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An integrated study of natural hydroxyapatite-induced osteogenic differentiation of mesenchymal stem cells using transcriptomics, proteomics and microRNA analyses
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Biomed. Mater. 9 045005 (http://iopscience.iop.org/1748-605X/9/4/045005) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 142.150.190.39 This content was downloaded on 18/11/2014 at 19:13
Please note that terms and conditions apply.
Biomedical Materials Biomed. Mater. 9 (2014) 045005 (13pp)
doi:10.1088/1748-6041/9/4/045005
An integrated study of natural hydroxyapatite-induced osteogenic differentiation of mesenchymal stem cells using transcriptomics, proteomics and microRNA analyses Zhiwei Zhang, Jiandan Wang and Xiaoying Lü1 State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, People’s Republic of China E-mail: [email protected] Received 27 January 2014, revised 14 May 2014 Accepted for publication 21 May 2014 Published 19 June 2014 Abstract
This work combined transcriptomics, proteomics, and microRNA (miRNA) analyses to elucidate the mechanism of natural hydroxyapatite (NHA)-induced osteogenic differentiation of mesenchymal stem cells (MSCs). First, NHA powder was obtained from pig bones and fabricated into disc-shaped samples. Subsequently, the proliferation and osteogenic differentiation of MSCs cultured on NHA were investigated. Then, proteomics was employed to detect the protein expression profiles of MSCs cultured on NHA, and the effect of NHA on MSCs was analyzed through an integrated pathway analysis (including proteomics and previous transcriptomics data) in which specific NHA-induced differentiation pathways were analyzed. The pathway nodes with expression data at both the mRNA and protein levels (mRNA–protein pairs) were filtered in differentiation-related pathways. miRNAs corresponding to these target mRNA–protein pairs were predicted, screened and tested, and the regulatory effects of miRNAs on mRNA–protein pairs were analyzed. Finally, the NHAinduced osteogenic pathways were verified. The results of an MTT assay and alkaline phosphatase (ALP) staining showed that the cell proliferation rate decreased and the osteogenic performance improved in the presence of NHA. By integrating transcriptomics and proteomics, the genes and proteins involved in 89 pathways were shown to be differentially expressed. Among them, 5 differentiation-associated pathways, in which 9 miRNAs and 8 regulated-target mRNA–protein zby inhibiting the target mRNA–protein pair HSPA8 in the MAPK signaling pathway, and miR26a and miR-26b might inhibit adipogenic differentiation by repressing the target mRNA–protein pair HMGA1 in the adipogenesis pathway. A verification experiment for the osteogenic pathway indicated that the ERK1/2 or JNK MAPK pathways might play an important role in NHA-induced osteogenic differentiation. In conclusion, NHA affected MSCs at both the transcriptional and translational levels, and MSC osteogenic differentiation eventually occurred through the MAPK and adipogenesis pathways, in which miRNAs and target mRNAs/proteins participated cooperatively. Keywords: natural hydroxyapatite, transcriptomics, proteomics, microRNA, osteoinduction, biological pathways (Some figures may appear in colour only in the online journal) S Online supplementary data available from stacks.iop.org/BMM/9/045005/mmedia 1
Author to whom any correspondence should be addressed.
1748-6041/14/045005+13$33.00
1
© 2014 IOP Publishing Ltd Printed in the UK
Z Zhang et al
Biomed. Mater. 9 (2014) 045005
1. Introduction
regulators of cell functions [26]. miRNAs function in the posttranscriptional and translational regulation of gene expression through a variety of mechanisms [27]. In addition, it has been found that miRNAs play an important role in the regulation of osteogenic differentiation [28, 29]. Therefore, miRNA research provides an interesting new perspective in the study of osteogenic differentiation mechanisms. To date, the biological effects of HA have been studied at the mRNA, protein and miRNA levels individually. However, mRNAs, proteins and miRNAs are interconnected parts of a whole and influence each other. mRNA, which is synthesized through gene transcription, serves as a template for protein synthesis; the order of the mRNA bases determines the protein that will be produced. miRNAs regulate the flow of genetic information by controlling protein translation or mRNA stability. Proteins, which eventually perform various biological functions, are the ultimate output of this genetic information flow; hence, mRNAs or miRNAs affect protein expression. Meanwhile, proteins are involved in the regulation of transcription and translation through complex feedback systems. Therefore, mRNAs, proteins and miRNAs form a complex network of reciprocal regulatory interactions [30]. By studying the expression and relationships of these three components (mRNAs, proteins and miRNAs) simultaneously, a more complete view of biological information flow from DNA to RNA to protein can be acquired. In addition, a panorama of the complex network of miRNAs, target mRNAs and target proteins involved in certain important biological pathways can be obtained. Therefore, each of the three components (mRNA, protein and miRNA) is an indispensable part of a complete osteogenic differentiation study. Integration of transcriptomics, proteomics and miRNA analyses could help elucidate the overall process of mesenchymal stem cells (MSCs) osteogenic differentiation more accurately and thoroughly. Due to its advantages of having extensive sources and being environmentally friendly and low cost, the preparation and biological properties of NHA have received attention from a growing number of researchers [2, 3, 10, 31]. Our group has been conducting a series of studies on NHA since 2001. NHA has been successfully extracted from pig bones, and the main chemical components of the prepared NHA were found to be identical to the standard HA characterized by Fourier transform infrared spectroscope (FTIR) and X-ray diffraction (XRD); however, there are several unique chemical compounds in NHA including CO32− and HPO42− [4]. A protein adsorption assay showed that the values of albumin/fibrinogen (RA/F) and albumin/IgG (RA/I) obtained for NHA were smaller than those obtained for synthesized HA, which indicated that NHA had better coagulation properties and would be favorable for bone repair [32]. In addition, NHA-induced osteogenic differentiation of MSCs was studied using transcriptomic technology combined with bioinformatics analysis, and 90 differentially expressed genes related to osteogenic differentiation were obtained [31], which indicated that NHA could regulate MSC osteogenic differentiation. Based on our previous research, this study aimed to further explore the osteoinduction mechanism of
Hydroxyapatite (HA), the main inorganic component of bone, has been widely used in human bone tissue restoration. Currently, there are two main methods of obtaining HA: 1) synthesis by chemical methods [1]; and 2) extraction from natural tissues such as bone, teeth, and scales [2–4], the product of which is defined as natural hydroxyapatite (NHA). Many studies have demonstrated that HA has good biocompatibility and osteoconductive performance [5, 6]. The osteoinductive properties of HA have also been reported widely. Using animal experiments, Heughebert first reported in 1988 the physicochemical characterization of the deposits associated with HA ceramics implanted in nonbone-forming sites; these deposits showed striking similarity to the inorganic bone phase [7]. Subsequently, studies have proven that HA has osteoinductive properties in many mammals [8, 9]. Commercial NHA (Bio-Oss) derived from bovine bone has also been reported to possess osteoinductive properties when implanted in rats, rabbits and other animals [3, 10]. Moreover, NHA derived from antler cancellous bone is efficacious in inducing neovascularization and osteogenesis within rabbit mandible defects [3]. In vitro experiments showed that HA, including but not limited to NHA derived from scales, significantly promotes osteogenic differentiation in the presence of osteogenic medium [2, 11, 12]. Both HA-composite materials [13, 14] and HA-modified materials [15, 16] have shown superior performance in inducing osteogenic differentiation than those without HA. Furthermore, some experiments have shown that osteogenic markers are induced only in the presence of HA [17]. With the development of scientific technology, methods for investigating HA-induced osteogenic differentiation have evolved from observing bone-like surface deposition on a material [7] to detecting the expression levels of bone-related mRNAs or proteins including alkaline phosphatase (ALP), osteocalcin, type 1 collagen (COL1), etc [11, 18] and further to analyzing global mRNA or protein expression at the transcriptomic or proteomic level comprehensively and systematically [19–22]. To date, transcriptomic technology has been used to show that HA causes differential expression of several osteogenic markers [19] and impacts signaling pathways [20]. Proteomic research on HA has primarily focused on biological functions including cell adhesion, proliferation and calcium regulation [21, 22] rather than osteogenic differentiation. Although transcriptomic and proteomic technology can be used to comprehensively reveal overall changes at the molecular level, there may be incongruent expression between mRNAs and proteins [23, 24]. Our group also found significant differences between mRNA and protein expression levels in both quantity and type compared with the results from transcriptomic and proteomic analysis of the molecular biocompatibility of gold nanoparticles with human dermal fibroblasts [25]. Apart from the defects of omic techniques and the incompleteness of mRNA/protein databases, the uncorrelated result may be caused by the complex regulatory mechanisms involved in translating mRNA into mature proteins. microRNAs (miRNAs), which were discovered in 1993, are major 2
Z Zhang et al
Biomed. Mater. 9 (2014) 045005
NHA, which mainly consisted of studying the NHA-induced osteogenic differentiation pathway and the interaction mechanisms between miRNA and target mRNAs/proteins in those pathways through an integrated study using transcriptomics, proteomics and miRNA analyses.
2.2.2. Flow cytometry. Flow cytometric analysis was per-
formed to evaluate cell surface markers and MSC homogeneity. Briefly, fourth-passage cells were trypsinized, washed with PBS three times, and incubated with antibodies against CD14, CD29, CD34 and CD44 [33] (Biolegend, USA) at the recommended concentrations for 15 min; the appropriate isotype controls were also included. After being washed twice, the cells were resuspended in 300 µl FACS buffer and immediately analyzed with a flow cytometer (BD FACSCalibur, USA). A total of 10,000 events were acquired for each sample, and data analysis was performed using BD CellQuest Pro software.
2. Materials and methods 2.1. Preparation and characterization of NHA
NHA powder was extracted from pig bones using high-temperature calcination, as previously established by our group [4]. The procedure is described briefly below. The fresh porcine bone were cleaned to remove most organic component and thoroughly dried, and subsequently cut into smaller pieces. After that, the bone pieces were calcinated at 650 ºC for 3 h, and then crushed sufficiently with a grinder to obtain NHA powder material. The powder was compressed into disc-shaped samples with diameters of 2 and 10 cm using selfdesigned molds and subsequently calcined at 600 °C for 4 h to obtain NHA disc-shaped samples. The surface morphology of the disc-shaped sample was observed using a scanning electron microscope (Zeiss Ultra Plus, Germany), and the chemical composition was determined through Fourier Transform Infrared Spectroscopy (Nicolet5700, Thermo-Fisher, USA).
2.3. Proliferation and osteoinduction assay for MSCs cultured on NHA 2.3.1. Cell proliferation assay. The cell proliferation rate
(P%) on NHA disc-shaped samples with diameters of 2 cm was evaluated after culture for 24, 48 and 72 h using the methylthiazoltetrazolium (MTT) assay [34]. Cells cultured on glass slides with diameters of 2 cm were used as a negative control, and cells treated with 0.7% acrylamide were used as a positive control. 2.3.2. Osteoinduction assay for different factors. To evaluate the osteoinduction potential of two factors (chemicalinducible factor and NHA), MSCs at the fourth passage were treated with these two factors for 7 and 21 days. In the chemical-induced group, the cells were seeded on glass coverslips in 12-well plates (Corning, USA) with osteogenic differentiation medium [complete medium with 0.1 µM dexamethasone, 5 mM β-glycerophosphate, and 50 µg/ml ascorbic acid [35] (Sigma-Aldrich, Germany)]. In the NHA group, the cells were seeded on NHA in 12-well plates with complete medium. Cells cultured on glass coverslips with complete medium were used as controls. ALP staining was performed after 7 and 21 days to examine osteogenic differentiation using the ALP staining kit (Jiancheng, China) according to the manufacturer’s instructions. The stained cells were observed using a light microscope (Olympus BX51, Japan).
2.2. Isolation, culture and characterization of MSCs 2.2.1. Isolation and culture of MSCs. Mouse bone marrow-
derived MSCs were isolated and cultured as described below. Four-week-old Kunming mice (regardless of their gender) were injected intraperitoneally with heparin (3000 U per mouse) and sacrificed 5 min later by cervical dislocation in accordance with the rules of the Institutional Ethical Board for experimental procedures. Bone marrow was collected from the mice by flushing femurs and tibias with complete medium consisting of low glucose Dulbecco’s modified Eagle medium (DMEM; HyClone, USA), 10% fetal bovine serum (FBS; ExCell, China), 12 µM L-glutamine (Gibco, USA), and 1% penicillin-streptomycin (HyClone, USA). The marrow was then layered onto a MSC separation medium (Haoyang Biological Manufacture Co. Ltd Tianjin, China) and centrifuged at 400 g for 20 min at room temperature. The cells at the intermediate layer were subsequently recovered, washed twice with media, and seeded on a culture dish (Corning, USA) at a density of 4 × 105 cells/cm2. The MSCs were cultured at 37 ºC under a humidified 5% CO2 atmosphere. Nonadherent cells were removed with two to three washes with phosphatebuffered saline (PBS) after 24 h. The adherent cells (passage 0) were further cultured, and the medium was changed every 3 days. Passages were performed when the cells approached 80% confluence. MSCs were recovered using 0.25% trypsin/0.02% EDTA (Beyotime, China) and were subcultured at a density of 1 × 104 cells/cm2 as passage 1 cells. Once cells reached 80% confluence, they were passaged and then used for all experiments at the fourth passage.
2.4. Gene expression profile microarray studies
We used transcriptomic data from our previous study on the osteogenic induction mechanism of NHA using microarray approaches [31]. Briefly, MSCs were cultured on NHA for 24, 48 and 72 h. Cells cultured in a Petri dish were used as a control. Total RNA was extracted, and Agilent mouse whole genome oligo microarrays were used to detect the gene expression profiles (i.e. global expression profiling of mRNA transcripts) of MSCs in each sample. A total of 8992 differentially expressed genes were obtained, and 90 differentially expressed genes related to osteogenic differentiation were identified. The 90 genes were subsequently analyzed with bioinformatics software. In the previous study, the 8992 differentially expressed genes were not analyzed by Gene Microarray Pathway Profiler (GenMAPP). Here, the transcriptomics data 3
Z Zhang et al
Biomed. Mater. 9 (2014) 045005
(8992 differentially expressed genes) coupled with proteomics data were analyzed to obtain a comprehensive understanding of the osteoinduction mechanism of NHA.
primer sequences used in this study. The data were normalized to the internal control U6. All experiments were repeated three times. Relative miRNA expression levels were calculated using the delta delta Ct (2–∆∆Ct) method [38]. The –∆∆Ct was calculated with the following formula.
2.5. Proteomics research
− ΔΔCt = − ⎡⎣ ( Ct target miRNA − CtU6 )test–material
MSCs were cultured on NHA for 24, 48 and 72 h. Cells cultured in a Petri dish were used as a control. Cell lysis was performed to extract total proteins. The proteomics analysis was performed by Shanghai Bo-Yuan Biological Technology Co. Ltd (Shanghai, China). The prepared samples were labeled with 8-plex iTRAQTM (Applied Biosystems, USA) and analyzed using 2D strong cationic exchange/reversed phase liquid chromatography matrix-assisted laser desorption/ionizationtandem mass spectrometry (MDS Sciex 4800 PLUS, Applied Biosystems Inc. SCX/RP-HPLC-ESI-MS/MS). The iTRAQ data were searched with ProteinPilot software v3.0 (Applied Biosystems/MDS-Sciex, USA) for protein identification and quantification. The experiment was performed in two independent runs. An Unused ProtScore of 1.3 (Confidence, 95%) was used as a threshold in all experiments. The proteins with fold changes >1.2 were considered differentially expressed.
− ( Ct target miRNA − CtU6 )control ⎤⎦
To identify those miRNAs that potentially play a regulatory role in osteogenic-related pathways, the expression levels of miRNAs and target mRNAs/proteins were compared. Then, the regulatory effects of miRNAs on mRNA–protein pairs were analyzed. 2.8. Validation of potential osteogenic differentiation pathways
Through the comprehensive analysis of proteomic, transcriptomic and miRNA experiments, it was suggested that the ERK1/2 and JNK MAPK pathways may be potential NHAinduced osteogenic differentiation pathways. To validate these predictions, we performed the following experiments to determine whether there were detectable differences when these pathways were inhibited. Pathway-specific inhibitors (ERK inhibitor, U0126; JNK inhibitor, SP600125) were added to the complete medium for MSCs cultured on NHA, and after a set period of time, the expression of osteogenic markers was detected to evaluate the effect of the pathways on osteogenic differentiation.
2.6. Biological pathway analysis of integrated transcriptomics and proteomics data
An integrated set of transcriptomic [31] and proteomic data was characterized using GenMAPP (Gene Microarray Pathway Profiler, http://www.genmapp.org [36]), a program designed to view and analyze mRNA/protein expression data in the context of biological pathways.
2.8.1. Selection of specific inhibitor concentrations. The
impact of different inhibitor concentrations (0, 2.5, 5, 10, 20, 40 µM) on cell growth and proliferation was studied using the real-time cell electronic sensing (RT-CES) microelectronic cell sensor system (ACEA, USA [39]). MSCs were seeded in an RT-CES device (16-well strip) at 5000 cells/ well in complete medium in triplicate. After 24 h, the cells were treated with various concentrations of SP600125 or U0126 for another 144 h to determine the maximum amount of inhibitor that did not affect normal growth. Cell growth was monitored continuously and recorded as a cell index (CI) every 30 min. The cell index is derived from the change in electrical impedance as the living cells interact with the microelectrode surface in the well, effectively measuring cell number, shape and adherence.
2.7. Analysis of the regulatory function of miRNA in differentiation-related biological pathways
A pathway node was selected for further investigation if the expression of the single pathway node (represented by rectangles) in differentiation-related pathway maps had been measured at both the mRNA and protein levels using transcriptomics and proteomics, respectively. The miRGen database [37] (http://www.diana.pcbi.upenn.edu/miRGen.html) was used to predict miRNAs that could potentially target the selected pathway nodes. Following that, miRNAs targeting at least two pathway nodes were selected for quantitative investigation. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression of selected miRNAs. Total RNA from MSCs cultured on NHA and glass for 24, 48 and 72 h was extracted using RNAiso plus (TaKaRa, Japan), and the concentration and purity of the RNA samples were detected with a UV-spectrophotometer OD-1000 (One Drop, China). Then, the RNA samples were diluted to a final concentration of 500 ng/µl. All primers were designed based on miRNA sequences and used to convert the RNA template into cDNA with a 2720 Thermal Cycler (Applied Biosystems Inc. USA). Then, PCR amplification was performed on a 7500 Real-Time PCR system (Applied Biosystems Inc. USA). Supplementary table S1 shows the
2.8.2. Effects of specific inhibitors on the expression of osteogenic markers. MSCs were seeded at a density of
2.5 × 104 cells/ml and cultured in complete medium containing SP600125 (JNK inhibitor) or U0126 (ERK inhibitor). Total RNA from MSCs cultured on NHA or glass for 21d was extracted and detected in the same manner as described above. qRT-PCR was used to detect the expression of mRNAs for Col1, Gpnmb (also referred to as osteoactivin) and Bmp2. In recent years, it has become clear that no single gene is constitutively expressed in all cell types and under all experimental 4
Z Zhang et al
Biomed. Mater. 9 (2014) 045005
Figure 1. FTIR spectrum of a disk-shaped NHA sample. Table 1. FTIR spectrum values of the standard HA and disk-shaped NHA samples (cm−1)
Sample
OH−
H2O
CO32−
PO43−
OH−
PO43−
HA standard spectrum Disk-shaped NHA samples
3570 3571
3441
1458
1090,1040,960 1092,1051,962
634 632
603,565 603,571
and retain the carbonate and poor crystalline apatite which exist in natural bone mineral, 650 ºC was selected in this study as the calcination temperature of powder material. In this study, compared with the standard HA FTIR spectrum, NHA exhibits an absorption peak of CO32− at 1458 cm−1 and not exhibits the absorption peak of organic component, which indicates that the prepared NHA only retained the inorganic component of natural bone. These are consistent with our previous results [4]. The presence of CO32− could potentially increase biodegradation properties and osteogenic capability [41]. Therefore, NHA may have a better biological function. Due to the crystallinity of HA increased with calcination temperatures from 600 to 900º C [42], there would be an increase in the crystallinity of NHA compared with natural bone mineral as a result of the calcination of 650 º C for 3 h. Figure 2 shows the SEM image of the NHA disc-shaped sample. There was clearly a loose and random porous microstructure. The microsphere-like particles were evenly distributed on the NHA disc-shaped surface, with cavities and pores of different sizes (approximately 100–500 nm).
conditions. Similarly, internal control gene expression has been reported to vary considerably. In this study, qRT-PCR data were normalized using the geometric average [40] of 6 internal control genes (Gapdh, β-actin, Oaz1, Rps29, Eef2 and Tbp). Supplementary table S2 shows the primer sequences used in this study. Relative mRNA expression levels were calculated using the delta delta Ct (2–ΔΔCt) method as described above [38]. 2.9. Statistical analyses
All quantitative results were expressed as the mean±SD from at least three experiments, except as indicated. A one-way analysis of variance (ANOVA) using the Origin6.0 software program was applied to assess the significance of the differences among the study groups. A p-value of less than 0.05 was considered significant, and a p-value of less than 0.01 was considered highly significant. 3. Results and discussion 3.1. Characterization of NHA samples
3.2. Flow cytometry
The FTIR spectrum of a disk-shaped sample is shown in figure 1 and table 1. The specific peaks of hydroxyapatite [e.g. the −OH peaks (3570 cm−1, 632 cm−1) and the –PO4 peaks (1092 cm−1, 1051 cm−1, 962 cm−1, 602 cm−1, and 571 cm−1)] were observed, confirming that the disk-shaped samples were HA. Three calcination temperatures (650, 850 and 1050 ºC) were chosen based on the curves of thermal gravimetric analysis in our previous study. The results showed that the organic component was effectively removed at all the three temperatures and the CO32− was present only at 650 and 850 ºC. The poor crystalline apatite structure was found at 650 ºC [4]. Therefore, in order to effectively remove organic component
Flow cytometric analysis showed that the tested cell samples typically expressed the cell adhesion molecules CD29 (>99%) and CD44 (>90%) but were negative for the typical lymphocytic marker CD14 (
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An integrated study of natural hydroxyapatite-induced osteogenic differentiation of mesenchymal stem cells using transcriptomics, proteomics and microRNA analyses
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Biomed. Mater. 9 045005 (http://iopscience.iop.org/1748-605X/9/4/045005) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 142.150.190.39 This content was downloaded on 18/11/2014 at 19:13
Please note that terms and conditions apply.
Biomedical Materials Biomed. Mater. 9 (2014) 045005 (13pp)
doi:10.1088/1748-6041/9/4/045005
An integrated study of natural hydroxyapatite-induced osteogenic differentiation of mesenchymal stem cells using transcriptomics, proteomics and microRNA analyses Zhiwei Zhang, Jiandan Wang and Xiaoying Lü1 State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, People’s Republic of China E-mail: [email protected] Received 27 January 2014, revised 14 May 2014 Accepted for publication 21 May 2014 Published 19 June 2014 Abstract
This work combined transcriptomics, proteomics, and microRNA (miRNA) analyses to elucidate the mechanism of natural hydroxyapatite (NHA)-induced osteogenic differentiation of mesenchymal stem cells (MSCs). First, NHA powder was obtained from pig bones and fabricated into disc-shaped samples. Subsequently, the proliferation and osteogenic differentiation of MSCs cultured on NHA were investigated. Then, proteomics was employed to detect the protein expression profiles of MSCs cultured on NHA, and the effect of NHA on MSCs was analyzed through an integrated pathway analysis (including proteomics and previous transcriptomics data) in which specific NHA-induced differentiation pathways were analyzed. The pathway nodes with expression data at both the mRNA and protein levels (mRNA–protein pairs) were filtered in differentiation-related pathways. miRNAs corresponding to these target mRNA–protein pairs were predicted, screened and tested, and the regulatory effects of miRNAs on mRNA–protein pairs were analyzed. Finally, the NHAinduced osteogenic pathways were verified. The results of an MTT assay and alkaline phosphatase (ALP) staining showed that the cell proliferation rate decreased and the osteogenic performance improved in the presence of NHA. By integrating transcriptomics and proteomics, the genes and proteins involved in 89 pathways were shown to be differentially expressed. Among them, 5 differentiation-associated pathways, in which 9 miRNAs and 8 regulated-target mRNA–protein zby inhibiting the target mRNA–protein pair HSPA8 in the MAPK signaling pathway, and miR26a and miR-26b might inhibit adipogenic differentiation by repressing the target mRNA–protein pair HMGA1 in the adipogenesis pathway. A verification experiment for the osteogenic pathway indicated that the ERK1/2 or JNK MAPK pathways might play an important role in NHA-induced osteogenic differentiation. In conclusion, NHA affected MSCs at both the transcriptional and translational levels, and MSC osteogenic differentiation eventually occurred through the MAPK and adipogenesis pathways, in which miRNAs and target mRNAs/proteins participated cooperatively. Keywords: natural hydroxyapatite, transcriptomics, proteomics, microRNA, osteoinduction, biological pathways (Some figures may appear in colour only in the online journal) S Online supplementary data available from stacks.iop.org/BMM/9/045005/mmedia 1
Author to whom any correspondence should be addressed.
1748-6041/14/045005+13$33.00
1
© 2014 IOP Publishing Ltd Printed in the UK
Z Zhang et al
Biomed. Mater. 9 (2014) 045005
1. Introduction
regulators of cell functions [26]. miRNAs function in the posttranscriptional and translational regulation of gene expression through a variety of mechanisms [27]. In addition, it has been found that miRNAs play an important role in the regulation of osteogenic differentiation [28, 29]. Therefore, miRNA research provides an interesting new perspective in the study of osteogenic differentiation mechanisms. To date, the biological effects of HA have been studied at the mRNA, protein and miRNA levels individually. However, mRNAs, proteins and miRNAs are interconnected parts of a whole and influence each other. mRNA, which is synthesized through gene transcription, serves as a template for protein synthesis; the order of the mRNA bases determines the protein that will be produced. miRNAs regulate the flow of genetic information by controlling protein translation or mRNA stability. Proteins, which eventually perform various biological functions, are the ultimate output of this genetic information flow; hence, mRNAs or miRNAs affect protein expression. Meanwhile, proteins are involved in the regulation of transcription and translation through complex feedback systems. Therefore, mRNAs, proteins and miRNAs form a complex network of reciprocal regulatory interactions [30]. By studying the expression and relationships of these three components (mRNAs, proteins and miRNAs) simultaneously, a more complete view of biological information flow from DNA to RNA to protein can be acquired. In addition, a panorama of the complex network of miRNAs, target mRNAs and target proteins involved in certain important biological pathways can be obtained. Therefore, each of the three components (mRNA, protein and miRNA) is an indispensable part of a complete osteogenic differentiation study. Integration of transcriptomics, proteomics and miRNA analyses could help elucidate the overall process of mesenchymal stem cells (MSCs) osteogenic differentiation more accurately and thoroughly. Due to its advantages of having extensive sources and being environmentally friendly and low cost, the preparation and biological properties of NHA have received attention from a growing number of researchers [2, 3, 10, 31]. Our group has been conducting a series of studies on NHA since 2001. NHA has been successfully extracted from pig bones, and the main chemical components of the prepared NHA were found to be identical to the standard HA characterized by Fourier transform infrared spectroscope (FTIR) and X-ray diffraction (XRD); however, there are several unique chemical compounds in NHA including CO32− and HPO42− [4]. A protein adsorption assay showed that the values of albumin/fibrinogen (RA/F) and albumin/IgG (RA/I) obtained for NHA were smaller than those obtained for synthesized HA, which indicated that NHA had better coagulation properties and would be favorable for bone repair [32]. In addition, NHA-induced osteogenic differentiation of MSCs was studied using transcriptomic technology combined with bioinformatics analysis, and 90 differentially expressed genes related to osteogenic differentiation were obtained [31], which indicated that NHA could regulate MSC osteogenic differentiation. Based on our previous research, this study aimed to further explore the osteoinduction mechanism of
Hydroxyapatite (HA), the main inorganic component of bone, has been widely used in human bone tissue restoration. Currently, there are two main methods of obtaining HA: 1) synthesis by chemical methods [1]; and 2) extraction from natural tissues such as bone, teeth, and scales [2–4], the product of which is defined as natural hydroxyapatite (NHA). Many studies have demonstrated that HA has good biocompatibility and osteoconductive performance [5, 6]. The osteoinductive properties of HA have also been reported widely. Using animal experiments, Heughebert first reported in 1988 the physicochemical characterization of the deposits associated with HA ceramics implanted in nonbone-forming sites; these deposits showed striking similarity to the inorganic bone phase [7]. Subsequently, studies have proven that HA has osteoinductive properties in many mammals [8, 9]. Commercial NHA (Bio-Oss) derived from bovine bone has also been reported to possess osteoinductive properties when implanted in rats, rabbits and other animals [3, 10]. Moreover, NHA derived from antler cancellous bone is efficacious in inducing neovascularization and osteogenesis within rabbit mandible defects [3]. In vitro experiments showed that HA, including but not limited to NHA derived from scales, significantly promotes osteogenic differentiation in the presence of osteogenic medium [2, 11, 12]. Both HA-composite materials [13, 14] and HA-modified materials [15, 16] have shown superior performance in inducing osteogenic differentiation than those without HA. Furthermore, some experiments have shown that osteogenic markers are induced only in the presence of HA [17]. With the development of scientific technology, methods for investigating HA-induced osteogenic differentiation have evolved from observing bone-like surface deposition on a material [7] to detecting the expression levels of bone-related mRNAs or proteins including alkaline phosphatase (ALP), osteocalcin, type 1 collagen (COL1), etc [11, 18] and further to analyzing global mRNA or protein expression at the transcriptomic or proteomic level comprehensively and systematically [19–22]. To date, transcriptomic technology has been used to show that HA causes differential expression of several osteogenic markers [19] and impacts signaling pathways [20]. Proteomic research on HA has primarily focused on biological functions including cell adhesion, proliferation and calcium regulation [21, 22] rather than osteogenic differentiation. Although transcriptomic and proteomic technology can be used to comprehensively reveal overall changes at the molecular level, there may be incongruent expression between mRNAs and proteins [23, 24]. Our group also found significant differences between mRNA and protein expression levels in both quantity and type compared with the results from transcriptomic and proteomic analysis of the molecular biocompatibility of gold nanoparticles with human dermal fibroblasts [25]. Apart from the defects of omic techniques and the incompleteness of mRNA/protein databases, the uncorrelated result may be caused by the complex regulatory mechanisms involved in translating mRNA into mature proteins. microRNAs (miRNAs), which were discovered in 1993, are major 2
Z Zhang et al
Biomed. Mater. 9 (2014) 045005
NHA, which mainly consisted of studying the NHA-induced osteogenic differentiation pathway and the interaction mechanisms between miRNA and target mRNAs/proteins in those pathways through an integrated study using transcriptomics, proteomics and miRNA analyses.
2.2.2. Flow cytometry. Flow cytometric analysis was per-
formed to evaluate cell surface markers and MSC homogeneity. Briefly, fourth-passage cells were trypsinized, washed with PBS three times, and incubated with antibodies against CD14, CD29, CD34 and CD44 [33] (Biolegend, USA) at the recommended concentrations for 15 min; the appropriate isotype controls were also included. After being washed twice, the cells were resuspended in 300 µl FACS buffer and immediately analyzed with a flow cytometer (BD FACSCalibur, USA). A total of 10,000 events were acquired for each sample, and data analysis was performed using BD CellQuest Pro software.
2. Materials and methods 2.1. Preparation and characterization of NHA
NHA powder was extracted from pig bones using high-temperature calcination, as previously established by our group [4]. The procedure is described briefly below. The fresh porcine bone were cleaned to remove most organic component and thoroughly dried, and subsequently cut into smaller pieces. After that, the bone pieces were calcinated at 650 ºC for 3 h, and then crushed sufficiently with a grinder to obtain NHA powder material. The powder was compressed into disc-shaped samples with diameters of 2 and 10 cm using selfdesigned molds and subsequently calcined at 600 °C for 4 h to obtain NHA disc-shaped samples. The surface morphology of the disc-shaped sample was observed using a scanning electron microscope (Zeiss Ultra Plus, Germany), and the chemical composition was determined through Fourier Transform Infrared Spectroscopy (Nicolet5700, Thermo-Fisher, USA).
2.3. Proliferation and osteoinduction assay for MSCs cultured on NHA 2.3.1. Cell proliferation assay. The cell proliferation rate
(P%) on NHA disc-shaped samples with diameters of 2 cm was evaluated after culture for 24, 48 and 72 h using the methylthiazoltetrazolium (MTT) assay [34]. Cells cultured on glass slides with diameters of 2 cm were used as a negative control, and cells treated with 0.7% acrylamide were used as a positive control. 2.3.2. Osteoinduction assay for different factors. To evaluate the osteoinduction potential of two factors (chemicalinducible factor and NHA), MSCs at the fourth passage were treated with these two factors for 7 and 21 days. In the chemical-induced group, the cells were seeded on glass coverslips in 12-well plates (Corning, USA) with osteogenic differentiation medium [complete medium with 0.1 µM dexamethasone, 5 mM β-glycerophosphate, and 50 µg/ml ascorbic acid [35] (Sigma-Aldrich, Germany)]. In the NHA group, the cells were seeded on NHA in 12-well plates with complete medium. Cells cultured on glass coverslips with complete medium were used as controls. ALP staining was performed after 7 and 21 days to examine osteogenic differentiation using the ALP staining kit (Jiancheng, China) according to the manufacturer’s instructions. The stained cells were observed using a light microscope (Olympus BX51, Japan).
2.2. Isolation, culture and characterization of MSCs 2.2.1. Isolation and culture of MSCs. Mouse bone marrow-
derived MSCs were isolated and cultured as described below. Four-week-old Kunming mice (regardless of their gender) were injected intraperitoneally with heparin (3000 U per mouse) and sacrificed 5 min later by cervical dislocation in accordance with the rules of the Institutional Ethical Board for experimental procedures. Bone marrow was collected from the mice by flushing femurs and tibias with complete medium consisting of low glucose Dulbecco’s modified Eagle medium (DMEM; HyClone, USA), 10% fetal bovine serum (FBS; ExCell, China), 12 µM L-glutamine (Gibco, USA), and 1% penicillin-streptomycin (HyClone, USA). The marrow was then layered onto a MSC separation medium (Haoyang Biological Manufacture Co. Ltd Tianjin, China) and centrifuged at 400 g for 20 min at room temperature. The cells at the intermediate layer were subsequently recovered, washed twice with media, and seeded on a culture dish (Corning, USA) at a density of 4 × 105 cells/cm2. The MSCs were cultured at 37 ºC under a humidified 5% CO2 atmosphere. Nonadherent cells were removed with two to three washes with phosphatebuffered saline (PBS) after 24 h. The adherent cells (passage 0) were further cultured, and the medium was changed every 3 days. Passages were performed when the cells approached 80% confluence. MSCs were recovered using 0.25% trypsin/0.02% EDTA (Beyotime, China) and were subcultured at a density of 1 × 104 cells/cm2 as passage 1 cells. Once cells reached 80% confluence, they were passaged and then used for all experiments at the fourth passage.
2.4. Gene expression profile microarray studies
We used transcriptomic data from our previous study on the osteogenic induction mechanism of NHA using microarray approaches [31]. Briefly, MSCs were cultured on NHA for 24, 48 and 72 h. Cells cultured in a Petri dish were used as a control. Total RNA was extracted, and Agilent mouse whole genome oligo microarrays were used to detect the gene expression profiles (i.e. global expression profiling of mRNA transcripts) of MSCs in each sample. A total of 8992 differentially expressed genes were obtained, and 90 differentially expressed genes related to osteogenic differentiation were identified. The 90 genes were subsequently analyzed with bioinformatics software. In the previous study, the 8992 differentially expressed genes were not analyzed by Gene Microarray Pathway Profiler (GenMAPP). Here, the transcriptomics data 3
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(8992 differentially expressed genes) coupled with proteomics data were analyzed to obtain a comprehensive understanding of the osteoinduction mechanism of NHA.
primer sequences used in this study. The data were normalized to the internal control U6. All experiments were repeated three times. Relative miRNA expression levels were calculated using the delta delta Ct (2–∆∆Ct) method [38]. The –∆∆Ct was calculated with the following formula.
2.5. Proteomics research
− ΔΔCt = − ⎡⎣ ( Ct target miRNA − CtU6 )test–material
MSCs were cultured on NHA for 24, 48 and 72 h. Cells cultured in a Petri dish were used as a control. Cell lysis was performed to extract total proteins. The proteomics analysis was performed by Shanghai Bo-Yuan Biological Technology Co. Ltd (Shanghai, China). The prepared samples were labeled with 8-plex iTRAQTM (Applied Biosystems, USA) and analyzed using 2D strong cationic exchange/reversed phase liquid chromatography matrix-assisted laser desorption/ionizationtandem mass spectrometry (MDS Sciex 4800 PLUS, Applied Biosystems Inc. SCX/RP-HPLC-ESI-MS/MS). The iTRAQ data were searched with ProteinPilot software v3.0 (Applied Biosystems/MDS-Sciex, USA) for protein identification and quantification. The experiment was performed in two independent runs. An Unused ProtScore of 1.3 (Confidence, 95%) was used as a threshold in all experiments. The proteins with fold changes >1.2 were considered differentially expressed.
− ( Ct target miRNA − CtU6 )control ⎤⎦
To identify those miRNAs that potentially play a regulatory role in osteogenic-related pathways, the expression levels of miRNAs and target mRNAs/proteins were compared. Then, the regulatory effects of miRNAs on mRNA–protein pairs were analyzed. 2.8. Validation of potential osteogenic differentiation pathways
Through the comprehensive analysis of proteomic, transcriptomic and miRNA experiments, it was suggested that the ERK1/2 and JNK MAPK pathways may be potential NHAinduced osteogenic differentiation pathways. To validate these predictions, we performed the following experiments to determine whether there were detectable differences when these pathways were inhibited. Pathway-specific inhibitors (ERK inhibitor, U0126; JNK inhibitor, SP600125) were added to the complete medium for MSCs cultured on NHA, and after a set period of time, the expression of osteogenic markers was detected to evaluate the effect of the pathways on osteogenic differentiation.
2.6. Biological pathway analysis of integrated transcriptomics and proteomics data
An integrated set of transcriptomic [31] and proteomic data was characterized using GenMAPP (Gene Microarray Pathway Profiler, http://www.genmapp.org [36]), a program designed to view and analyze mRNA/protein expression data in the context of biological pathways.
2.8.1. Selection of specific inhibitor concentrations. The
impact of different inhibitor concentrations (0, 2.5, 5, 10, 20, 40 µM) on cell growth and proliferation was studied using the real-time cell electronic sensing (RT-CES) microelectronic cell sensor system (ACEA, USA [39]). MSCs were seeded in an RT-CES device (16-well strip) at 5000 cells/ well in complete medium in triplicate. After 24 h, the cells were treated with various concentrations of SP600125 or U0126 for another 144 h to determine the maximum amount of inhibitor that did not affect normal growth. Cell growth was monitored continuously and recorded as a cell index (CI) every 30 min. The cell index is derived from the change in electrical impedance as the living cells interact with the microelectrode surface in the well, effectively measuring cell number, shape and adherence.
2.7. Analysis of the regulatory function of miRNA in differentiation-related biological pathways
A pathway node was selected for further investigation if the expression of the single pathway node (represented by rectangles) in differentiation-related pathway maps had been measured at both the mRNA and protein levels using transcriptomics and proteomics, respectively. The miRGen database [37] (http://www.diana.pcbi.upenn.edu/miRGen.html) was used to predict miRNAs that could potentially target the selected pathway nodes. Following that, miRNAs targeting at least two pathway nodes were selected for quantitative investigation. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression of selected miRNAs. Total RNA from MSCs cultured on NHA and glass for 24, 48 and 72 h was extracted using RNAiso plus (TaKaRa, Japan), and the concentration and purity of the RNA samples were detected with a UV-spectrophotometer OD-1000 (One Drop, China). Then, the RNA samples were diluted to a final concentration of 500 ng/µl. All primers were designed based on miRNA sequences and used to convert the RNA template into cDNA with a 2720 Thermal Cycler (Applied Biosystems Inc. USA). Then, PCR amplification was performed on a 7500 Real-Time PCR system (Applied Biosystems Inc. USA). Supplementary table S1 shows the
2.8.2. Effects of specific inhibitors on the expression of osteogenic markers. MSCs were seeded at a density of
2.5 × 104 cells/ml and cultured in complete medium containing SP600125 (JNK inhibitor) or U0126 (ERK inhibitor). Total RNA from MSCs cultured on NHA or glass for 21d was extracted and detected in the same manner as described above. qRT-PCR was used to detect the expression of mRNAs for Col1, Gpnmb (also referred to as osteoactivin) and Bmp2. In recent years, it has become clear that no single gene is constitutively expressed in all cell types and under all experimental 4
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Figure 1. FTIR spectrum of a disk-shaped NHA sample. Table 1. FTIR spectrum values of the standard HA and disk-shaped NHA samples (cm−1)
Sample
OH−
H2O
CO32−
PO43−
OH−
PO43−
HA standard spectrum Disk-shaped NHA samples
3570 3571
3441
1458
1090,1040,960 1092,1051,962
634 632
603,565 603,571
and retain the carbonate and poor crystalline apatite which exist in natural bone mineral, 650 ºC was selected in this study as the calcination temperature of powder material. In this study, compared with the standard HA FTIR spectrum, NHA exhibits an absorption peak of CO32− at 1458 cm−1 and not exhibits the absorption peak of organic component, which indicates that the prepared NHA only retained the inorganic component of natural bone. These are consistent with our previous results [4]. The presence of CO32− could potentially increase biodegradation properties and osteogenic capability [41]. Therefore, NHA may have a better biological function. Due to the crystallinity of HA increased with calcination temperatures from 600 to 900º C [42], there would be an increase in the crystallinity of NHA compared with natural bone mineral as a result of the calcination of 650 º C for 3 h. Figure 2 shows the SEM image of the NHA disc-shaped sample. There was clearly a loose and random porous microstructure. The microsphere-like particles were evenly distributed on the NHA disc-shaped surface, with cavities and pores of different sizes (approximately 100–500 nm).
conditions. Similarly, internal control gene expression has been reported to vary considerably. In this study, qRT-PCR data were normalized using the geometric average [40] of 6 internal control genes (Gapdh, β-actin, Oaz1, Rps29, Eef2 and Tbp). Supplementary table S2 shows the primer sequences used in this study. Relative mRNA expression levels were calculated using the delta delta Ct (2–ΔΔCt) method as described above [38]. 2.9. Statistical analyses
All quantitative results were expressed as the mean±SD from at least three experiments, except as indicated. A one-way analysis of variance (ANOVA) using the Origin6.0 software program was applied to assess the significance of the differences among the study groups. A p-value of less than 0.05 was considered significant, and a p-value of less than 0.01 was considered highly significant. 3. Results and discussion 3.1. Characterization of NHA samples
3.2. Flow cytometry
The FTIR spectrum of a disk-shaped sample is shown in figure 1 and table 1. The specific peaks of hydroxyapatite [e.g. the −OH peaks (3570 cm−1, 632 cm−1) and the –PO4 peaks (1092 cm−1, 1051 cm−1, 962 cm−1, 602 cm−1, and 571 cm−1)] were observed, confirming that the disk-shaped samples were HA. Three calcination temperatures (650, 850 and 1050 ºC) were chosen based on the curves of thermal gravimetric analysis in our previous study. The results showed that the organic component was effectively removed at all the three temperatures and the CO32− was present only at 650 and 850 ºC. The poor crystalline apatite structure was found at 650 ºC [4]. Therefore, in order to effectively remove organic component
Flow cytometric analysis showed that the tested cell samples typically expressed the cell adhesion molecules CD29 (>99%) and CD44 (>90%) but were negative for the typical lymphocytic marker CD14 (
