Biomimetic modification of synthetic hydrogels by incorporation of adhesive peptides and calcium phosphate nanoparticles: in vitro evaluation of cell behavior

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Biomimetic modification of synthetic hydrogels by incorporation of adhesive peptides and calcium phosphate nanoparticles: in vitro evaluation of cell behavior
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  359www.ecmjournal.orgM Bongio et al  .  Biomimetic modi   fi cation of synthetic hydrogels   European Cells and Materials Vol. 22 2011 (pages 359-376) ISSN 1473-2262 Abstract The ultimate goal of this work was to develop a biocompatible and biomimetic in situ  crosslinkable hydrogel scaffold with an instructive capacity for bone regenerative treatment. To this end, synthetic hydrogels were functionalized with two key components of the extracellular matrix of native bone tissue, i.e. the three-amino acid peptide sequence RGD (which is the principal integrin-binding domain responsible for cell adhesion and survival of anchorage-dependent cells) and calcium phosphate (CaP) nanoparticles in the form of hydroxyapatite (which are similar to the inorganic phase of bone tissue). Rat bone marrow osteoblast-like cells (OBLCs) were encapsulated in four different biomaterials (plain oligo(poly(ethylene glycol) fumarate) (OPF), RGD-modi fi ed OPF, OPF enriched with CaP nanoparticles and RGD-modi fi ed OPF enriched with CaP nanoparticles) and cell survival, cell spreading, proliferation and mineralized matrix formation were determined via cell viability assay, histology and biochemical analysis for alkaline phosphatase activity and calcium. This study showed that RGD peptide sequences promoted cell spreading in OPF hydrogels and hence play a crucial role in cell survival during the early stage of culture, whereas CaP nanoparticles signi fi cantly enhanced cell-mediated hydrogel mineralization. Although cell spreading and proliferation activity were inhibited, the combined effect of RGD peptide sequences and CaP nanoparticles within OPF hydrogel systems elicited a better  biological response than that of the individual components. Speci fi cally, both a sustained cell viability and mineralized matrix production mediated by encapsulated OBLCs were observed within these novel biomimetic composite systems. Keywords : Synthetic polymers, biomimetic hydrogels, RGD peptide, calcium phosphate nanoparticles, cell-material interactions, in vitro  mineralization.*Address for correspondence:John A Jansen, DDS, PhD Radboud University Nijmegen Medical Center Department of Biomaterials (309)PO Box 91016500 HB NijmegenThe NetherlandsPhone: +31-24-3614006Fax: +31-24-3614657E-mail: Introduction Over the last decades, a more re fi ned understanding of the biological properties relevant to bone substitute materials encouraged the scienti fi c community to develop increasingly powerful scaffolds that are able to tolerate and support the damaged tissue, to interact with the  physiological environment, and even to guide new bone formation (Bongio et al. , 2010). Among these, hydrogels are currently being broadly explored for bone tissue engineering applications. Hydrogels are hydrophilic networks of synthetic or natural polymer chains that can approximate the viscoelastic properties of native tissue (Peppas et al  ., 2006). Due to these and other distinctive features, such as biocompatibility, biodegradability, injectability and ability to release entrapped drugs, these  polymeric hydrogels are generally recognized as highly attractive candidates for use in rengenerative medicine. Oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels have been explored as synthetic matrices for  bone and cartilage tissue engineering applications (Guo  et al. , 2010; Park   et al. , 2005; Park   et al. , 2007; Temenoff   et al. , 2004b). OPF polymers consist of two repeating units,  poly(ethylene glycol) (PEG) and fumaric acid, which are alternately linked by ester bonds and can be degraded in aqueous solutions (Shin et al. , 2003). Although many advantages for this material exist, including versatile swelling characteristics and tailorable structural as well as mechanical properties (Brink   et al. , 2009; Guo  et al. , 2010; Holland  et al. , 2003; Holland  et al. , 2005; Jo  et al. , 2001b; Temenoff   et al. , 2002; Temenoff   et al. , 2004a), the purely synthetic components of OPF do not possess biospeci fi c cell adhesion sites, which limits cell-material interactions and potentially leads to anoikis ( i.e ., cell death due to lack of matrix interactions) of anchorage-dependent cells (Salinas and Anseth, 2009). To overcome such a drawback, biomimetic approaches have  been developed to functionalize synthetic matrices with  bioactive molecules, such as growth factors or adhesive  peptide sequences. Over the past decades, the tri-amino acid sequence RGD (arginine-glycine-aspartic acid) has  been identi fi ed to be the principal integrin-binding domain  present within adhesive proteins of the extracellular matrix (ECM), including fi  bronectin, vitronectin, fi  brinogen and osteopontin (Barczyk   et al. , 2010; Pierschbacher and Ruoslahti, 1984) . More recently, immobilization of “pro-adhesive” ligands on cell repellent surfaces has been shown to be pivotal in the interaction with neighboring cells and regulation of cell functions associated with regeneration of lost or damaged tissues (Bellis, 2011). BIOMIMETIC MODIFICATION OF SYNTHETIC HYDROGELS BY INCORPORATION OF ADHESIVE PEPTIDES AND CALCIUM PHOSPHATE NANOPARTICLES:  IN VITRO  EVALUATION OF CELL BEHAVIOR M. Bongio 1 , J.J.J.P. van den Beucken 1 , M.R. Nejadnik  1 , S.C.G. Leeuwenburgh 1 , L.A. Kinard 2 , F.K. Kasper  3 ,A.G. Mikos 2,3  and J.A. Jansen 1, * 1 Department of Biomaterials, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands 2  Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas, USA 3 Department of Bioengineering, Rice University, Houston, Texas, USA  360www.ecmjournal.orgM Bongio et al  .  Biomimetic modi   fi cation of synthetic hydrogels Shin and co-workers have already shown the potential of RGD peptides tethered to OPF-based hydrogels to improve cell adhesion and hence to control cell-functions. More speci fi cally, cell migration, osteogenic differentiation and mineralization were modulated by varying either the tether  peptide concentration or the nature of the peptide sequence (Shin et al. , 2002; Shin et al. , 2004b; Shin et al. , 2004a). Taking inspiration from the mineral component and nanostructure of natural bone, which itself is a nanocomposite, we have previously combined OPF hydrogels with calcium phosphate (CaP) in the form of carbonated apatite nanocrystals (Leeuwenburgh et al. , 2007). The major advantage for developing a composite material is the possibility of combining multiple properties associated with the natural tissue. While the polymeric network resembles the architecture of natural ECM, and hence acts as a supportive environment for cell functions (Drury and Mooney, 2003), homogeneously dispersed CaP nanoparticles render the resulting composite osteoconductive, encourage protein adsorption (Kilpadi  et al. , 2001) and increase matrix stiffness, thereby exerting strong effects on lineage speci fi cation and commitment of naive mesenchymal stem cells (Engler   et al. , 2006). To date, only few attempts to design a biomimetic hydrogel by means of combining adhesive peptides and nanosized CaP particles to elicit an osteogenic response from precursor cells have been reported (Paxton  et al. , 2009; Xu  et al. , 2010). The overall objective of the present study was to assess the biological behavior of encapsulated  bone marrow derived osteoblast-like cells (OBLCs) within a novel three-dimensional (3D) OPF-based hydrogel system in vitro . Experimental groups consisted of OPF hydrogels either modi fi ed with GRGD (glycine-arginine-glycine-aspartic acid), CaP nanoparticles or a combination thereof (Fig. 1a,b). Speci fi c objectives of this study were (1) to assess whether GRGD peptides affect cell spreading and hence cell viability, (2) to investigate the role of CaP nanoparticles in potential biomimetic mineralization of OPF-based hydrogels, thus stimulating encapsulated OBLCs to mineralize their environment, and fi nally, (3) to evaluate the potential combined effect between tethered GRGD peptides and CaP nanoparticles on the overall  biological performance of the scaffolds compared to plain OPF, with the ultimate goal of developing biomimetic composite systems for bone tissue engineering. Materials and MethodsReagents and culture supplements Fumaryl chloride, phosphoric acid (H 3 PO 4 ) and calcium hydroxide (Ca(OH) 2 ) were obtained from Acros (Pittsburgh, PA, USA). Poly(ethylene glycol) (PEG) was purchased from Aldrich (Milwaukee, WI, USA). Poly(ethylene glycol) diacrylate (PEGDA, MW 3400 Da) was purchased  by Glycosan Biosystems (Salt Lake City, UT, USA). GRGD peptide (MW 403.40 Da) was obtained from Bachem (Torrance, CA, USA). Acrylate-PEG-succinimidyl carboxymethyl (acrylate-PEG-SCM; MW 3400 Da) was  purchased from Laysan Bio (Arab, AL, USA).  N  ,  N  ,  N  ΄  ,  N  ΄  -tetramethylethylenediamine (TEMED) was obtained from Fluka (Buchs, Switzerland). Ammonium persulfate (APS), ninhydrin reagent, phosphate-buffered saline, pH 7.4 (PBS)  , Dulbecco’s modi fi ed Eagle medium (DMEM), fetal  bovine serum (FBS), dexamethasone, β -glycerophosphate, ascorbic acid, fungizone, ampicillin, gentamicin, trypsin-EDTA, and 10 % neutral-buffered formalin were purchased from Sigma-Aldrich ( St. Louis, MO, USA) . OPF synthesis OPF macromer was synthesized using fumaryl chloride and poly(ethylene glycol) (PEG) with an initial molecular weight of 10,000 g/mol (designated OPF 10K), following established procedures (Jo  et al. , 2001b). The puri fi ed macromer was stored at -20 °C. Synthesis of acrylate-PEG-peptide conjugates Synthesis of acrylated peptide was conducted by reacting GRGD peptide with acrylate-PEG-SCM, as previously described (Hern and Hubbell, 1998). Speci fi cally, the  peptide was dissolved to a fi nal aqueous concentration of 1 mg/mL in 50 mM sodium bicarbonate buffer, pH 8.2. The acrylate-PEG-SCM was dissolved separately in 50 mM of  bicarbonate buffer in a scintillation vial, such that the fi nal molar ratio of acrylate-PEG-SCM to peptide was 2. The PEG solution (2 mL) was added dropwise to the peptide solution (10 mL) using a syringe pump positioned on a magnetic stir plate over 20 min (0.1 mL/min). The solution was reacted at room temperature for 2.5 h and dialyzed using 2000 MWCO dialysis membranes (Slide-A-Lyzer Dialysis cassette, Thermo Scienti fi c) for 2 d using distilled deionized water with periodic changes. The primary goal of puri fi cation was the removal of non-acrylated peptide. The fi nal dialysis product was frozen in liquid nitrogen and lyophilized to remove water for 48 h, then stored at -20 °C until use. Characterization of acrylate-PEG-peptide conjugates The ef  fi ciency of peptide conjugation was determined  by the ninhydrin assay according to the manufacturer’s instructions. Briefly, working solution was freshly  prepared on the day of the assay by adding 4 M lithium acetate buffer (4.5 mL) in ninhydrin reagent solution (13.5 mL) and stirring under a nitrogen purge for 10 min. Five standards (range: 0-0.033 μ mol/mL) were prepared in 1.5 mL tubes from a stock solution of GRGD at a concentration of 0.05 μ mol/mL (50 μ M) in 0.05 % glacial acetic acid. Standards and samples (acrylated PEG-GRGD pre-dialysis and acrylated PEG-GRGD post-dialysis) were added to MilliQ H 2 O and ninhydrin reagent ( fi nal volume 2 mL) in 15 mL tubes. The tubes were immediately capped, gently mixed and heated in boiling water for 10 min to allow the reaction to proceed. After cooling, 5 mL of 95 % ethanol was added to each tube. Samples and standards (300 μ L) were transferred to a 96-well plate and run in triplicate. The absorbance of each solution was measured on a UV spectrophotometer at 570 nm, and the concentration of free GRGD peptide in the samples was calculated from the standard calibration curve.  361www.ecmjournal.orgM Bongio et al  .  Biomimetic modi   fi cation of synthetic hydrogels Table 1. Compounds and relative amounts used to prepare OPF hydrogel scaffolds.* Cell-laden hydrogels** Cell-free hydrogels CaP nanoparticles preparation Stable sus  pensions of homogeneously dispersed needle-like apatite nanoparticles were prepared using a conventional wet-chemical precipitation method. Speci fi cally, 25 mL of a solution of H 3 PO 4 (3.56 M) was added dropwise at a rate of about 1-2 drops per second into 25 mL of a basic suspension of Ca(OH) 2  (5.92 M), while stirring at a temperature of 60 °C. The amounts of reagents were used at a stoichiometric Ca/P ratio of 1.67 to obtain an apatite content of ~0.3 g/mL   (CaP 30 % w/v). The product was left to age for 15-18 h. After aging, the pH of the suspensions was adjusted to 7.4 and then autoclaved for sterilization. Since the aim of this study was to maximize the amount of CaP nanoparticles in the hydrogel composite, the CaP content was also maximized in the suspension up to a value of 30 % w/v. At higher CaP contents, stable and homogeneous suspensions could not be obtained anymore using the described reaction. Characterization of CaP nanoparticles Prior to characterization, the prepared CaP nanoparticles were washed with ultrapure water in three subsequent series of centrifugation and rinsing steps. Subsequently, the nanoparticles were dried for X-ray diffraction analysis (XRD, Philips, PW 1830, Almelo, the Netherlands) and attenuated total reflection-Fourier transform infrared spectroscopy (ATR–FTIR, Perkin Elmer, Spectrum One, Groningen, the Netherlands). Specifically, XRD was  performed in a re fl ection geometry using a Cu K  α  radiation source (1.54056 Å) at a voltage of 40 kV and a current of 30 mA. Spectra were collected at a scanning rate of 0.01 °/s   in the 2 Θ  range of 20° to 60° (0.02° interval). ATR–FTIR was performed in the range of 500 to 2000 cm -1  at 22 °C. To record spectra with satisfying peak-to-noise ratio, 10 scans were averaged for each sample. Finally, transmission electron microscopy (TEM, Jeol, JEM 1010, Tokyo, Japan) was used to image the CaP nanoparticles at an acceleration voltage of 60 kV. In order to prepare samples for TEM, a drop of CaP suspension was deposited onto a TEM grid and left to dry in air. Bone marrow stromal cell isolation and preculture Primary cells were freshly isolated from 8 Fischer 344 male rats (6-8 weeks old, 150-165 g), as previously described (Peter et al  ., 2000).   Brie fl y, femora and tibiae were excised from the rats under aseptic conditions, and the bone marrow was fl ushed from the bone with primary media. Freshly isolated cells from two femora and two tibiae were seeded in three T-75 culture fl asks. The cells were  precultured for 6 d in DMEM supplemented with 10 % v/v FBS, 10 -8  M dexamethasone, 10 mM β -glycerophosphate, 50 mg/L ascorbic acid, 250 μ g/L fungizone, 100 mg/L ampicillin, and 50 mg/L gentamicin, at 37 °C, 95 % relative humidity and 5 % CO 2  to obtain osteoblast-like cells (OBLCs). Non-adherent cells were removed and the media were refreshed three times per week. After 6 d, the OBLCs were washed twice with PBS, enzymatically lifted with exposure to 0.25 % trypsin-EDTA for 5 min, and resuspended in osteogenic medium. OBLCs encapsulation and culture OPF, PEGDA and acrylated-PEG-GRGD (PEG-R) were sterilized by exposure to ethylene oxide for 14 h and  physically mixed as outlined in Table 1. Hydrogels were fabricated using 2 μ mol of PEG-R per mL of precursor solution. This concentration was demonstrated to be optimal for cell proliferation, alkaline phosphatase (ALP) activity and mineralized matrix formation in previous studies (Shin  et al. , 2005; Shin  et al. , 2004b). The approximate ratio of the double bonds in PEGDA or PEGDA plus PEG-R to those in OPF was 6. Subsequently, either sterile PBS (for CaP-free hydrogels) or CaP suspension (for CaP-enriched hydrogels) was added to the polymers, and the mixtures were shaken at room temperature for 30 min. The polymer/CaP ratio was calculated to be 50/50 w/w. Equal volumes of sterile initiator solutions, 300 mM APS and 300 mM TEMED, were added. Next, 10 million cells/mL were added to the OPF solution to prepare cell-laden hydrogels. After gentle mixing, 30 μ L of the polymer mixture (containing 300,000 cells) was quickly injected SCAFFOLDSOPFOPF-ROPF-CaPOPF-R-CaP OPF (g) (g)0.0750.0660.0750.066PEG-R(g)-0.018-0.018PBS ( μ L)692692--CaP suspension ( μ L)--6926920.3 M APS ( μ L)707070700.3 M TEMED ( μ L)70707070Cell suspension*/culture medium** ( μ L)168168168168Total volume ( μ L)1000100010001000  362www.ecmjournal.orgM Bongio et al  .  Biomimetic modi   fi cation of synthetic hydrogels in pre-sterilized Te fl on molds, followed by incubation at 37 ºC for 30 min. Previous studies indicated that RGD is successfully incorporated into the gel network using the described method (Jo  et al. , 2001a; Shin  et al. , 2005). The newly formed cross-linked gels were transferred to 24-well  plates. 2 mL of medium including osteogenic supplements was added and changed 1 h and 1 d after encapsulation to ensure the removal of unreacted components. During the culture period, the medium was changed three times  per week to prevent nutrient exhaustion. Constructs were collected after 4, 8, 12, 16 and 24 d in culture following initial seeding. In addition, scaffolds without cells were made as described above, except for the fact that culture medium was added instead of the cell suspension. These cell-free hydrogels, soaked in complete osteogenic medium, were subjected to the same analyses as cell-laden hydrogels in order to be used as background value (blank) and to evaluate the cell-mediated calcium accumulation in cellular gels compared to the passive mineralization in acellular gels. Hydrogel composite characterization Cell-free hydrogels from OPF and OPF-CaP groups ( n  = 3) were used for material characterization analyses. First, the hydrated samples were subjected to rheological studies using an AR2000ex rheometer (TA Instrument, New Castle,  NJ, USA) equipped with a fl at, steel-plate geometry (20 mm diameter) at 22 °C. Storage modulus (G´) and loss modulus (G´´) were determined employing an oscillatory time sweep test for 10 min at a constant strain of 1 % and constant frequency of 1 Hz. Analyses of the results revealed that the effects of the evaporation on measurements were negligible given the low temperature and short duration of the experiment. In addition, samples were freeze-dried (for 48 h) and coated with a thin gold layer for Field Emission Scanning Electron Microscopy (FESEM, Jeol6330, Tokyo, Japan)  performed at 5 kV. Separate samples were used to collect ATR-FTIR spectra. Furthermore, the fold swelling ratios of the hydrogels (swollen in MilliQ H 2 O overnight) were calculated accord ing to the following formula:  Fold swelling = (Ws-Wd)/Wd (1)Ws and Wd are the weight of the disc in swollen and dry (i.e. lyophilized) states, respectively. Cell survival Cell survival was determined using a LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR, USA), which measures the membrane integrity of cells. Constructs were washed in sterile PBS for 30 min at 37 °C prior to incubation for 30 min at room temperature with 2 mM calcein-AM and 4 mM ethidium homodimer in PBS solution. After incubation, constructs were rinsed again in PBS for confocal microscopy. Cell viability was assessed after 4 and 16 d of culture. Percentage of live cells was quanti fi ed using Image J software (Zeiss  LSM  Image Browser  , Version, Jena, Germany) for a minimum of four images from two samples for each condition. Cell behavior At days 4, 8, 12, 16 and 24, samples and blanks were washed in PBS for 30 min at 37 °C and homogenized with a pellet grinder (Kontes Pellet Pestle, Daigger, Vernon Hills, IL, USA) in 1000 μ L double distilled water. The samples were frozen at -20°C before analysis. At the time of analyses, samples were subjected to three freeze-thaw cycles including sonication with ice for 10 min after each cycle. A total of n  = 3 replicates per time point for each experimental condition were used.  Proliferation  Double-stranded DNA content from all homogenates at each time point was measured using a fl uorometric PicoGreen DNA kit (Molecular Probes, Junction City, KS, USA) following the protocol of the manufacturer. Brie fl y, 100 μ L of samples or DNA standards were incubated with 100 μ L of PicoGreen working solution and allowed to incubate at room temperature for 10 min in the dark. The excitation of the solution at 485 nm and fl uorescence measurement at 530 nm was performed using a FLx800 fl uorescence microplate reader (Bio-Tek Instruments, Winooski, VT, USA). The values of cell-free hydrogels were subtracted from the sample values of corresponding cell-laden hydrogels.  Differentiation ALP activity was measured by using Sigma diagnostic kit 104, according to instructions of the manufacturer. Brie fl y, 80 μ L of sample and 20 μ L of alkaline buffer were added to 100 μ L of substrate solution (5 mM paranitrophenyl  phosphate). A standard curve was made (range: 0-25 nmol/mL) of the stock solution (4-nitrophenol). Samples and standards were added to a 96-well plate and incubated at 37 °C for 60 min. The reaction was terminated by the addition of 100 μ L of 0.3 M NaOH to each well. The absorbance of each well was read at 405 nm using a PowerWave X340 microplate spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA). Absorbance of the samples was adjusted with cell-free hydrogels and the values were normalized to the amount of DNA.  Mineralization Before analysis of calcium content for each homogenate, 1.0 N acetic acid was added to the samples and placed on a shaker table overnight to dissolve mineral deposits. A volume of 20 μ L samples or standards were incubated with 300 μ L of working solution (Genzyme Diagnostics, Cambridge, MA, USA). The standards (range: 0-100 μ g/mL) were prepared using a CaCl 2  stock solution. The absorbance of each well was measured at 570 nm on a PowerWave X340 microplate spectrophotometer (Bio-Tek Instruments). The readout values of cell-containing gels were compensated for the calcium uptake as measured for cell-free hydrogels. Cyt ological Analysis Two specimens ( n  = 2) for each group at day 4, 16 and 24 were fi xed in 10 % neutral-buffered formalin, serially dehydrated in ethanol (35 % and 70 %), paraf  fi n-embedded, and cut on a microtome in 6 μ m-thick cross sections. The  363www.ecmjournal.orgM Bongio et al  .  Biomimetic modi   fi cation of synthetic hydrogels sections were subsequently mounted on glass slides and stained with Von Kossa reagent to visualize the mineralized matrix and counterstained with Nuclear Fast Red. The resulting slides were imaged with a light microscope (Axio Imager Microscope Z1, Carl Zeiss Micro imaging GmbH, Göttingen, Germany) equipped with a digital camera. Roundness Roundness index was used to evaluate cell spreading of encapsulated cells, according to a previously described method (Kim et al  ., 2010). The analysis was based on Von Kossa-stained histological sections (2 specimens per experimental group, 2 histological sections per specimen, 3 views per histological section) at day 4, 16 and 24. A minimum number of 50 cells were counted per histological section. The roundness of cells was computed (Leica Qwin Pro-image analysis software; Leica imaging system, Cambrige, UK) by the formula: Roundness index = Perimeter 2 / 4 x π  x Area x 1.064 (2)Perimeter is the total length of the boundary of the cell and Area is the total number of pixels within the cell. The adjustment factor of 1.064 corrects the parameter for the effect of the corners produced by the digitization of the image. This equation gives a minimum value of 1 for a true circle. Therefore, the roundness index of a cell with less cellular extensions is closer to 1. On the contrary, the cells with more cellular extensions have a roundness greater than 1. Statistical analysis Analysis of quantitative data consisted of one way ANOVA combined with a post-hoc Tukey-Kramer Multiple Comparisons Test to detect statistically signi fi cant differences at a signi fi cance level (or  p -value) of  p  < 0.05. Results are reported as means ± standard deviations. Results To test the reproducibility, the entire experiment was  performed for two independent runs with a separate isolation of pooled cells for each run. Both experimental runs showed similar results. Fig. 1. (a)  Schematic drawing of OBLCs encapsulated in OPF hydrogels involved in this study, and (b)  representative images of CaP-free (OPF and OPF-R) and CaP-enriched (OPF-CaP and OPF-R-CaP) hydrogels.
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