Control over the Gradient Differentiation of Rat BMSCs on a PCL

Article pubs.acs.org/Biomac

Control over the Gradient Differentiation of Rat BMSCs on a PCL Membrane with Surface-Immobilized Alendronate Gradient Yang Zhu,†,§ Zhengwei Mao,† and Changyou Gao*,†,‡ †

MOE of Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China ‡ State Key Laboratory of Diagnosis and Treatment for Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China S Supporting Information *

ABSTRACT: Gradient biomaterials can offer progressively changing signals to specific tissue interface, and thereby modulate the conjunction between different tissues. A linear density gradient of alendronate (Aln), a molecule that is capable of promoting osteogenic differentiation of bone mesenchymal stem cells (BMSCs), was created on an aminolyzed poly(ε-caprolactone) (PCL) membrane. X-ray photoelectron spectroscopy and quartz crystal microbalance with dissipation revealed the linear increase of the Aln amount as a function of the position on the PCL membrane. By contrast, the surface wettability and energy were kept unchanged. The surface-grafted Aln showed a stronger ability to induce the osteogenic differentiation of rat BMSCs than its counterpart in culture medium of the same amount, and the osteo-inductive culture medium. On the Aln-grafted gradient surface, the BMSCs showed gradient osteogenic differentiation as a function of membrane position in terms of cell morphology, alkaline phosphatase activity, calcium deposition, and the expression of osteogenesis marker proteins including collagen type I (COL I), Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN).

1. INTRODUCTION The interface between tissues is characterized with gradually changing properties including types of residing cells and matrix components of complex hierarchical structures to fulfill specific functions. For example, at the interface of the epidermis and dermis lies the transition bilayered basement membrane, which binds to the epithelium and connective tissue.1 Within the musculoskeletal system, graded interfaces between bone and soft tissue facilitate the transmission of complex mechanical loads across limb joints by minimizing stress concentrations at the junction of two tissue types.2,3 The interface tissue engineering is a promising novel strategy aiming at repairing and regenerating graded tissue interfaces to restore native tissue architectures and functions, extending the applications to a more complex scenario.4 Current approaches mainly rely on the integration of multiple cell types or growth factor gradients within multilayered 3D scaffolds.5,6 Strategies based on laminated/layered constructs have restricted potentials for generating continuous, graded interfaces due to inherent discontinuities across the dissimilar materials.7 By contrast, the gradient biomaterials with gradually varying physiochemical properties are thought to be the ideal candidates to guide the formation of alternates for continuous tissue interfaces. Phillips et al. described a method of engineering continuous bone-soft tissue-mimetic interface via gradient gene transfer.8 A linear gradient in calcium phosphate content was obtained across the surface of the nanofiber mat by © 2012 American Chemical Society

Li et al., which can be potentially used in engineering the interfaces from tendon to bone.9 Shi et al. incorporated a protein gradient into the electrospun nanofibers via simple adsorption.10 For more strategies of gradient materials fabrication and their applications, please refer to the following reviews and articles.4,11−17 The versatile differentiation capacity of bone mesenchymal stem cells (BMSCs) into various lineages including osteocytes, chondrocytes, and adipocytes has drawn intensive attention and backed their extensive applications in tissue engineering.18−22 Previous studies have shown that the properties of materials exert great influence on BMSC differentiation in terms of direction and level.23−28 For example, BMSCs display characteristics of neurogenic, myogenic, and osteogenic phenotypes after being cultured on substrates mimicking the stiffness of neural, muscle, and bone tissues, respectively.29,30 In addition, the surface morphology of materials has a significant impact on BMSC differentiation too.26,31,32 Moreover, small functional groups (on silicon wafer and in poly(ethylene glycol) (PEG) hydrogels) as well as immobilized peptides and proteins can also modulate BMSC differentiation.24,33−37 These findings suggest the possibility of spatiotemporally modulating BMSCs fate by gradient physical, chemical, or Received: September 28, 2012 Revised: December 2, 2012 Published: December 11, 2012 342

dx.doi.org/10.1021/bm301523p | Biomacromolecules 2013, 14, 342−349

Biomacromolecules

Article

membrane was cut into pieces with diameters fitting the tissue culture plates (6-well, 24-well, and 96-well plates). The membranes were aminolyzed in 0.43 mol/L 1,6-hexanediamine/isopropanol at 30 °C for 1 min, 5, 15, and 30 min, respectively. To generate the −NH2 gradient on the PCL membrane, 1,6hexanediamine/isopropanol solution (0.43 mol/L) was continuously injected into a tube with a vertically fixed PCL membrane (10 mm ×40 mm) by a microinfusion pump (WZS-50F2, Zhejiang University Medical Instrument, China), as shown in Scheme S1. The injection rate was adjusted to precisely generate a 30 mm gradient in 30 min, and a water bath was used to maintain the temperature of solution in the tube at 30 °C. After aminolysis, the PCL membranes were rinsed with copious water for 4 h at room temperature to remove free 1,6hexanediamine, and then dried under a nitrogen gas flow. 2.3. Grafting of Aln. After the aminolyzed PCL membrane (including both the uniform and the gradient ones) was treated with 2% (v/v) glutaraldehyde (GA) solution at 37 °C for 4 h and then washed five times with water,16,48,49 it was immersed into 1% (w/v) Aln solution at 37 °C for 2 h. Via the coupling reaction between the derived aldehyde groups and −NH2 groups of Aln molecules, Aln molecules were covalently attached onto the PCL membrane (Scheme 1). Finally, the Aln-grafted membrane was washed five times in water. 2.4. Determination of Density of −NH2 and Aln. The ninhydrin assay was used to quantitatively determine the amount of −NH2 groups on the aminolyzed PCL membrane.48 Briefly, the membrane was placed into a glass tube containing 2 mL 0.1 mol/L ninhydrin/ethanol solution, and treated at 80 °C for 15 min to accelerate the reaction between ninhydrin and −NH2 groups. Eight milliliters of 1,4-dioxane was subsequently added into the tube to dissolve the membrane, and the absorbance at 538 nm was recorded on a UV−vis spectrophotometer (Shimadzu UV-2550, Japan) to calculate the density of −NH2 groups by referring to a calibration curve. To analyze the surface Aln density, X-ray photoelectron spectroscopic (XPS) spectra were recorded on an Axis Ultra spectrometer (Kratos Analytical, UK) employing Al Kα excitation radiation. The charging shift was referred to the C 1s line emitted from the saturated hydrocarbon (284.6 eV). The weight alteration of PCL membrane during Aln grafting was recorded by a quartz crystal microbalance with dissipation (QCM-D E4, Q-Sense, Sweden) according to the change of resonance frequency (Supporting Information). 2.6. Cell Culture. The bone marrow stem cells (BMSCs) were isolated from the bone marrow of young adult male Sprague−Dawley rats as previously described.21 The procedures were performed in accordance with the “Guidelines for Animal Experimentation” by the Institutional Animal Care and Use Committee, Zhejiang University. Briefly, the bone marrow cells were obtained from the femoral shafts of rats by flushing out with 10 mL of culture medium (low glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, New York, USA), 100 μg/mL penicillin and 100 U/mL streptomycin). The released cells were collected into a 9 cm cell culture dish (Corning, USA) containing 10 mL of culture medium and incubated in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. After the cells reached about 80% confluence, they were detached and serially subcultured. The BMSCs at passage 2 (P2) were used in this study. The pristine and Aln-grafted PCL membranes were sterilized in 75% ethanol for 1 h, followed with washes in phosphate buffered saline (PBS, pH 7.4) five times. The membranes were then placed in the wells of corresponding tissue culture plate. BMSCs were trypsinized and seeded into the wells at a density of 3 × 104 cells/cm2 and incubated at 37 °C under a 5% CO2 humidified atmosphere. If not otherwise mentioned, low glucose DMEM supplemented with 10% FBS was used as the culture medium, which was changed with fresh one every 3 d. The BMSCs seeded on the pristine membranes and cultured with osteogenic differentiation medium (Cyagen Biosciences, Guangzhou, China) were set as controls. 2.7. Cell Morphology. After 24 h culture the PCL membranes with BMSCs were washed twice with PBS and stained with fluorescein

biological cues. For example, Tse et al. prepared a hydrogel containing a physiological gradient of 1.0 ± 0.1 kPa/mm and found that the BMSCs cultured up to 21 d migrated up the stiffness gradients and then differentiated into a more contractile myogenic phenotype.38 Moore et al. prepared a density gradient surface of a peptide-encoding active sequence of bone morphogenetic protein 2 (BMP-2), and found that BMSCs overexpressed osteogenic marker proteins on the surface, positively correlating to the local BMP-2 peptide density.39 However, despite the successful concept-proving results achieved in those model systems, limited effort has been made on the biodegradable materials, which can be eventually applied in vivo for tissue regeneration hence are of great importance. Furthermore, the approaches based on the growth factors are severely limited by suboptimal immobilization chemistry, short protein half-life, and the cost prohibitive supraphysiologic concentrations required to initiate a cellular response.40,41 In this study we shall create a density gradient of robust small molecules on the surface of biodegradable poly(ε-caprolactone) (PCL) membrane in order to spatially modulate the differentiation of rat BMSCs (Scheme 1). Generation of the gradient Scheme 1. Aminolysis and Aln Immobilization on a PCL Membrane, and Influence on the Differentiation of BMSCs in a Gradient Manner

-NH2 groups is implemented by dynamically controlling the gradual prolongation of aminolysis time of the PCL membrane with an injection method (for more details of aminolysis, see refs 42−45). Alendronate (Aln), a small molecule that is able to promote osteogenic differentiation of BMSCs via MAPK pathway (Mitogen-activated protein kinase pathway, a chain of proteins which transduce extracellular signals into nucleus) in a dose-dependent manner,46,47 is conjugated via aldehydeactivated coupling to obtain the Aln density gradient on the PCL membrane. In vitro cell culture is finally conducted to reveal the gradient osteogenic differentiation of BMSCs on the gradient material. Here we focus on the efficacy of this strategy on the scale of the entire gradient, and shall pay no attention to investigate the influence of gradient on a scale comparable to a cell.

2. EXPERIMENTAL SECTION 2.1. Materials. PCL (Mw 80 kDa) was obtained from SigmaAldrich. Aln was purchased from Spectrum Chemicals (USA). Hexadecylpyridinium chloride was obtained from Aladdin (China). All other chemicals were of analytical grade and purchased from Sinopharm Chemical Reagent (China). The water used in the experiments was purified by a Milli-Q water system (Millipore, USA). 2.2. Aminolysis of PCL Membranes. The translucent PCL membrane with a thickness of 0.2 mm was prepared by casting a 10% PCL/1,4-dioxane solution into a glass Petri dish, which was covered with a lid, allowing solvent evaporation at 35 °C for 48 h. The 343

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Figure 1. (a) Surface density of amino groups on the PCL membrane as a function of aminolysis time or gradient position. (b) Grafting density of Aln on the uniform PCL membrane as a function of aminolysis time. Data were measured by QCM-D. (c) XPS spectra of P 2p on the Aln-grafted PCL membranes with variable aminolysis time. (d) Atomic concentration of P element on the Aln-grafted PCL membrane as a function of aminolysis time. The measured thickness is about 10 nm. 0.5% Triton at 4 °C for 10 min to increase the permeability of the cell membrane. After being rinsed three times with PBS, they were incubated in 1% BSA/PBS at 37 °C for 30 min to block the nonspecific interactions. Then the samples were divided into three groups and incubated with corresponding mouse monoclonal antibody (Abcam, USA) against COL I, Runx2, and OCN for 1 h, respectively. After being washed twice in 1% BSA/PBS, they were further stained with FITC-labeled goat antimouse IgG (Beyotime, Haimen, China) and 4′,6-diamidino-2-phenylindole (DAPI) (Sigma) at room temperature for 1 h, followed by three washes in PBS. The cells were observed under confocal laser scanning microscopy (TCS SP5 II, Leica, Germany). Cells in the captured images were analyzed with Image J (background subtracted, gray value of the green channel calculated). 2.11. Cell Adhesion Force. The average cell adhesion force of over 104 BMSCs on the pristine and Aln-grafted PCL membranes were determined by a centrifugation method as reported previously.15 2.12. Statistical Analysis. Experimental data from all the cell culture studies were analyzed using One-Way ANOVA with a Tukey posthoc method, n ≥ 3, with a significant level of p < 0.05. As supplement to ANOVA, a few comparisons were also made between individual groups with t-test, as pointed out in the text. Results are reported as mean ± standard deviation.

diacetate (FDA, Sigma-Aldrich). The morphology of the stained cells was observed under a fluorescence microscope (Axovert 200, Carl Zeiss, Germany). Image Pro Plus was used to quantitatively analyze the aspect ratio of BMSCs on the membranes. 2.8. Alkaline Phosphatase (ALP) Activity. The BMSCs were cultured on pristine and Aln-grafted PCL membranes for 1 and 7 d. The samples were washed with PBS three times and treated with 0.5% Triton/PBS at 4 °C for 24 h. The ALP activity was assayed with Alkaline Phosphatase Kit (KeyGEN Biotech). The total protein was determined using a BCA assay kit (KeyGEN Biotech). The physically adsorbed proteins on the membranes were subtracted from the total amount. The ALP activity per microgram of protein was reported. The BMSCs on the gradient PCL membranes (Aln density gradient) were washed with PBS twice and stained with a Leukocyte Alkaline Phosphatase Kit (Sigma) to reveal the relative ALP activity on the gradient. 2.9. Calcium Deposition. After 21 days of culture the samples were washed three times with PBS and stained in Alizarin Red solution (Cyagen Biosciences) at room temperature for 5 min. The stained samples were subsequently transferred into a new plate and washed three times with PBS. The adsorbed Alizarin Red was dissolved in 150 μL 10 wt % hexadecylpyridinium chloride/PBS solution. The absorbance at 570 nm was measured by a microplate reader (Model 680, Bio-Rad). 2.10. Expression Level of Osteogenesis-Related Proteins. Immunofluorescence assay was adopted to stain collagen type I (COL I), Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN). Briefly, after the BMSCs were cultured on the gradient PCL membranes for 21 days, they were carefully washed with PBS three times, and then fixed with 4% paraformaldehyde at 37 °C for 30 min, followed by three washes in PBS. The cells were further treated in

3. RESULTS AND DISCUSSION 3.1. Fabrication of Aln Gradient. The preparation methodology of surface-grafted Aln density gradient is illustrated in Scheme 1. Aminolysis is an effective surface modification method to introduce primary amine groups to polyester materials, providing versatile reactive sites for 344

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Biomacromolecules

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Figure 2. Morphology of BMSCs cultured on PCL membranes with Aln gradient at positions of (a) 1 mm, (b) 5 mm, (c) 15 mm, and (d) 30 mm, respectively. The cells were cultured on a single piece of membrane (10 mm ×30 mm) for 1 day and stained by FDA. (e) Aspect ratio of BMSCs on the Aln gradient surface as a function of gradient position. Scale bar, 200 μm.

subsequent functionalization.16,43,48,50 Here the 1,6-hexanediamine concentration was fixed at 0.43 mol/L since the resultant density of −NH2 groups is decreased with still higher concentration of 1,6-hexanediamine such as 0.86 mol/L due to the interplay between generation of −NH2 groups and erosion of the PCL membranes.51 Combined with an injection method, the 30 mm long −NH2 density gradient was first fabricated on the PCL membrane via the gradient aminolysis. Here the upper end with the smallest density of −NH2 groups is defined as the 0 position, and the total length of the membrane is fixed at 30 mm. Since 1,6-hexanediamine solution was injected into the tube in a constant and fast enough rate, and the reaction temperature was kept constant, the −NH2 density was supposed to increase in a linear pattern along the membrane.51 Figure 1a shows that the density of the primary amine groups almost linearly increased along with aminolysis time, i.e., the gradient position, confirming the good control over the quality of the aminolyzed membrane. The highest density of primary amine groups was achieved after 30 min of aminolysis on the end of the gradient surface. At each given reaction time (i.e., the gradient position), the −NH2 density on the gradient membrane is close to its counterpart on the uniformly aminolyzed one, which is consistent with the results of previous kinetic studies.51 The gradient −NH2 are then used to covalently graft Aln molecules, which are known to enhance BMSCs differentiation toward osteoblast-like cells and thereby the gradient differentiation of BMSCs on the Aln gradient (Scheme 1). One of the important advantages of this preparation strategy is that the reaction processes are basically thermodynamically controlled except for the initial aminolysis reaction, leading to the good control over the quality of gradients in different batches. The Aln grafting process on the uniformly aminolyzed PCL membrane with different time was first followed by QCM-D (Figure S1). Basically, the grafting mass of Aln increased continuously along with the reaction time in 2 h regardless of the surface density of −NH2 groups. Figure 1b shows that the obtained surface Aln grafting density increased with the aminolysis time almost in a linear trend, and reached 28, 55, 111, and 164 ng/cm2 on the PCL membranes aminolyzed for 1, 5, 15, and 30 min, respectively. Since the gradient aminolysis results in a similar −NH2 density with the corresponding uniform surface when the reaction time is same, it is reasonable to assume that the Aln densities on the gradient surfaces at 1, 5,

15, 30 mm are equal to their counterparts on the uniform surfaces. XPS was used to characterize the relative density of Aln molecules on the gradient surface. The characteristic peak of P element, which is solely attributed to the immobilized Aln, appeared near 131 eV on the XPS spectra of Aln-grafted PCL membranes, as shown in Figure 1c. Figure 1d reveals that the relative atomic concentration of P on the gradient surface (calculated from the peak areas) increased linearly along with the aminolysis time or the extension of position in the outermost ∼10 nm layer. All the results are consistent with each other regardless of the gradient or uniform given that the aminolysis time is the same. This enables the convenient use of the data measured on the uniform samples. Static contact angles (SCA) of different positions on the gradient surface were measured by using different liquids, upon which the corresponding surface energy was calculated (Table S1). It shows that the surface hydrophilicity (SCA around 78°) and surface energy (around 49 mN/m) did not vary significantly after Aln grafting. Although the aminolysis lowers down the water contact angle to some extent, after reaction with GA the value recovers to that of the pristine PCL again.51,52 In this regard, the similar value of surface energy would imply also that the immobilization of the Aln molecules does not have significant impact on the surface wettability. Moreover, no significant change was observed for the surface morphology either (Figure S2). Therefore, the possible impact of surface topography and wettability of the Aln-grafted gradient PCL membrane on the differentiation of BMSCs can be safely ruled out. 3.2. Gradient Differentiation of BMSCs. The osteogenic differentiation of BMSCs and its degree can be revealed by several characteristics such as morphology and production of marker products (ALP, calcium, etc). The BMSCs cultured on the Aln gradient surface for 24 h post cell seeding are shown in Figure 2a−d. More cells were found on the positions of a higher density of Aln. During the washing step after staining, the cells on the regions of lower Aln density were easier to detach, leading to a smaller cell number on these regions. Figure 2a−d shows also the BMSCs were progressively elongated along with the extension of position. Quantitative analysis confirms that the aspect ratio (longer axis/shorter axis of ellipse equivalent to cell) gradually increased along with the Aln density on both the gradient (Figure 2e) and uniform surfaces (Figure S3) from