Osteogenic differentiation and mineralization of human exfoliated deciduous teeth stem cells on modified chitosan scaffold

Materials Science and Engineering C 41 (2014) 152–160

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Osteogenic differentiation and mineralization of human exfoliated deciduous teeth stem cells on modified chitosan scaffold Wen-Ta Su a,⁎, Pai-Shuen Wu a, Chih-Sheng Ko b, Te-Yang Huang c a b c

Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan PhytoHealth Corporation, Maywufa Biopharma Group, Taipei, Taiwan Mackay Memorial Hospital, Taipei, Taiwan

a r t i c l e

i n f o

Article history: Received 12 January 2014 Received in revised form 13 April 2014 Accepted 18 April 2014 Available online 26 April 2014 Keyword: Human exfoliated deciduous teeth stem cells (SHEDs) Strontium Osteogenic differentiation Chitosan scaffold Dynamic culture

a b s t r a c t Stem cells from human exfoliated deciduous teeth (SHEDs) have been considered as alternative sources of adult stem cells in tissue engineering because of their potential to differentiate into multiple cell lineages. Strontium has an important function in bone remodeling because it can simulate bone formation and decrease bone resorption. In this study, the effects of strontium phosphate on the osteogenic differentiation of SHEDs were investigated. Strontium phosphate was found to enhance the osteogenic differentiation of SHEDs with up-regulated osteoblast-related gene expression. The proliferation of SHEDs was slightly inhibited by chitosan scaffolds; however, type-I collagen expression, alkaline phosphatase activity, and calcium deposition on chitosan scaffolds containing strontium were significantly enhanced. Furthermore, cells seeded in a 3D scaffold under dynamic culture at an optimal fluid rate might enhance cellular differentiation than static culture in osteoblastic gene expression. This experiment might provide a useful cell resource and dynamic 3D culture for tissue engineering and bone repair. © 2014 Elsevier B.V. All rights reserved.

1. Introduction An anti-osteoporotic drug called strontium ranelate is composed of an organic moiety with two stable strontium (Sr) atoms. Pre-clinical studies have shown that strontium ranelate dissociates bone resorption from formation, thereby increasing bone mass and strength [1–3]. In vivo, strontium ranelate induces a positive bone balance, which decreases the risk of vertebral and non-vertebral fractures in patients with postmenopausal osteoporosis [4]. In vitro, strontium ranelate can reduce osteoclast activity and bone resorption [5]. Moreover, the amount of several osteoblast markers increases, such as alkaline phosphatase, type-I collagen, bone sialoprotein, and osteocalcin in murine mesenchymal steoprogenitor cells and immature osteoblasts [6,7]. Strontium-containing bone cement was also demonstrated to have good bioactivity and good bone binding strength [8]. Previous studies have shown that strontium could induce prostaglandin production and cyclooxygenase expression to increase the osteoblastic differentiation of human mesenchymal stem cells (MSCs) and rat bone marrowderived MSCs [9,10]. The beneficial effects of strontium on promoting bone formation are closely related to its capability of increasing bone formation and decreasing bone resorption [11,12]. Nevertheless, only few investigations have been conducted on the effect of strontium⁎ Corresponding author at: 1 Sec. 3, Chung-Hsiao E. Rd, Taipei 10608, Taiwan. Tel.: +886 2 27712171x2554; fax: +886 2 27317117. E-mail address: [email protected] (W.-T. Su).

http://dx.doi.org/10.1016/j.msec.2014.04.048 0928-4931/© 2014 Elsevier B.V. All rights reserved.

containing biomaterial on the osteogenic differentiation of SHEDs in tissue engineering. SHEDs have recently attracted attention as a novel multi-potential stem cell source [13,14]. The isolation of SHEDs is simple, painless, and convenient, with little or no trauma. Every child loses primary teeth; thus, recovering and storing this convenient source of stem cells become easier. Immature stem cells are capable of extensive proliferation and differentiation, which make them an important resource of stem cells for the regeneration and repair of craniofacial defects, tooth loss, and bones [15]. SHEDs generate rapidly and grow much faster than adult stem cells, which suggest that they are less mature and can develop into a wide variety of tissues in a process called trans differentiation or plasticity [16]. SHEDs have been identified as a population of postnatal stem cells that are capable of differentiating into osteogenic, odontogenic, adipogenic, and neural cells [17]. The multipotential differentiation of SHEDs makes them a good candidate to renew degenerating tissues and to restore their functions. SHEDs are anchorage-dependent cells that require a supportive matrix to survive and grow. The matrix must provide an appropriate environment for cellular proliferation and differentiation. In tissue engineering, implanted scaffold gradually degrades into small molecules that can be easily metabolized and eventually eliminated from the body [18]. Furthermore, an ideal tissue scaffold should have appropriate mechanical strength, suitable degradation rate, adequate porosity, interconnectivity, and permeability to allow cells to attach and subsequently proliferate. The most popular materials used for biodegradable

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scaffolds include chitosan-based materials [19,20]. These materials can be fabricated as a supportive structure for culture and transplantation, which make them ideal three-dimensional (3D) scaffolds for tissue regeneration [21]. Chitosan, an amino-polysaccharide natural biodegradable cationic polymer, is biologically renewable, biocompatible, nonantigenic, non-toxic, antimicrobial activity, wound healing ability, low immunogenicity, low cost and biofunctional characteristic [22]. In contrast with several synthetic polymers, chitosan has a hydrophilic and surface charge that promotes cell adhesion, proliferation, and differentiation. Thus, the use of chitosan evokes minimal foreign body reaction upon implantation [23]. The development of a medical scaffold using chitosan, a class of biodegradable polymers with various attractive properties, was clinical requirement. 3D chitosan scaffolds have been successfully developed to enhance the mineralization of osteoblastlike MG-63 cell by a perfusion culturing system [24]. Culture techniques in vitro of three-dimensional (3D) tissue engineering scaffolds exhibit nutrient transfer limitations that should be overcome to increase the feasibility of cell-based tissue engineering strategies. Dynamic cell culture techniques have been proposed to overcome these limitations [25]. The aim of the present study is to confirm the effect of strontium phosphate in promoting the osteogenic differentiation of SHEDs, as well as to compare the osteogenic characteristics of SHEDs under chitosan scaffolds supplemented with β-tricalcium phosphate or hydroxyapatite. The strontium phosphate was Sr-fortified tricalcium phosphate powder, which was synthesized by partially substituting calcium (Ca) in tricalcium phosphate with Sr. Cellular growth was measured via scanning electron microscopy (SEM) and MTT assays. Alkaline phosphatase (ALP) activity, real-time polymerase chain reaction (RT-PCR), and Alizarin red S staining were performed to determine matrix maturation and mineralization. 2. Materials and methods 2.1. Isolation of SHEDs and fluorescence-activated cell sorting (FACS) analysis Human exfoliated deciduous molars were obtained as discarded biological samples from three different seven-year-old children at the Dental Clinic of the Kaohsiung Medical University with informed consent and followed the approved Institutional Review Board (IRB) guidelines, and were isolated according to the method used by Miura et al. [17]. Pulps were gently separated from the crown and then digested in a solution of 3 mg/mL type I collagenase and 4 mg/mL dispase for 1 h at 37 °C. A single cell was suspended in alpha-modified Eagle's medium (α-MEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum, 20 μg/mL ascorbic acid, and 5 ng/mL basic fibroblast growth factor (bFGF) for ordinary culture. To confirm the attribution of isolated cells, SHEDs were suspended in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin at a concentration of 5.82 × 106 cells/mL. The cells were then incubated for 30 min on ice with FITC-conjugated anti-human CD45, PE-conjugated anti-human CD105, FITC-conjugated anti-human CD34, PE-conjugated anti-human CD73, and PE-conjugated anti-human CD90 (BD Biosciences, USA). Analyses were performed with BD-FACS calibur flow cytometer (Becton Dickinson) using the WinMDI software. 2.2. Preparation of porous chitosan scaffolds containing induced supplements Strontium phosphate was synthesized by wet precipitation using 0.8 M SrCl2 with 0.2 M Na2HPO4 agitation for 1 d, then rinsed with distilled water fully and dried in the oven. Chitosan (MW 400,000, 98.5% deacetylation) was obtained from C&B Industrial Co., Ltd. (Taiwan). Porous chitosan scaffolds were prepared using the freeze-drying method. Chitosan (4%) has better cellular proliferation and mechanical

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characteristics than those of 2% and 3% chitosan in our previous study, so choose the 4% chitosan as our experimental concentration. A 4% (w/v) chitosan solution was prepared by dissolving chitosan in 0.167 N acetic acid. CS scaffold, as a control, was fabricated from this solution. SH scaffold, TH scaffold and HA scaffold were separately prepared by adding 0.7% strontium phosphate + 0.3% hydroxyapatite, 0.7% βtricalcium phosphate + 0.3% hydroxyapatite, and 1% hydroxyapatite into the chitosan solution, respectively. All the solutions were poured into a mold and kept in a freezer at −20 °C for 1 d. The frozen samples were then lyophilized in a freeze-dryer (FDU-1200 EYELA, Japan) at −50 °C and 11.2 Pa for 8 h. Subsequently, all scaffolds were neutralized with 1.25 M sodium hydroxide solution in a shaker for 30 min and then thoroughly rinsed with deionized water. The characteristics of the scaffold, such as swelling ratio, porosity, and mechanical strength, were assayed. The swelling ratio of the scaffolds was calculated using the following Eq. (1) Swelling ratioð%Þ ¼ ½ðWs−WdÞ=Wd  100%

ð1Þ

where Wd is the dry weight, Ws is the weight of the swollen scaffold, and V is the volume of the swollen scaffold. The porosity was determined by water intrusion porosimeter (PMI's Porous Material Inc. USA). The mechanical property of all the scaffolds was measured using BOSE Electro Force 3100. All experiments were performed three times for different samples. Data were expressed as mean ± SD. All samples were sterilized with 70% ethanol and washed with PBS (pH 7.4) prior to use. 2.3. Cellular morphology observation The morphology of the cells in the scaffold was examined via SEM. SHEDs grown in chitosan scaffolds with and without induced supplements were fixed with 4% paraformaldehyde for 30 min at room temperature. All the samples were rinsed with PBS, dehydrated in sequentially increasing ethanol solutions to 100 vol%, and then dried in a CO2 critical-point dryer (Pelco CPD#2400). The specimens were then sputter-coated with a thin layer of gold and examined via SEM (FEI QUANTA 200). 2.4. Cellular viability assay An indirect method that measures the metabolic activity of mitochondrial enzymes was performed to estimate cellular viability in the chitosan scaffolds containing induced supplements. The assay was based on 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). The tetrazolium salts were transformed using cellular mitochondrial dehydrogenase into visible dark blue formazan deposits so that the amount of color produced was directly proportional to the number of viable cells. The whole scaffold that contains the proliferated cells was treated with 5 mg/mL MTT at 37 °C for 24 h. The cell culture medium was removed, and formazan was solubilized in DMSO. The metabolized MTT was evaluated in terms of its optical density in a spectrophotometer at 540 nm (Thermo Scientific Multiskan FC). 2.5. Osteogenic differentiation For the evaluation of the effects of SHEDs cultured in scaffolds containing induced supplements of bone differentiation, cells from passage number 15 to 22 were seeded into the scaffolds at 2 × 104 cells/scaffold with the proliferated medium. After 2 days of culture, differentiation was initiated using an osteogenic medium (α-MEM, Gibco) containing 10% heat-inactivated fetal bovine serum, 20 μg/mL ascorbic acid, 8 mM β-glycerol phosphate, 10−8 M dexamethasone and 1% penicillin/ streptomycin solution. The medium was replaced every 3 days for 21 days.

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Table 1 The sequences of primers used for the real time PCR. Gene name

Primer sequences

Type I collagen

F: 5′-TGCTTGAATGTGCTGATGACAGGG-3′ R: 5′-TCCCCTCACCCTCCCAGTAT-3′ F: 5′-AGGTATCTGTGGGAGCTAATC-3′ R: 5′-ATTGCTGCACACCTTCTC-3′ F: 5-ACCACATCGGCTTTCAG-3′ R: 5′-CAAGGGCAAGGGGAAGA-3′ F: 5′-ATGAGAAGTATGACAACAGCC-3′ R: 5′-AGTCCTTCCACGATACCAA-3′

Osteonectin Osteocalcin GAPDH

2.6. Reverse transcription-polymerase chain reaction (RT-PCR) The cetyltrimethylammonium bromide (CTAB) extraction buffer was modified using the methods described by Wang and Stegemann [26], and consisted of 2.0% CTAB (Sigma), 2.0% polyvinylpyrrolidone (PVP 40; Sigma), 1.4 M sodium chloride (Sigma), 100 mM Tris–HCl (pH 8.0; Sigma), 20 mM ethylenediaminetetraacetic acid (Sigma), and 1.0% beta-mercaptoethanol (Sigma) in RNase-free water. The total RNA from SHEDs was extracted using CTAB buffer extraction. The quantity and purity of the RNA were determined at an absorbance of 260/280 nm. First-strand cDNA was synthesized from 500 ng of the RNA by using Super Script TM III Reverse Transcriptase kit, and real-time PCR reactions were performed using Invitrogen protocols. RT-PCR was performed using Smart Quant Green Master Mix with dUTP & ROX according to Protech Technology Enterprise Co., Ltd. (Taiwan) protocols. Forty-five cycles of Q-RT-PCR were performed for the target genes and housekeeper GAPDH. The PCR primers of type I collagen (COL I), osteonectin (ON), osteocalcin (OC), and GAPDH are listed in Table 1.

2.7. Alkaline phosphatase assay Alkaline phosphatase (ALP) assay was performed by first rinsing the cultured cells with PBS to remove the remaining culture medium. Approximately 500 μL of PBS containing 0.1 M glycine, 1 mM MgCl2, and 0.5% Triton X-100 (pH 10.5) were added to each sample to rupture the cell membranes and release the ALP molecules. The samples were then incubated for 1 h, after which 100 μL of the supernatant was extracted and transferred into microcentrifuge tubes wrapped in aluminum foil. About 200 μL of p-nitrophenyl phosphate solution (pNPP, Sigma) was added to each of the microcentrifuge tubes. The microcentrifuge tubes were then placed in a water bath at 37 °C for 30 min, followed by an ice bath at 0 °C for 10 min to reduce the reaction rate. About 50 μL of 3 N NaOH solution was added to each

microcentrifuge tube to stop the reaction. The collected solutions were then placed into a 96-well microplate for ALP tests, which were performed in triplicate. The plate was measured using an ELISA reader (Thermo Scientific Multiskan FC) at a wavelength of 405 nm. 2.8. Calcium quantification The differentiated cells in the scaffolds were removed for calcium quantification on 7, 14, and 21 days of culture. The accumulated calcium in the secreted mineral matrix of the osteoblasts was quantified via Alizarin red S staining. The samples were washed twice with PBS and then immersed into 95% ethanol solution for 30 min. The samples were stained with 1% Alizarin red S for 10 min, washed three times with PBS, and solubilized with 10% cetylpyridinum chloride. Total calcium was calculated using standard solutions and the absorbance at 570 nm was measured (Thermo Scientific Multiskan FC). 2.9. Perfusion dynamic culture The perfusion culture system consisted of peristaltic pump, reservoir, and silicon tube. Cell-seeded scaffolds were transferred into a 50 mL serum vial connected by a gas-permeable silicon tube to a medium reservoir with a peristaltic pump. The culture medium flowed through the cell/scaffold vessel and re-circulated back to the medium reservoir. All scaffolds were soaked overnight in culture medium before use. Excess medium of scaffold was squeezed out using sterile tweezers, and then transferred to 24-well culture plates. Subsequently, 100 μL of the cell suspension (2.0 × 105 cells/mL) was injected slowly into each scaffold using a 1 mL syringe with a 23-gauge needle. The cell-seeded scaffolds were incubated in an atmosphere of 5% CO2 for 1 h in order to ensure adhesion of seeding cells to chitosan scaffolds, and then positioned cell-seeded scaffolds into reservoir for perfusion culture under different flow rate (0.1, 0.4 and 1.0 mL/h, respectively). In addition, some cell-seeded scaffolds were positioned into 24-well culture plates for static culture, as control. All samples were allowed to incubate for a period of 7 days. The entire flow perfusion circuit was maintained under an environment of 37 °C with 5% CO2. Prior to use, the perfusion circuit was sterilized by autoclaving for 20 min. 2.10. Statistical analysis All experiments were performed three times for different samples. The data are presented as means ± standard deviation (SD) measured by ANOVA. Statistical comparisons were performed, and P values smaller than 0.05 were considered significant.

Fig. 1. The composition analysis of synthesized strontium phosphate by energy dispersive X-ray (EDX), and the element ratio of Sr/P is 1.49 (0.42/0.29).

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a

b

c

d

e

f

g

155

Compression Modulus kpa

180 160 140 120 100

*

*

SH

TH

80 60 40 20 0 CS2%

CS3%

CS4%

HA

Type of scaffolds Fig. 2. Images of chitosan scaffold (a). Microstructures of the CS scaffold (b), SH scaffold (c), TH scaffold (d), HA scaffold (e) and the swollen state of scaffold (f). The mechanical strength of the chitosan scaffold with and without bio-ceramic supplements (g). Values are expressed as mean ± SD (n = 3), * p b 0.05.

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Table 2 The composition, swelling ration and porosity of chitosan scaffold with and without bio-ceramic supplements. Composition (4% chitosan + bio-ceramic supplements) CS SH TH HA

– 0.7% strontium phosphate + 0.3% hydroxyapatite 0.7% β-tricalcium phosphate + 0.3% hydroxyapatite 1% hydroxyapatite

3. Results

Swelling ratio (%)

Porosity (%)

854.41 564.84 614.96 648.14

95.16 89.67 88.74 90.89

± ± ± ±

21.54 22.52 25.86 38.80

± ± ± ±

2.38 0.97 1.91 0.62

SHEDs were fabricated from enzyme-disaggregated deciduous dental pulp obtained from three different seven-year-old children. After isolating the cells from a single colony, the subcultured cells were gradually cultivated as an adherent monolayer and had a long and thin form fibroblast-like morphology. As determined by flow cytometry, the important mesenchymal stem cell (MSC) markers CD90 (99.34%) and CD73 (99.17%) were highly expressed in SHEDs, and the endothelial progenitor marker CD105 (44.45%) was moderately expressed, whereas the hematopoietic markers CD34 and CD45 were not expressed at all (0 and 0.03%, respectively). Similar to MSCs, these findings support that SHEDs can also differentiate into multiple cell lineages and are the potential source of cells for tissue engineering.

inferred to be Sr3(PO4)2 based on the Sr/P ratio of 1.49 (0.42/0.29). The surface morphology and microstructure image of the fabricated scaffold are shown in Fig. 2. Chitosan scaffold has a diameter of 8 mm and a height of 6 mm (Fig. 2a). The SEM micrograph of the scaffold cross-section exhibits a homogenous structure with regular porosity, a pore size of approximately 150 μm, and good pore interconnections (Fig. 2b–e). The swollen state of scaffold was shown in Fig. 2 f. Fig. 2 g illustrates the mechanical properties of the four types of scaffold with respect to compression modulus. The mechanical properties of scaffold were enhanced with increasing chitosan concentration, and bioceramic supplements significantly improved the mechanical strength of the pure chitosan scaffold. Table 2 shows the all used scaffolds composition, swelling ratio and porosity. Bio-ceramic supplements slightly reduced the swelling and porosity of the prepared scaffolds. However, porous void space of the modified scaffolds was still enough for the cellular attachment, spreading and growth.

3.2. Characterization of modified chitosan scaffolds

3.3. Cell proliferation

The energy-dispersive X-ray spectroscopy pattern of the crystal strontium phosphate is shown in Fig. 1. The molecular formula was

SHEDs cells proliferated on the chitosan scaffolds were measured via MTT assay. The MTT value is directly proportional to the number of cells

3.1. Characterization of SHED cells

2.5

a

*

Cell activity OD570

2

1.5

*

*

*

SH

*

1

CS

TH HA

0.5

0 3 days

b

7 days

14 days

21 days

c

Fig. 3. Growth curve of SHEDs plated at 2 × 104 cells/mL in the chitosan scaffold and cultured with a regular cell culture medium for 21 days via MTT assay at 570 nm (a). Morphology of cellular spreading on pure chitosan scaffold (b) and SH-chitosan scaffold (c). Values are expressed as mean ± SD (n = 3), *p b 0.05.

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present in the sample [27]. Fig. 3a shows the graph of cellular viability on the scaffolds after 3, 7, 14, and 21 days of culture with and without bio-ceramic supplement. The number of cells in the scaffolds increased with time, which indicates that the scaffolds are biocompatible for the cell cultures. However, the bio-ceramic supplements slightly inhibited cellular proliferation. Fig. 3b shows the cellular attachment and spreading on pure 4% chitosan scaffold, and Fig. 3c exhibits the morphology of cellular spreading on SH-chitosan scaffold for 2 days of culture.

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hydroxyapatite, which appeared highest in SH-chitosan scaffolds at 7 days of culture and in HA-chitosan scaffolds at 14 days of culture (Fig. 4b). The terminal marker gene of osteoblast differentiation (OC) was notably upregulated in the SH-scaffold compared with other scaffolds (Fig. 4c). The mRNA had a markedly higher expression in the SH-scaffold for 14 days of culture, and was approximately three-fold higher than the pure chitosan scaffold. The RT-PCR data confirms that osteogenic differentiation at the SH-scaffold is superior to the TH- and HA-scaffolds.

3.4. Quantitative real-time PCR analysis of gene expression 3.5. ALP activity The RT-PCR results show that bone-related genes can be detected in the culture system. Fig. 4 shows the expression levels quantified in the folds by the housekeeping gene GAPDH as a control gene in the scaffolds after 7, 14, and 21 days of culture. Cell-seeded scaffolds containing bioceramics expressed higher osteoblast maturation and mineralization compared with pure chitosan scaffolds. In general, bio-ceramic enhanced differentiated SHEDs cells produced higher level of type I collagen (Fig. 4a), and the best expression is observed in the HA-induced sample. The ON gene expression of SHEDs was affected by Sr and

Collagen type 1 relation expression

a

3.6. Quantification of calcium in mineralized matrix

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

CS

*

*

SH TH

*

HA

14 days

7 days

21 days

Time

b

1.4

Osteinectin relation expression

1.2 1

*

* CS

0.8

* *

0.6

SH TH HA

0.4

0 7 days

14 days

Calcium deposits are found in the body, especially in bone tissue. Alizarin red S stained the intra-cellular calcium as well as calciumbinding proteins and proteoglycans. This method is a useful way of evaluating bone differentiation at early phases. Fig. 6a shows the increase in calcium content during the 21 days of differentiation period, and the SH-chitosan scaffold exhibited the largest increase. The gene expression in TH-and HA-chitosans had a better effect compared with pure chitosan; however, calcium content was not remarkably enhanced. Detecting the formation of mineralized nodules in the osteogenic cell culture provides a means of assessing mature osteoblast cell function and culture status. At day 21, three-dimensional nodule formation was observed in SH-chitosan scaffold cultures, as illustrated by SEM images in Fig. 6b. These particles were characterized as calcium-rich in nature via EDAX (Fig. 6c). The formation of calcium phosphate salts or mineral deposition is a primary function of osteoblast cells [23]. The results suggest that SHEDs cells seeding in SH-chitosan scaffolds can produce mineralized nodules in osteogenic differentiation. 3.7. Dynamic culture The effect of fluid flow of the dynamic cultivated system for SHEDs osteogenic differentiation was examined. RT-PCR results show that bone-related genes can be detected in a dynamic system. Fig. 7 shows the ALP activity and expression levels quantified in the folds by the

0.2

c

A common marker of early bone cell differentiation is ALP activity. In this study, ALP activity was measured in SHEDs differentiation between different scaffolds containing bio-ceramics for 21 days of culture. Among all tested scaffolds, Sr up-regulated ALP activity shows the highest expression for 7 days of culture, and then decreased with increasing cultivation period (Fig. 5). Furthermore, ALP activity was significantly down-regulated by TH and HA. The results imply that the SH-scaffold can affect the expression of ALP activity of osteoblasts at any culture period.

21 days

16 0.6

*

12 10

0.5

* CS

*

SH

*

8

TH

6

HA

ALP activity OD405

Osteocalcin relation expression

14

0.4

*

* *

* * *

0.3

CS

*

SH TH

0.2

4

HA

0.1

2

0

0 7 days

14 days

21 days

3 days

7 days

14 days

21 days

Time Fig. 4. Gene expressions of type I collagen, osteonectin and osteocalcin for 21 days in the differentiated culture in different scaffolds, as determined via RT-PCR assay. Values are expressed as mean ± SD (n = 3), *p b 0.05.

Fig. 5. Expression of ALP activity for 21 days differentiated culture in different scaffolds. Values expressed are the mean ± SD (n = 3), *p b 0.05.

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a

350

*

Calcium content (µ µg)

300 250

* CS

200

*

SH

150 100

TH

*

HA

50 0 7 days

14 days

21 days

Time

b

nodules

c

Fig. 6. Calcium deposits on the scaffolds during 21 days of differentiated period via Alizarin red S staining (a). Formation of mineralized nodules on the SH-chitosan scaffold (b) and the nodules were analyzed via EDAX (c). Values are expressed as mean ± SD (n = 3), *p b 0.05.

housekeeping gene GAPDH as the control gene for bone-specific proteins in the scaffolds after 7 days of dynamic culture. Cell-seeded SH-chitosan scaffolds in the dynamic culture had higher osteoblast maturation and mineralization compared with scaffolds in the static culture. The cells produced maximal levels of ALP activity, ON and OC expression in the dynamic culture at a flow rate of 0.4 mL/min. In this study, the perfusion culture generated fluid shear stresses in 0.0369 to 0.3686 dyn/cm2 range, which can improve mass transport throughout the scaffolds. A shear stress is induced at the interface between the fluid and the structure, which is supposed to have a key function in mechanobiology. 4. Discussion Osteoblastic differentiation from human mesenchymal stem cell is an important step for bone formation and repair. Strontium is a boneseeking element, and has a dual action by improving bone formation as well as inhibiting bone resorption [28]. In SHEDs culture, strontium could enhance the induction of SHEDs so that they are differentiated into osteoblasts. The increase in the gene expression of osteonectin and osteocalin, ALP activity, and calcium deposition indicate the effect of strontium in enhancing bone remodeling and bone structure

stabilization. We proposed that strontium is an important factor for inducing SHEDs to differentiate into osteoblasts and increase bone formation further. This study might provide a useful cell source for tissue engineering in bone repair. As determined through flow cytometry, mesenchymal surface markers CD73 and CD90 were highly expressed in the isolated cells, and CD105 was moderately expressed, whereas the hematopoietic stem cell markers CD34 and CD45 were not expressed at all. The high expressions of CD73, CD90, and CD105 confirm that SHEDs comprise a unique undifferentiated stem cell lineage. The results show that SHEDs are promising and abundantly available autologous cell source for tissue engineering applications. Similar to human bone marrow stromal cells, SHEDs can also differentiate into various cell types such as neural cells, adipocytes, and odontoblasts [29,30]. Researchers recognize porous biomaterials as having potential in tissue regeneration applications and can function as substitute systems that mimic the important features of in vivo environments [22]. Chitosan is widely used as a scaffold for bone tissue engineering, and might enhance the adhesion and osteoblast differentiation of MSC [31]. Chitosan scaffold fabricated via lyophilization appeared to have a high interconnected channel (Fig. 2b–e), and its mechanical strength can be reinforced by adding bio-ceramic supplements such as SH, TCP and

a

14

Gene expression relation expression

W.-T. Su et al. / Materials Science and Engineering C 41 (2014) 152–160

12

*

10 8

SH

6

SH(P0.1)

*

SH(P0.4)

4

SH(P1)

2

*

0 COL-I

ON

OC

Genes

b

0.6

*

ALP activity OD405

0.5 0.4

*

1%SH 1%SH(P0.1)

0.3

1%SH(P0.4)

0.2

1%SH(P1)

0.1 0 7 days Fig. 7. Gene expressions of type I collagen, osteonectin, and osteocalcin for 7 days of dynamic differentiated culture in the SH-chitosan scaffold, as determined via RT-PCR assay (a), and the expression of ALP activity for 7 days of dynamic differentiated culture in the SH-chitosan scaffold. SH is static culture, SH (P0.1) is dynamic culture at 0.1 mL/min flow rate, SH (P0.4) is dynamic culture at 0.4 mL/min flow rate, SH (P1) is dynamic culture at 1.0 mL/min flow rate. Values are expressed as mean ± SD (n = 3), *p b 0.05.

HA (Fig. 2f). Further, cells adhered well and proliferated continuously with time, which indicate that chitosan scaffolds with and without modification are suitable for SHEDs growth (Fig. 3b and c). Osteoblast proliferation and differentiation can be evaluated by estimating the expression of several osteo-specific genes, which include type I collagen, ALP, ON, and OC. Type I collagen is a marker of early stage osteogenesis, whereas OC is a late stage marker. In general, ascorbic acid, β-glycerol phosphate, and dexamethasone are essential compounds in osteogenic medium. However, in this study, strontium phosphate significantly upregulated osteogenic gene expression, ALP activity, and calcium deposition, which indicate that strontium phosphate has an enhancing function in osteoblastic differentiation and bone formation. Strontium is a naturally occurring mineral found in water and food, and trace amounts of strontium can be found in human skeletons. Since 1950s, strontium was believed to influence bone metabolism by promoting bone formation while decreasing bone resorption [32]. In the present study, bio-ceramics can enhance type I collagen expression (Fig. 4a). However, the earlier maximal expression of osteonectin on the SH-scaffold is larger than the TH- or HA-scaffolds after 7 days of culture only (Fig. 4b). Osteocalcin is a major noncollagenous protein component of bone extracellular matrix, which is synthesized and secreted exclusively by osteoblasts in the late stage of maturation. Therefore, it is considered as late stage marker of osteoblastic differentiation. Fig. 4c indicates the maximal osteocalcin expression at day 14 of culture at strontium-induced conditions. The result corresponds to Fig. 6a, where the maximal deposition of mineralized calcium content in a strontium-induced environment was observed. Generally, nodule formation in the bone cell culture takes a longer period of up to 6 weeks. In our strontium-inducing model, several nodule formations were observed in SH-scaffold cultures, as shown by the SEM images taken after three weeks (Fig. 6b). Furthermore, similar osteogenic induction was also observed in the TH- and HA-scaffold

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groups. However the influence of TH and HA is significantly weaker and occurs at a later time compared with SH. The experimental findings suggest that strontium phosphate is an effective inductor of SHEDs osteogenic differentiation. ALP, a useful marker of osteoblast activity, is an ectoenzyme produced by osteoblasts and is involved in the degradation of inorganic pyrophosphates to provide sufficient local concentration of phosphate or inorganic pyrophosphate for mineralization. Fig. 5 showed that strontium phosphate can promote ALP activity compared with the other supplements, which is further confirmation that strontium affects the osteogenic action of SHEDs. Conventional cellular culture systems are in static media in vitro, thereby leading to 3D structures with seemingly low cellularity and a non-homogenous distribution of cells located in the periphery of the scaffolds [33]. The drawbacks of the static culture include the negative influences of concentration gradients of regulatory molecules, transport limitations of nutrients, and accumulation of harmful metabolic products within the culturing scaffolds [34,35]. Thus, providing a culture medium through the spaces between the scaffolds is a method that can effectively address these difficulties. Perfusion culture of cells seeded into a 3D scaffold can both potentially provide mobile fluid-induced mechanical stimulation and permit multilayered 3D cellular growth and organization. Thus, bone-related gene and phenotypic expression are enhanced [36,37]. When cells were seeded in SH-scaffolds in a perfusion culture, the gene expressions (COL-I, ON and OC) are superior compared with those in a static culture, and the flow rate at 0.4 mL/min is best than those of static culture and other flow rate 0.1 and 1.0 mL/min (Fig. 7). Therefore, the suitable fluid flow rate of the dynamic culture is an alternative option for future tissue engineering applications. 5. Conclusions This study demonstrated that modified chitosan scaffold is a suitable material for MSC differentiation, and strontium phosphate can be an effective inducer for SHEDs osteogenesis. The induced effect of strontium phosphate on the osteoblastic differentiation of SHEDs is stronger than those of β-tricalcium phosphate and hydroxyapatite. The addition of low-dose Sr mixed with HA into chitosan scaffolds would affect the osteoblastic differentiation of SHEDs in ON and OC gene expressions, ALP activity, and calcium deposition. This study confirmed that strontium is an important component for osteoblastic differentiation. In additional, dynamic culturing is a valuable and convenient tool for applications in the generation of three-dimensional bone tissue in vitro, and flow rate is an important operational factor. Understanding these different behaviors will be useful for the design and development of strategies for tissue engineering. Acknowledgment The authors would like to thank the NTUT-MMH Joint Research Program for their financial support under NTUT-MMH-No.10115. References [1] P.J. Marie, Strontium as therapy for osteoporosis, Curr. Opin. Pharmacol. 5 (2005) 633–636. [2] P.J. Marie, Strontium ranelate: a dual mode of action rebalancing bone turnover in favour of bone formation, Curr. Opin. Rheumatol. 18 (2006) S11–S15. [3] P.J. Marie, D. Felsenberg, M.L. Brandi, How strontium ranelate, via opposite effects on bone resorption and formation, prevents osteoporosis, Osteoporos. Int. 22 (2011) 1659–1667. [4] M.E. Arlot, Y. Jiang, H.K. Genant, J. Zhao, B. Burt-Pichat, J.P. Roux, P.D. Delmas, P.J. Meunier, Histomorphometric and μCT analysis of bone biopsies from postmenopausal osteoporotic women treated with strontium ranelate, J. Bone Miner. Res. 23 (2008) 215–222. [5] R. Baron, Y. Tsouderos, In vitro effects of S12911-2 on osteoclast function and bone marrow macrophage differentiation, Eur. J. Pharmacol. 450 (2002) (2002) 11–17. [6] E. Bonnelye, A. Chabadel, F. Saltel, P. Jurdic, Dual effect of strontium ranelate: stimulation of osteoblast differentiation and inhibition of osteoclast formation and resorption in vitro, Bone 42 (2008) 129–138.

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