1. Introduction
Bone is a highly vascularized organ, providing shape, mechanical support and protection to vital organs and facilitating movement [
1]. The functional restoration of bone defects resulting from trauma or bone diseases such as osteoporosis, arthritis and cancer is required for functional and aesthetic reasons [
2]. The use of an appropriate material onto which osteoblastic cells attach, proliferate and differentiate is a major challenge in the replacement of the damaged tissue and the restoration of the impaired functionality [
3]. During the last decades, a wide range of biomaterials have been employed as bone substrates, including bioactive ceramics, bioactive glasses, natural or chemical/synthetic polymers and their composites [
4]. Biodegradable materials, both of natural or synthetic origin are particularly attractive for use in many tissue engineering applications, because a second surgery, after implantation, is avoided [
5].
Chitosan (CS) is a natural, linear polysaccharide, derived from fully or partially deacetylated chitin [
6]. A characteristic of chitosan is the presence of amino groups along the polymer chain which allows it to form complexes with anionic molecules such as lipids, DNA, proteins and negatively charged synthetic polymers [
7]. Moreover, it is a non-toxic, non-immunogenic, biodegradable and biocompatible polymer with antibacterial and analgesic properties [
8,
9,
10]. Therefore, chitosan constitutes an ideal material for wound dressing, drug delivery and tissue engineering applications such as skin and nerve regeneration [
5,
11]. On the other hand, its poor mechanical properties, plasticity and brittleness are the main limitations for its use in many applications [
5]. Graft copolymerization [
12,
13] and blending with other polymers [
14,
15,
16] have been employed as common approaches to overcome these limitations.
Polycaprolactone (PCL) is a synthetic biodegradable and biocompatible polyester with good mechanical properties, high plasticity, non-toxicity and gradual resorption following implantation [
5]. Despite the lack of bioactive functional groups and the hydrophobic character of PCL, several studies propose the use of PCL scaffolds to promote the proliferation of cells and extracellular matrix production [
17,
18,
19]. PCL copolymers and blends with other natural or synthetic polymers have been frequently used as a biocompatible material both in soft and hard tissue engineering applications including cartilage and bone regeneration [
18,
19].
Previous studies have shown that CS/PCL blends are promising for use in skin and osteochondral tissue engineering, because they combine the biocompatibility and biological interactions of chitosan with the suitable mechanical properties of polycaprolactone [
20]. Moreover, our group has shown that CS-
g-PCL graft copolymers promote the viability and proliferation of Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) and could be used in tissue regeneration [
21]. In another study from our group we report on the immunomodulatory potential of CS-
g-PCL compositions 50/50 and 78/22 wt %, showing that both indicate an anti-inflammatory activity that was higher by increasing the chitosan content from 50 to 78 wt %.
The present study focuses on the evaluation of the in vitro biological response of MC3T3-E1 pre-osteoblastic cells cultured onto CS-g-PCL surfaces and their osteogenic potential in bone tissue engineering. First, the adhesion and morphology of the pre-osteoblastic cells on the CS-g-PCL surfaces were investigated using scanning electron microscopy (SEM) and confocal laser fluorescence microscopy. Moreover, the viability and proliferation of the cells at different time periods of culture were evaluated using the colorimetric assay PrestoBlue® (ThermoFisher Scientific, Waltham, MA, USA) . Finally, the ability of the graft copolymer to promote the differentiation capacity of the pre-osteoblastic cells was assessed by determining specific early and late markers of osteogenesis, such as alkaline phosphatase activity, collagen production, calcium biomineralization and endogenous osteopontin expression.
2. Materials and Methods
2.1. Materials
A CS-g-PCL copolymer, with 78 wt % CS content, was used to coat 15 mm diameter glass coverslips. For the synthesis of the CS-g-PCL graft copolymer, low molecular weight chitosan (Sigma-Aldrich, St. Louis, MO, USA), with degree of deacetylation (DD) of ~85%, as determined by proton nuclear magnetic resonance (1H NMR) spectroscopy, was used. Poly(ε-caprolactone) bearing a carboxylic acid end-group (PCL-COOH), with a degree of polymerization of 45, as determined by 1H NMR analysis, was synthesized by ring opening polymerization using stannous octoate as the catalyst and glycolic acid as the initiator, and was chemically grafted along the chitosan backbone via the primary amino groups. Phosphate buffer saline (PBS), bovine serum albumin (BSA), Triton X-100, acetic acid, Direct red 80, Alizarin red S, cetylpyridinium chloride (CPC), p-nitrophenyl phosphate, Bradford reagent, paraformaldehyde, ascorbic acid, β-glycerolphosphate and dexamethasone, 4′,6-Diamidino-2-Phenylindole (DAPI) dihydrochloride, FITC-conjugated anti-vinculin antibody (F7053) and TRITC-phalloidin conjugated antibody (P1951) were purchased from Sigma-Aldrich (St. Louis, MO, USA). PrestoBlue® reagent for cell viability and proliferation, minimum essential Eagle’s medium (α-MEM), L-glutamine, trypsin/EDTA, penicillin/streptomycin, fetal bovine serum (FBS), To-Pro®-3 iodide, actin from rabbit muscle were purchased from Molecular Probes by Life Technologies, ThermoFisher Scientific Waltham, MA, USA).
2.2. Preparation of the CS-g-PCL Samples
CS-g-PCL surfaces on glass substrates were prepared by spin coating using a SPS spin coater (model Spin 150). 140 μL of a 1% w/v copolymer solution in H2O/CF3COOH 50% v/v was spun at 2000 rpm for 160 s. The coating was dried under high vacuum at 60 °C for 24 h before being neutralized by rinsing with 0.1 M NaOH solution for several minutes, washed with water and dried under a N2 flow.
2.3. Biomaterial Characterization
The synthesized graft copolymer was characterized by 1H NMR spectroscopy and Attenuated Total Reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. 1H NMR spectra were obtained on a Bruker AMX-500 spectrometer (Billerica, MA, USA) by dissolving the samples in a CF3COOD:D2O (1:1) solvent mixture at 45 °C. The ATR-FTIR spectra were recorded on a Nicolet 6700 spectrometer (ThermoFisher Scientific, Waltham, MA, USA).
2.4. Cell Culture of Pre-Osteoblasts
MC3T3-E1 pre-osteoblastic cells were isolated from newborn mouse calvaria. Cells were obtained from DSMZ GmbH (DSMZ No: ACC 210 Braunschweig, Germany) and have the capacity to differentiate into osteoblasts and osteocytes in vitro. Pre-osteoblastic cells were cultured in α-ΜΕΜ medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1% penicillin/streptomycin. Once a week, when the cells reached confluence, they were passaged using trypsin/EDTA and were resuspended in culture medium. Next, 3 × 104 and 5 × 104 MC3T3-E1 cells were used for viability/proliferation and differentiation assays, respectively and were seeded onto the sterilized CS-g-PCL surfaces. Samples were placed in 24-well Corning® plates, where tissue culture polystyrene were used as control substrates. All experiments were carried out using cells between passage 8 and 15.
2.5. Adhesion and Morphology of the Pre-Osteoblasts on the CS-g-PCL Surfaces
The morphology of the pre-osteoblast cells on the CS-g-PCL surfaces was monitored by means of scanning electron microscopy (SEM) (JEOL JSM-6390 LV, Tokyo, Japan) after 3 days of incubation. At each time point, cells were rinsed twice with 1 M PBS buffer and were fixed with 2% v/v para-formaldehyde and 2% v/v glutaraldehyde for 15 min at room temperature and dehydrated in increasing concentrations (30% v/v–100% v/v) of ethanol. Samples were then dried in a critical point drier (Baltec CPD 030, Baltec, Los Angeles, CA, USA), sputter-coated with a 20 nm thick layer of gold (Baltec SCD 050, Baltec, Los Angeles, CA, USA) and observed under a microscope at an accelerating voltage of 15 kV.
Actin distribution and focal adhesion points of the cells were observed using confocal laser fluorescence microscopy. A suspension of 2 × 104 cells were cultured on the polymer surface and after 3 days the medium was removed and the samples were washed twice with PBS. Cells were fixed with 4% para-formaldehyde for 15 min and permeabilized with 0.1% Triton X-100 in PBS. The non-specific binding sites were blocked with 2% BSA solution in PBS for 30 min. Samples were stained using fluorescein isothiocyanate-conjugated anti-vinculin antibody (1/100) (TRITC-phalloidin conjugate, Sigma-Aldrich, St. Louis, MO, USA) and tetramethyl rhodamine isothiocyanate-conjugated phalloidin (1/40) (Molecular Probes by Life Technologies, ThermoFisher Scientific, Waltham, MA, USA) for 50 and 30 min, respectively. For cell nuclei staining 100 μL of a DAPI dihydrochloride solution (1/100) were added on top for another 10 min. Samples were rinsed twice with PBS and were observed under a Leica DM IRBE laser scanning confocal microscope using a 40-fold magnification objective lens (Leica, Wetzlar, Germany).
2.6. Viability and Proliferation of Pre-Osteoblasts on the CS-g-PCL Surfaces
3 × 104 pre-osteoblastic cells were used for the viability and proliferation assay. The metabolic activity of the cells on the CS-g-PCL surfaces was assessed with the PrestoBlue® assay. When cells are viable, the nontoxic metabolic indicator resazurin, is reduced to a red product resorufin, which can be detected photometrically. At each time point, namely after 1, 3 and 7 days in culture, 400 μL PrestoBlue® reagent, diluted in primary culture medium (a-MEM) (1:10), was added directly to each well and was incubated at 37 °C for 30 min. The supernatants of the samples were transferred to another 24-well plate and the absorbance (570 nm and 600 nm) was measured using a spectrophotometer (Synergy HTX Multi-Mode Microplate Reader, BioTek, Winooski, VT, USA). Absorbance units were translated to cell number after using a calibration curve. Two independent experiments were performed in triplicates.
2.7. Osteogenic Response of the Pre-Osteoblastic Cells on the CS-g-PCL Surfaces
2.7.1. Alkaline Phosphatase (ALP) Activity
An enzymatic activity assay was used to measure the levels of alkaline phosphatase activity expressed from the pre-osteoblastic cells cultured on the CS-g-PCL surfaces. Cells were cultured for 4, 7 and 14 days in osteogenic medium (primary medium supplemented with ascorbic acid, sodium glycerophosphate and dexamethasone (50 μg/mL, 0.1 μΜ, and 10 nM, respectively) and at each time point they were harvested by trypsin-EDTA and collected by centrifugation. Pellets were dissolved in 100 μL lysis buffer (0.1% Triton X-100 in 50 mM Tris-HCl pH 10.5) and were subjected to two freeze-thaw cycles from −80 °C to room temperature. Then, 100 μL of a 2 mg/mL p-nitrophenyl phosphate (pNPP, Sigma, St. Louis, MO, USA) substrate in 50 mM Tris-HCl at pH 10 with 2 mM MgCl2 were added to each sample and incubated at 37 °C for 60 min. The reaction was stopped with the addition of 50 μL 1 N NaOH. Absorbance was measured using a Synergy HTX plate reader (BioTek, Winooski, VT, USA) at 405 nm and was correlated to equivalent amounts of para-nitrophenol using a calibration curve. Alkaline phosphatase activity was normalized to cellular protein levels and was measured by the Bradford assay.
2.7.2. Alizarin Red Staining
Alizarin red was used to stain the calcium deposits in the extracellular matrix of the pre-osteoblasts. Cells were cultured for 7 and 14 days in osteogenic medium (as described in
Section 2.7.1) and at each time point of 7 and 14 days they were fixed with 4% paraformaldehyde for 15 min, rinsed twice with PBS and stained with 300 μL of 2% alizarin red S at pH 4.1 for 30 min. Then, cells were rinsed three times with H
2O in order to remove the excess stain. Cetylpyridinium chloride (CPC) was used to quantify the accumulation of calcium deposits by dye extraction. 300 μL of 10% CPC in 10 mM sodium phosphate buffer solution (pH 7) were added to each sample and was incubated for 1 h under shaking. Absorbance was measured using a Synergy HTX plate reader at 550 nm. Absorbance measurements were normalized to the cell number, measured by the PrestoBlue
® assay prior to staining.
2.7.3. Collagen Production in the ECM
The Sirius Red Dye assay (Direct red 80, Sigma-Aldrich, St. Louis, MO, USA) was used to measure total collagen levels secreted from the pre-osteoblastic cells in the culture medium. Briefly, at each time point, 100 μL of culture medium was mixed with 1 mL 0.1% Sirius Red Dye and were incubated for 30 min at room temperature. After centrifugation of the samples at 15,000 g for 15 min, the pellets were washed with 0.1 N HCl in order to remove the non-bound dye. Samples were finally centrifuged at 15,000 g for 15 min and were dissolved in 500 μL 0.5 N NaOH. Absorbance was measured using a Synergy HTX plate reader at 530 nm. The absorbance measurements were correlated to the concentration of collagen type I using a calibration curve.
2.7.4. Endogenous Expression of Osteopontin Using in-Cell Enzyme-Linked Immunosorbent Assay (ELISA)
Osteopontin is a phosphorylated glycoprotein, which is involved in bone mineralization. In our study, the levels of endogenous osteopontin expressed from cells cultured on the CS-g-PCL surfaces and the tissue culture treated polystyrene (TCPS) control were measured using the in-cell enzyme-linked immunosorbent assay (ELISA). First, 5 × 104 cells/well were cultured on the two materials surfaces for 10 days. At this time point, cells were fixed with 4% v/v paraformaldehyde for 15 min, before permeabilization with 0.1% v/v and blocking with 2% w/v BSA for 1 h at room temperature. Samples were incubated with anti-mouse primary antibody (1/1000) in 2% BSA/PBS for 2 h at 4 °C (300 μL) and after washing away the unbound antibody, an anti-rabbit IgG H&L antibody was added on top at 1/1000 dilution for 2 h (300 μL). Then, the enzyme substrate (TMB) (100 μL) is added and the reaction produces a color change signal, in proportion to the amount of the osteopontin levels. Finally, 100 μL of sulfuric acid stock solution was added, which changes the color from blue to yellow. The absorbance was measured using a Synergy HTX plate reader at 450 nm and valued by normalization to the cell number as percentage over the background.
2.7.5. Endogenous Expression of Osteopontin and Visualization by Means of Confocal Laser Fluorescence Scanning Microscopy
The endogenous expression of osteopontin (OPN) from cells cultured on the CS-g-PCL copolymer for 10 days was examined using a confocal laser scanning fluorescence microscope (CLSM, Leica DM IRBE, Wetzlar, Germany). At this time point, cells were fixed with 4% paraformaldehyde for 15 min, before permeabilization with 0.1% v/v Triton and blocking with 2% w/v BSA for 1 h at room temperature. Samples were incubated with anti-mouse primary antibody at a dilution of 1:1000 in 2% BSA/PBS for 2 h at 4 °C. A FITC-conjugated anti-rabbit IgG H&L was used as a secondary antibody at a 1:1000 dilution. For this experiment cells cultured on cover slips for 10 days were used as a control.
2.8. Statistical Analysis
Statistical analysis was performed using the student’s t-test (GraphPad Prism version 5 software, GraphPad, La Jolla, CA, USA) to evaluate the significance of the differences between the CS-g-PCL surfaces and the TCPS control surfaces. A p value of <0.05 was considered significant.
4. Discussion
The development of biomaterials for bone tissue engineering aims to provide favorable substrates for osteoblastic and mesenchymal stem cells to facilitate their functions such as cellular viability, proliferation, migration and differentiation towards tissue regeneration [
22,
23]. Ceramics were widely used as bone implants due to their stability and mechanical strength, but their bioactivity and compatibility are under investigation [
24,
25]. On the other hand, many studies have indicated the enhancement of osteogenesis both in vitro and in vivo using biodegradable polymeric materials [
26,
27]. All these factors determine the material selection for the fabrication of the suitable scaffolds. Chitosan is a well-known natural polymer derived from chitin, the second most abundant polymer in nature. Chitosan has been extensively employed in tissue engineering applications due to its advantageous properties [
7]. Many studies have shown that following blending or graft polymerization of chitosan with other polymers, the biological, mechanical and degradation characteristics are enhanced [
28]. In this direction, PCL is a suitable synthetic polymer for blending with chitosan due to its low melting point and good tensile properties [
11]. Young et al. have shown that CS-PCL blends can form membranes, which were used to fabricate a bioengineered corneal endothelium and therefore, facilitated the corneal endothelial cell (CEC) transplantation in vivo [
29]. Moreover, Prasad et al. showed that the adhesion, viability and proliferation of human keratinocytes (HaCaT) and mouse fibroblasts (L-929) cultured on electrospun chitosan/PCL blends were enhanced and proposed the use of these blends as appropriate biomaterials for skin tissue engineering [
11].
Previous studies from our group have focused on the synthesis of a CS-
g-PCL copolymer, bearing PCL chains grafted along the chitosan backbone. This copolymer was used as a matrix for the development of Wharton’s jelly mesenchymal stem cells from three different donors and the results showed excellent cellular response [
21]. In addition, evaluation of the immunomodulatory potential of CS-
g-PCL copolymers with different compositions by investigating the differentiation of primary bone marrow derived macrophages (BMDM) showed that the materials promote the anti-inflammatory activity and the transition of M1 to M2 macrophages [
30]. Interestingly, the observed anti-inflammatory effect increased with increasing chitosan content from 50 to 78%, a finding that makes the latter composition attractive for further investigations as the immunomodulation is crucial for the prediction of the fate of a developed biomaterial to be used as an implant.
Based on our previous work, the aim of this study was to evaluate the in vitro biological response of MC3T3-E1 pre-osteoblastic cells on the CS-
g-PCL surfaces. These cells, which can be easily obtained and cultured, have the capacity to differentiate into osteoblasts and osteocytes in vitro [
31]. First, the adhesion of the cells on the CS-
g-PCL surfaces after 2 and 7 days of culture was examined by SEM analysis (
Figure 3). Cells reached confluence on day 7 and retained their characteristic fibroblastic phenotype with a highly elongated spindle-like shape. This result is in accordance with previous studies reporting the promoting of the adhesion of bone marrow mesenchymal stem cells, fibroblasts, keratinocytes and corneal endothelial cells on CS-PCL blends [
5,
11,
28,
32]. Initial cell adhesion is a critical step in tissue engineering because it mediates subsequent events such as cell viability and proliferation [
33]. In our study, the viability and proliferation of pre-osteoblastic cells cultured on the copolymeric surfaces were significantly increased compared to TCPS after 3 and 7 days in culture (
Figure 5). Similarly to our copolymeric material composition that supports the pre-osteoblastic proliferation increase, Young et al. showed a good growth of corneal endothelial cells reaching confluence on CS-PCL membranes with a composition of 75–25% demonstrating sufficient strength and suitability as a carrier in culture [
29]. In another study, He et al. report that the growth of bone marrow mesenchymal stem cells cultured on CS-PCL membranes was better on the 30–70% composition compared to the 100% PCL, which could be attributed to amino groups on the CS-PCL composites which impart more hydrophilic sites than on the pure PCL, thus resulting in more suitable conditions for cell growth [
5].
Our results indicate that the CS-
g-PCL graft copolymer supports the osteogenic differentiation of the pre-osteoblastic cells. This was confirmed by a significant increase in the ALP activity of cells cultured on the CS-
g-PCL surfaces compared to TCPS (
Figure 6a), which is in line with the notion that chitosan promotes the osteogenic differentiation of cells as reported by He et al. who show that the ALP activity of bone marrow mesenchymal stem cells cultured on CS-PCL 30–70% membranes was higher compared to pure PCL [
5]. Moreover, increased levels of extracellular collagen at early stages of culture were measured (
Figure 6b), indicating a healthy extracellular matrix formation on the copolymeric surface, which is essential for tissue regeneration. Interestingly, Young et al. showed that bovine corneal endothelial cells cultured on a CS-PCL 75–25% membrane exhibited the highest production of collagen type IV, compared to the CS-PCL 85–15% and 90–10% blends [
29]. Finally, the increased expression of osteopontin as well as the higher calcium deposits for the cells cultured on the CS-
g-PCL surfaces indicate that the copolymeric material promotes the mineralized matrix formation.
Previous studies have reported that the composition of chitosan and polycaprolactone in composites made from these two components is crucial as high concentrations of chitosan can favor the proliferation, the osteogenic differentiation potential and the anti-inflammatory activity [
5,
28,
29,
30]. We therefore focus in this study on the in vitro biological assessment of a CS-
g-PCL copolymeric material containing 78%
w/
w chitosan, which was employed as two-dimensional substrates in an osteogenic cell model. Our data demonstrate that the CS-
g-PCL copolymeric substrates are capable to promote the viability, proliferation and osteogenic differentiation of pre-osteoblastic cells in vitro. Since our next goal is the in vivo application of customized three-dimensional CS-
g-PCL copolymeric scaffolds as attractive candidates in clinical translation, our efforts focus on the fabrication of scaffolds, which can elicit a strong osteogenic response in vivo, however, this is the subject of another work. Preliminary data of our group in this direction indicate promising results on the in vitro biological response of human bone marrow derived mesenchymal stem cells cultured on CS-
g-PCL substrates.