundance of water and PEG in the matrix. Further improvements in material design should be performed in order to improve peptide presentation to the cells. The metabolic activity of the cells in the hydrogels was examined using AlamarBlue reagent, and the DNA content was determined using the PicoGreen assay. DNA content of control cells (grown in monolayer) was lower than DNA content found in most of the other culture conditions tested, probably due to space limitations in the 2D culture. Metabolic activity, however, was similar in most of the groups, indicating that cells were active in the confluent monolayer, up to 30 days post seeding. After 1 week, all of the peptide-modified hydrogel groups displayed AlamarBlue reduction (consistent with higher cell metabolic activity), whereas no reduction was noticeable in the unmodified hydrogels. In addition, the results of the PicoGreen assay demonstrated higher DNA content in peptide-modified hydrogel groups than in all other groups on day 7. From this data we can infer that in the peptide-modified groups, cell death occurred between days 3 and 7, at which time point a steady state was reached, whereas in the unmodified groups, cells proliferated between day 3 and day 7 and began to die after that time, as can be deduced from the significantly lower DNA content found in these groups on day 30. One exception to this dynamics was the PMPI-CGRGDSY 0.3 mM group. In this group, the DNA content and AlamarBlue reduction significantly increased with time from day 7 to 30. This finding indicates that following an initial period of cell death, the remaining cells proliferated and were active in this hydrogel. On day 30, both the metabolic activity (measured by AlamarBlue reduction) and the DNA content were significantly higher in the PMPICGRGDSY 0.3 mM group than in almost all other groups. The data especially denote PMPI-CGRGDSY 0.3 mM as a material that contains hydrolytically stable peptide-anchoring group, and might suggest better efficiency and supporting for cell survival and activity over a prolonged culture period. When a comparison was done between the peptide containing groups to the F127dMA groups it seems that while DNA content was higher in F127-dMA groups than in most of the peptide groups (except for the PMPI-CGRGDSY 0.3 mM group), metabolic activity was similar, again with the exception of the PMPI-CGRGDSY 0.3 mM group. It is also interesting to note that the linker group of the peptide influenced strongly on cell survival through time, as noted from DNA results at day 30. The hydrolytically stable peptide-anchoring group found in the PMPI-CGRGDSY 0.3 mM group could explain this observation, but a thorough investigation should be done to elucidate the reason for this phenomenon. In addition, further characterization of peptide attachments and toxicity are due to fully reveal the influence of culture conditions on cell activity. Nevertheless, the data obtained in the present study support our claim of an advantage of peptide-modified hydrogels as an efficient tool for three-dimensional cell cultures and formation of in situ biologically functional implants.


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5. Conclusions Our results suggest that peptide-modified reverse thermoresponsive hydrogels and hydrolytically stable peptide anchoring (such as in the case of F127-di-PMPI-CGRGDSY 0.3 mM in particular) present an attractive modality for tissue engineering. These materials allow a minimally invasive approach and can carry and support cell survival and viability. Moreover, they form a sturdy scaffold in situ, providing a necessary temporary mechanical performance and sufficient support for cell proliferation as well as eventual tissue regeneration aimed at a full healing response and new tissue formation. Further investigation into the application of those hydrogels is required to analyze their efficiency in vivo. Based on the in vitro results, we envision that these materials will have a variety of applications for soft and hard tissue engineering. Acknowledgment. This research was supported by research grants from the National Institutes of Health: R01AR056694 (G.P. and D.G.); the Telemedicine and Advanced Technology Research Center (TATRC), U.S. Army Medical Research and Materiel Command No. 08217008 (G.P. and D.G.); the Eshkol Scholarship for Ph.D. students, The Ministry of Science, Culture and Sport; State of Israel (N.K.) and the Foulkes Foundation Fellowship, London, U.K. (N.K.); Dean’s Excellence Fellowship for Ph.D. students, Faculty of Science, The Hebrew University of Jerusalem, Israel (S.G.).

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