The synergistic effect on osteogenic differentiation of human mesenchymal stem cell by diode laser-treated stimulating human umbilical vein endothelial cell
Chia-Tze Kao1,2, Tuan-Ti Hsu3, Tsui-Hsien Huang1,2, Yu-Tin Wu1,2, Yi-Wen Chen4,5,6, Ming-You Shie5,6
1School of Dentistry, Chung Shan Medical University, Taichung City 40447, Taiwan
2Department of Stomatology, Chung Shan Medical University Hospital, Taichung City 40447, Taiwan
3Institute of Oral Science, Chung Shan Medical University, Taichung City 40447, Taiwan
4Graduate Institute of Clinical Medical Science, China Medical University, Taichung City 40447, Taiwan
53D Printing Medical Research Center, China Medical University Hospital, Taichung City 40447, Taiwan
6Author to whom any correspondence should be addressed
Short title: Diode laser treated HUVEC stimulate the osteogenesis of hMSCs Classification numbers: 87
Correspondence:
Ming-You Shie, School of Dentistry, China Medical University, Taichung City 40447, Taiwan (E-mail: [email protected]; tel: +886-4-22052121; fax: +886-4- 24759065)
Abstract
Angiogenesis play an important role in determining the biostimulation of bone regeneration, either in new bone and blood vessel formation. Human umbilical cord cell (HUVEC) is the important effector cell in angiogenesis, which are indispensable for osteogenesis and their heterogeneity and plasticity. However, there are very few studies about the effects of HUVEC on diode laser stimulated-regulated osteogenesis. In this study, we used diode laser as a model biostimulation to examine the role of HUVEC on the laser stimulated osteogenesis. Several bone formation-related proteins were also significantly up regulated by the diode laser stimulation, indicating that HUVEC may participate in the diode laser stimulated osteogenesis. Interestingly, when human mesenchymal stem cells (hMSCs) cultured with HUVEC-diode laser-treated, the osteogenesis differentiation of hMSCs was significantly promoted, indicating the important role of HUVEC in diode laser-enhanced osteogenesis. Adequately activated HUVEC are vital for the success of diode laser-stimulated hard tissue regeneration. These findings provided valuable insights into the mechanism of diode laser-stimulated osteogenic differentiation, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of laser treatment in periodontal repair.
Keywords: Diode laser, human mesenchymal stem cell, human umbilical vein endothelial cell, co-culture, osteogenesis.
1. Introduction
Laser is classified as light amplification by stimulated involves the modification of the environment to stimulate existing bacteria capable of bioremediation [1]. It is believed that the main structures responsible for absorption of light are proteins and their photo-biomodulation involve reactions in receptor on cell membrane and organelles, which would generate affected the behavior of cellular metabolism [1,2]. Beyond the common effects of laser stimulation as improving tissue regeneration in skin, mucosa, and anti-inflammatory, due to its greater penetrating power is also used in inflammatory diseases of the joints, to treat muscle injuries, to repair peripheral nerves and to promote hard tissue regeneration [3-5]. The laser light was used in many fields including medical, industrial, and the military. In the clinical, there are several commercial lasers, including diode, CO2, and erbium (Er):yttrium aluminum garnet (YAG) lasers [6,7]. For some otorhinolaryngological indications, fiber controlled diode lasers are preferred over conventional laser systems for reasons of practicability [8]. In recent year, certified medical 1,470 nm diode laser system has been introduced clinically [8,9]. Due to its absorbance profile within human tissue, the system is expected to offer both ablative and coagulative tissue effects. In addition, some studies shown that lasers affect cell proliferation, collagen synthesis and reduce inflammation [6,10]. In addition, the laser light can promote periodontal cell differentiation and it has potentially be used to enhance periodontal tissue regeneration [5,6].
Human umbilical vein endothelial cells (HUVEC) and other endothelial cells contribute to osteogenic through the secretion of a wide range of regulatory biomolecules, such as osteoprotegerin (OPG) [11], vascular endothelial growth factor
(VEGF) [12] and bone morphogenetic proteins (BMP-2) [13]. These cytokines, growth factors, and hormones result in an imbalance between osteoblast and osteoclast activities and can result in skeletal abnormalities, such as osteopetrosis and osteoporosis [14]. In addition, it is well known that bone remodeling is regulated by the replacement of old bone with new bone through sequential osteoblastic bone formation and osteoclastic resorption [15]. The selective and cultivation differentiation of human mesenchymal stem cells (hMSCs) could supply further understanding of this important progenitor of multiple type tissue and the potential of novel therapeutic methods for the restoration of injured or unhealthy tissue [16]. Given the important roles of HUVEC in the bone formation, some studies have analyzed the interactions between laser treatment and HUVEC. However, these studies are focused on either the inflammatory contributions or the differentiation into osteoclasts under various laser treatment.
It has recently been reported that the osteogenesis of hMSCs is greatly promoted by being co-cultured with HUVEC in 3D scaffolds [17-19]. These studies have confirmed that the extent of cell-cell communication between hMSCs and HUVEC in a co-culture system in combination with secreted cytokines, such as BMP, VEGF and fibroblast growth factor (FGF), may increase the osteogenic differentiation of hMSCs.
Interestingly, a paracrine effect through osteogenic factors has also been observed in the indirect contact co-culture of macrophage cells and bone cells in previous studies [20- 22]. However, implantation of a host inflammatory and immune reaction generally described as the foreign body response. Several pro-inflammatory cytokines secreted by HUVEC are immediately up-regulated post-injury in the presence of a foreign materials [23,24]. These soluble growth factors are recognized by the same cells in autocrine, and
neighboring cells in paracrine [24]. HUVEC and hMSCs during bone formation via soluble autocrine and paracrine signals as well as juxtacrine signals associated with direct cell-cell contacts [20].
The diode laser treatment is well recognized as the osteoconductive stimulation and has been widely used for clinical dentistry regeneration application. Although there are many studies about the in vitro and in vivo osteogenesis of diode laser, the effect of diode laser treatment on the cellular behavior of HUVEC is unclear, and the effect of HUVEC on laser-coordinated osteogenesis of hMSCs has not been demonstrated. Therefore, the aim of this study, the diode laser is used as a model to investigate the interactions between laser-treatment and HUVEC, and to further reveal whether HUVEC participate in the laser-stimulated osteogenesis of hMSCs.
2. Materials and Methods 2.1 Cell culture
Human mesenchymal stem cells and human umbilical vein endothelial cells were obtained from Bioresources Collection and Research Center (BCRC, Taiwan). Cells were cultured in T-25 cultured flasks with different medium (hMSCs: StemPro® MSC SFM basal medium requires supplementation with StemPro® MSC SFM xenoFree supplement; HUVEC: Medium 200 (Gibco) supplementation with low serum growth supplement), which were comprised of L-glutamine, and penicillin–streptomycin mixture. All cells were subculture by successive passaging at a 1:3 ratio until they were used for experiments (passages 3–8).
2.2 Fabrication of cell co-cultured device
The 3D printed cell co-cultured devices were designed using AutoCAD 2013 software (Autodesk, Inc., San Rafael, CA). The 3D CAD model was created using software and saved as stereolitography (.stl) file allowing direct import into the printer software (Fig. 1A). The co-cultured devices were printed on a Connex 500 printer (Stratasys, Edina, MN, USA) using biocompatible material (Objet Med610, Stratasys) (Fig. 1B).
2.3 Cytotoxicity
To compare the cytotoxicity of hMSCs and HUVEC between tissue culture plate (TCP) and 3D printed device (3DP), the cells were resuspended in 48 well (GeneDireX) with a DMEM for 1, 2, and 3 days. After different culture times, cell viability was
evaluated by the PrestoBlue® assay (Invitrogen, Grand Island, NY). Briefly, at the end of the culture period, the medium was discarded and the wells were washed with cold PBS twice. Each well was filled with medium with a 1:9 ratio of PrestoBlue® in fresh DMEM and incubated at 37°C for 30 min. The solution in each well was transferred to a new 96- well plate. Plates were read in a multi-well spectrophotometer (Hitachi, Tokyo, Japan) at 570 nm with a reference wavelength of 600 nm. The results were obtained in triplicate from three separate experiments for each test.
2.4 HUVEC viability
After HUVEC culture on 3DP device for 1 day, then treatment with diode laser system (SIROLaser Advance, Sirona Dental Systems GmbH, Bensheim, Germany) that emits energy at 980 nm for 1 w, 0.002s with 1, 2, and 3 times. And, we controlled the diameter of the laser spot was 5 mm. After different culture times, cell viability was evaluated by the PrestoBlue® assay (Invitrogen, Grand Island, NY). Briefly, at the end of the culture period, the medium was discarded and the wells were washed with cold PBS twice. Each well was filled with medium with a 1:9 ratio of PrestoBlue® in fresh DMEM and incubated at 37°C for 30 min. The solution in each well was transferred to a new 96- well plate. Plates were read in a multi-well spectrophotometer (Hitachi, Tokyo, Japan) at 570 nm with a reference wavelength of 600 nm. The results were obtained in triplicate from three separate experiments for each test.
2.5 The inflammatory protein of HUVEC
After HUVEC culture on 3DP device for 1 day, the culture medium was removed and treatment with diode laser (1 w, 0.002s, 1 and 2 times). Cells without treatment with diode laser was used as a control. After cultured for different time-points, the cell lysates of all groups was analysis IL-1, TNF-α, and IL-10 proteins by enzyme-linked immunosorbent assay kit (ELISA, Invitrogen).
2.6 The osteogenic protein secretion from HUVEC
After HUVEC culture on 3DP device for 1 day, the culture medium was removed and treatment with diode laser (1 w, 0.002s, 1 and 2 times). Cells without treatment with diode laser was used as a control. After treatment for different time-points, OPG, MMP- 3, and VEGF protein released from cells were determined by ELISA kit (Invitrogen) following the manufacturer’s instruction. The protein concentration was measured by correlation with a standard curve. All experiments were done in triplicate.
2.7 The proliferation of hMSCs co-cultured with diode laser-tread HUVEC
After hMSCs and HUVEC culture on 3DP device for 1 day, the culture medium was removed and HUVEC treatment with diode laser (1 w, 0.002s, 1 and 2 times). The hMSCs cultured without HUVEC was used as a control. At the end of the culture period, the medium was discarded and the wells were washed with cold PBS twice. The proliferation of hMSCs was evaluated by PrestoBlue® assay.
2.5 Effects of HUVEC with diode laser on the osteogenic differentiation of hMSCs
Cells were seeded in co-cultured device at a density of 4 x 10 cells per well for 1 day, and the culture medium was removed and HUVEC treatment with diode laser (1 w, 0.002s, 1 and 2 times). The detection of bone-related gene [alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC)] and protein expression (ALP, OC) of hMSCs cells at the end of the culture period. Total RNA of all four groups was extracted using TRIzol reagent (Invitrogen) after 3 days and analyzed by RT-qPCR. Total RNA (500 ng) was used for the synthesis of complementary DNA using cDNA Synthesis Kit (GenedireX) following the manufacturer’s instructions. RT-qPCR primers (Table 1) were designed based on cDNA sequences from the NCBI Sequence database. SYBR Green qPCR Master Mix (Invitrogen) was used for detection and the target mRNA expressions were assayed on the ABI Step One Plus real-time PCR system (Applied Biosystems, Foster City, California, USA). Each sample was performed in triplicate.
In addition, the ALP activity was determined after cell cultured for 7 days. The process was as follows: the cells were lysed from dish using 0.2 % NP-40, and centrifuged for 10 min at 2000 rpm after washing with PBS. ALP activity was determined using p-nitrophenyl phosphate (pNPP, Sigma) as the substrate. Each sample was mixed with pNPP in 1 M diethanolamine buffer for 15 min, after which the reaction was stopped by the addition of 5 N NaOH and quantified by absorbance at 405 nm. All experiments were done in triplicate. The OC protein released from cells were determined by ELISA kit (Invitrogen) following the manufacturer’s instruction. The protein concentration was measured by correlation with a standard curve.
2.6 The mineralization of hMSCs
The accumulated calcium deposition after 7 and 14 days was analyzed using Alizarin Red S staining, as in a previous study [25]. In brief, the cells were fixed with 4%
paraformadedyde (Sigma-Aldrich) for 15 min and then incubated in 0.5% Alizarin Red S (Sigma-Aldrich) at pH 4.0 for 15 min at room temperature in an orbital shaker (25 rpm).
After the cells were washed with PBS, photographs were observed using an optical microscope (BH2-UMA, Olympus, Tokyo, Japan) equipped with a digital camera (Nikon, Tokyo, Japan) at 200x magnification. To quantify the stained calcified nodules after staining, samples were immersed with 1.5 mL of 5% SDS in 0.5N HCl for 30 min at room temperature. After that, the tubes were centrifuged to 5,000 rpm for 10 min and the supernatant was transferred to the new 96-well plate (GeneDireX); absorbance was measured at 405 nm (Hitachi).
2.7 Statistical Analysis
A one-way variance statistical analysis was used to evaluate the significance of the differences between the groups in each experiment. Scheffe’s multiple comparison test was used to determine the significance of the deviations in the data for each specimen. In all cases, the results were considered statistically significant with a p value < 0.05.
3. Results
3.1 Cytotoxicity of 3D printed substrate
The cell viability of hMSCs (Fig. 1C) and HUVEC (Fig. 1D) was similar (p > 0.05) between the 3DP substrate and TCP after cultured for different time-points. It means the 3D printed co-cultured device is the biocompatibility material.
3.2 HUVEC viability
HUVEC were treated with the diode laser for various times (1, 2, and 3 times) for different days. The results showed that the HUVEC viability was similar (p > 0.05) between the diode laser-treatment for 1 times groups and Ctl (Fig 2) for all time-points.
Interesting, the cell proliferation of HUVEC treated with diode laser for 2 times was significantly increased than other groups. PrestoBlue® analysis showed that the overall metabolic activity of most groups with laser-treatment increased in a time-dependent manner. However, the viability of HUVEC in the presence of laser-treatment for 3 times significantly decreased than all groups (p < 0.05).
3.3 Inflammatory protein expression of HUVEC
The inflammatory protein of TNF-α (Fig 3A) was showed no significant change after diode laser stimulation with HUVEC. On the contrary, IL-1 (Fig 3B) was down regulated significantly with the treatment with diode laser for 1 and 2 times (p < 0.05).
The anti-inflammatory protein IL-10 expression was showed similar trend after stimulation (Fig. 3C).
3.4 Osteogenic protein secretion from HUVEC
OPG, MMP-3, and VEGF proteins secretion were up regulated by the stimulation of diode laser (p < 0.05) (Fig 4). The OPG, MMP-3, and VEGF secretion from HUVEC cells with laser-treated for 2 times were promoted 44%, 31%, and 34% (p < 0.05) than Ctl. In addition, these proteins were increased in a time-dependent manner.
3.5 The proliferation of hMSCs in co-cultured system
Fig. 5 shows the proliferation of hMSCs cultured with laser treated HUVEC on co- cultured system after culture for 1, 2, and 3 days. The hMSCs tend to increase with culture time from 1 to 3 days. Such enhanced efficiency of hMSCs proliferation with laser-treated HUVEC compared to hMSCs alone is consistent with the enhanced trend in the amount of cytokines release from HUVEC. Furthermore, cell proliferation of hMSCs was significantly (p < 0.05) promoted with HUVEC-laser treatment for 1 and 2 times.
3.6 Osteogenic differentiation of hMSCs
In the present study, the mRNA expression of ALP (Fig. 6A), BSP (Fig. 6B) and OC (Fig. 6C) in hMSCs was detected to be improved by co-cultured with laser treated- HUVEC, especially in 1 and 2 times group (p < 0.05). Because of the extended time of HUVEC-later treatment, the expression of ALP mRNA on hMSCs was up-regulated with HUVEC for 3 days. The expression of BSP and OC was up-regulated from day 3 and 7 with laser-treated HUVEC. Both ALP quantitative activity (Fig. 7A) and OC (Fig. 7B) assays showed that the ALP and OC were promoted by HUVEC-laser treated for 1 times at day 3. At day 7, though ALP activity and OC of hMSCs were up-regulated cultured
with HUVEC. Moreover, the secretion of OC from hMSCs cultured with laser treated- HUVEC for 0, 1, and 2 times was significantly increased 5.3%, 17.1%, and 25.4% (p <
0.05) than Ctl. Alizarin Red S staining showed evidence of calcium deposition and nodule formation. Mineralized nodules formation was observed in all groups at day 14 (Fig. 8A). In addition, the calcium deposits are found in the body, particularly in the hard tissues. Fig. 8B demonstrates that the quantified calcium content increased after 14 d of differentiation. More distinct nodules were observed in hMSCs stimulated by HUVEC with laser-treatment relative to cell alone (p < 0.05). The calcium deposits in HUVEC- laser treated for 2 time groups were the highest among the other groups. The formation of calcium phosphate salts or mineral deposits is the primary function of the osteoblast cells.
4. Discussion
Dental caries is one of the most common chronic disorders throughout the world [26]. In the oral environment, there were several types stem cells include dental pulp stem cell, periodontal ligament stem cells, mesenchymal stem cells and stem cells from the apical papilla [27-29]. Gronthos et al. was first proved that stem cells isolated from adult dental pulp tissue could be cultured in vitro, and this type of cell could differentiate into osteoblasts and could form dentine-like tissue [30]. The hMSCs are thought to be multi- potent cells that have the ability to proliferate extensively, which can replicate maintain the potential to differentiate into various cell types in vitro, including bone, cartilage, and fat [19,31]. In addition, several studies have showed the use of angiogenic growth factors such as VEGF and FGF-2 in promoting osteogenesis during dentin/pulp regeneration [32,33]. Thus, the endothelial cells play an important role in the interaction between bone regeneration and angiogenic differentiation. HUVEC belongs to the human endothelial cell line with great abilities to differentiate toward vascular tube. During the incubation period, HUVEC populating in this 3DP substrate exhibited a satisfying growth status as expected. Therefore, diode laser can simulate the significant proliferation of HUVEC.
HUVEC and other endothelial cells were both expressed OPG. Analysis of human tissues showed that endothelial cells in normal surrounding tissue express OPG. There is a strong positive correlation between endothelial OPG expression and ER expression [11].
Previous study had shown that VEGF release (MMP-dependent) results in the formation of a more regular vasculature compared with passive VEGF release via diffusion [12].
Several studies on multi-type cell co-cultured have been proved to achieve promoted cellular differentiation-related expression that deeply relies on the interactive
biological stimulation between cells [34]. For example, during the bone regeneration process, osteoblast differentiation can be stimulated by introducing a blood vessel forming cell system that in the meanwhile can be stimulated by surrounding osteoblast−ECM environment to develop into blood vessel formation [35]. Therefore, this interaction also existed between hMSCs and diode laser-treated HUVEC in our modular system and promoted cell-specific differentiation. The effects are commonly attributed to an inflammatory response and diode laser stimulation. While these effects are undoubtedly important, our results further revealed that in response to the diode-laser stimulation, leading to the secretion of osteoinductive proteins and anti-inflammatory cytokines from HUVEC, which promoted the osteogenesis of hMSCs. These findings demonstrate that HUVEC participated and made important contributions to the diode- laser stimulated osteogenesis. Although HUVEC have been confirmed to be involved in the osteogenesis, there is still no consensus on which phenotype is more useful for the osteogenic differentiation. In the current study, immune response was evaluated by measuring the expression of TNF-α, IL-1 and IL-10, and HUVEC decreased IL-1 expression when irradiated with diode-laser as compared to those in the control environment. IL-1 is the early “immune” cytokine identified to be involved in the control of bone regeneration and promoted pre-osteoclast cell behavior. It is also a typical example of multifunctional cytokines that are activated by environmental stress [36]. In addition, IL-1 activation of the iNOS pathway in cells, and iNOS is only expressed in responses to inflammatory stimuli [37]. Similar studies have shown that inflammatory responses to laser irradiation was shown to inhibit prostaglandin E2 and IL-1β production
and gene expression; the inhibition of the prostaglandin E2 and IL-1β might be of therapeutic value [38].
During bone formation, the endothelial cells participate in both ends, via secreting relating cytokines. They may contribute osteoinductive and osteogenic cytokines (BMP2, VEGF) to promote osteogenesis, but also assist inflammatory and fibrous agents (TNF-α, VEGF, TGF-β) to increase pathological fibrosis [38]. In present results, HUVEC played an important role in regulating osteogenesis. Possible effects of HUVEC in the process of diode-laser promote osteogenesis are summarized in Fig. 9. The diode-laser stimulated HUVEC were result in the down-regulation inflammatory cytokines (IL-1). The laser stimulated HUVEC also up-regulated OPG, MMP-3, and VEGF protein secretion.
Osteogenic differentiation is generally accompanied by the production of ALP, BSP, OC, and in vitro mineralization [39]. In the early stage of bone formation progress, ALP played a major role in the initiation of osteogenic differentiation, and ALP expression was up regulated after the start of differentiation. As an example, ALP protein expression of hMSCs was similar to gene expression [40]. The explanation for the low number of increments seen in ALP activity with culture time may be an indicator of the cell progressing into biomineralization. OC is the most abundant non-collagenous bone- matrix protein and is secreted at the late stage in osteoblast differentiation and mineralization [41]. The hMSCs cultured with laser treatment-HUVEC demonstrated significantly increased BSP and OC expression levels with increased treatment time compared to those without HUVEC. The results of the current study consistently indicated that the presence of diode-laser-stimulated HUVEC was effective in supporting the differentiation of hMSCs and strongly enhanced a biological response in cells through
the production of bone formation-relative proteins [42]. Therefore, initiation of the osteogenesis effect could be observed after cellular inflammation lessened. It is reasonable to assume that the alveolar bone had good osteogenesis abilities on the diode laser treatment.
The confirmation that HUVEC behavior actively during the osteogenic differentiation caused by diode laser is meaningful. Inadequately activated HUVEC will influence failure of bone formation, leading to undesired inflammatory fibrosis. The HUVEC should be taken into consideration for evaluating the in vitro osteogenic capacity of diode laser-treatment therapy. At low-power densities, the energy fluence range from 1 to 10 Jcm−2, and frequently, the photochemical interactions can influence the biostimulation of soft tissues. In dental practice, the orthodontist treatment is an increasingly daily occurrence. From our present study, the diode laser can be used a non- invasive, painless and a non-thermal therapy that repaired tissue functionality through its biostimulation, regenerative and anti-inflammatory effects. Moreover, we provide that although most diode laser increased the cytokine secretion from HUVEC to influence behavior of hMSCs. Consequently, diode laser alters cell signal transduction and outcomes through similar mechanoreceptors and signaling effectors in cells, and it is therefore deduced that common signaling mechanisms are involved in laser-transduction pathways.
5. Conclusion
HUVEC played important roles in the diode laser treatment-induced osteogenesis.
The product from diode laser-treated HUVEC inhibits the inflammation and promotes the osteogenic differentiation of hMSCs. Adequately activated HUVEC are vital for the success of diode laser-stimulated hard tissue regeneration. The interaction with endothelial cells, especially HUVEC, should be elucidated when evaluating the in vitro osteogenic capacity of laser treatment in periodontal repair.
Acknowledgements
The authors acknowledge receipt of a grant from the National Science Council grants (NSC 102-2314-B-040-007-MY3) of Taiwan.
.
Author disclosure statement
The authors declare no competing financial interests.
References
[1] Tosun E, Tasar F, Strauss R, Kıvanc D G and Ungor C 2012 Comparative evaluation of antimicrobial effects of Er:YAG, diode, and CO2 lasers on titanium discs: an experimental study J Oral Maxillofac Surg 70 1064–9
[2] de Oliveira A M, Castro-Silva I I, de Oliveira Fernandes G V, Melo B R, Alves A T N N, Silva Júnior A, Lima I C B and Granjeiro J M 2013 Effectiveness and acceleration of bone repair in critical-sized rat calvarial defects using low-level laser therapy Lasers Surg Med 46 61–7
[3] Hsu T T, Kao C T, Chen Y W, Huang T H, Yang J J and Shie M Y 2015 The synergistic effects of CO2 laser treatment with calcium silicate cement of antibacterial, osteogenesis and cementogenesis efficacy Laser Phys Lett 12 055602
[4] Kuo C L, Kao C T, Fang H Y, Huang T H, Chen Y W and Shie M Y 2015 Antiosteoclastogenesis activity of a CO2 laser antagonizing receptor activator for nuclear factor kappaB ligand-induced osteoclast differentiation of murine
macrophages Laser Phys Lett 12 035681
[5] Huang T H, Chen C C, Liu S L, Lu Y C and Kao C T 2014 A low-level diode laser therapy reduces the lipopolysaccharide (LPS)-induced periodontal ligament cell inflammation Laser Phys Lett 11 075602
[6] Huang T H, Liu S L, Chen C L, Shie M Y and Kao C T 2013 Low-level laser effects on simulated orthodontic tension side periodontal ligament cells Photomed Laser Surg 31 72–7
[7] Hsu T T, Yeh C H, Kao C T, Chen Y W, Huang T H, Yang J J and Shie M Y 2015 Antibacterial and odontogenesis efficacy of mineral trioxide aggregate combined with CO2 laser treatment J Endod 41 1073–80
[8] Havel M, Betz C S, Leunig A and Sroka R 2014 Diode laser-induced tissue effects: in vitro tissue model study and in vivo evaluation of wound healing following non-contact application Lasers Surg Med 46 449–55
[9] Havel M, Sroka R, Englert E, Stelter K, Leunig A and Betz C S 2012
Intraindividual comparison of 1,470 nm diode laser versus carbon dioxide laser for tonsillotomy: A prospective, randomized, double blind, controlled feasibility trial Lasers Surg Med 44 558–63
[10] Huang T H, Chen C L, Hung C J and Kao C T 2012 Comparison of antibacterial activities of root-end filling materials by an agar diffusion assay and Alamar blue assay J Dent Sci 7 336–41
[11] Weichhaus M, Chung S T M and Connelly L 2015 Osteoprotegerin in breast cancer: beyond bone remodeling Mol. Cancer 14 117
[12] Browne S and Pandit A 2014 Multi-modal delivery of therapeutics using biomaterial scaffolds J Mater Chem B 2 6692–707
[13] Pettit A R, Chang M K, Hume D A and Raggatt L-J 2008 Osteal macrophages: a new twist on coupling during bone dynamics Bone 43 976–82
[14] Boyle W J, Simonet W S and Lacey D L 2003 Osteoclast differentiation and activation Nature 423 337–42
[15] Eriksen E F 2010 Cellular mechanisms of bone remodeling Rev Endocr Metab Disord 11 219–27
[16] Eskildsen T, Taipaleenmäki H, Stenvang J, Abdallah B M, Ditzel N, Nossent A Y, Bak M, Kauppinen S and Kassem M 2011 MicroRNA-138 regulates
osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo.
Proc Natl Acad Sci USA 108 6139–44
[17] LI H and Chang J 2013 Bioactive silicate materials stimulate angiogenesis in fibroblast and endothelial cell co-culture system through paracrine effect Acta Biomater 9 6981–91
[18] Kang Y, Kim S H, Fahrenholtz M, Khademhosseini A and Yang Y J 2013 Osteogenic and angiogenic potentials of monocultured and co-cultured human- bone-marrow-derived mesenchymal stem cells and human-umbilical-vein endothelial cells on three-dimensional porous beta-tricalcium phosphate scaffold Acta Biomater 9 4906–15
[19] Yeh C H, Chen Y W, Shie M Y and Fang H Y 2015 Poly(dopamine)-assisted immobilization of Xu Duan on 3D printed poly(lactic acid) scaffolds to up- regulate osteogenic and angiogenic markers of bone marrow stem cells Materials 8 4299–315
[20] Chen Z, Wu C, Gu W, Klein T, Crawford R and Xiao Y 2014 Osteogenic differentiation of bone marrow MSCs by β-tricalcium phosphate stimulating macrophages via BMP2 signalling pathway Biomaterials 35 1507–18
[21] Schröder H C, Wang X H, Wiens M, Diehl-Seifert B, Kropf K, Schloßmacher U and Müller W E G 2012 Silicate modulates the cross-talk between osteoblasts (SaOS-2) and osteoclasts (RAW 264.7 cells): Inhibition of osteoclast growth and differentiation J Cell Biochem 113 3197–206
[22] Tu M G, Chen Y W and Shie M Y 2015 Macrophage-mediated osteogenesis activation in co-culture with osteoblast on calcium silicate cement J Mater Sci:
Mater Med 26 276
[23] Holt D J, Chamberlain L M and Grainger D W 2010 Cell–cell signaling in co- cultures of macrophages and fibroblasts Biomaterials 31 9382–94
[24] Schutte R J, Xie L, Klitzman B and Reichert W M 2009 In vivo cytokine- associated responses to biomaterials Biomaterials 30 160–8
[25] Chen Y W, Yeh C H and Shie M Y 2015 Stimulatory effects of the fast setting and degradable Ca–Si–Mg cement on both cementogenesis and angiogenesis differentiation of human periodontal ligament cells J Mater Chem B 3 7099–108 [26] Qu T and Liu X 2013 Nano-structured gelatin/bioactive glass hybrid scaffolds
for the enhancement of odontogenic differentiation of human dental pulp stem cells J Mater Chem B 1 4764–72
[27] Li X, Hou J, Wu B, Chen T and Luo A 2014 Effects of platelet-rich plasma and cell coculture on angiogenesis in human dental pulp stem cells and endothelial progenitor cells J Endod 40 1810–4
[28] Ju Y, Ge J, Ren X, Zhu X, Xue Z, Feng Y and Zhao S 2015 Cav1.2 of L-type calcium channel Is a key factor for the differentiation of dental pulp stem cells J Endod 41 1048–55
[29] Qin Z, Li Y, Li Y and Liu G 2015 Tumor necrosis factor alpha stimulates proliferation of dental pulp stem cells via Akt/glycogen synthase kinase- 3β/cyclin D1 signaling pathway J Endod 41 1066–72
[30] Gronthos S, Mankani M, Brahim J, Robey P G and Shi S 2000 Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo Proc Natl Acad Sci USA 97 13625–30
[31] Kao C T, Lin C C, Chen Y W, Yeh C H, Fang H Y and Shie M Y 2015
Poly(dopamine) coating of 3D printed poly(lactic acid) scaffolds for bone tissue engineering Mater Sci Eng C Mater Biol Appl 56 165–73
[32] Liu C H, Huang T H, Hung C J, Lai W Y, Kao C T and Shie M Y 2014 The synergistic effects of fibroblast growth factor-2 and mineral trioxide aggregate on an osteogenic accelerator in vitro Int Endod J 47 843–53
[33] Park J Y, Shim J H, Choi S A, Jang J, Kim M, Lee S H and Cho D W 2015 3D printing technology to control BMP-2 and VEGF delivery spatially and
temporally to promote large-volume bone regeneration J Mater Chem B 3 5415–
25
[34] Chen Y J, Shie M Y, Hung C J, Liu S L, Huang T H and Kao C T 2015 Osteoblasts subjected to tensile force inducing osteoclastic differentiation of murine macrophage in a co-culture system J Dent Sci 10 81–7
[35] Huang S C, Wu B C, Kao C T, Huang T H, Hung C J and Shie M Y 2015 Role of the p38 pathway in mineral trioxide aggregate-induced cell viability and angiogenesis-related proteins of dental pulp cell in vitro Int Endod J 48 236–45 [36] Kyriakis J M and Avruch J 2001 Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation Physiol Rev 81 807–69
[37] Hung C J, Kao C T, Shie M Y and Huang T H 2014 Comparison of host
inflammatory responses between calcium-silicate base material and intermediate restorative material J Dent Sci 9 158–64
[38] Nomura K, Yamaguchi M and Abiko Y 2001 Inhibition of interleukin-1beta production and gene expression in human gingival fibroblasts by low-energy laser irradiation Lasers Med Sci 16 218–23
[39] Lai W Y, Chen Y W, Kao C T, Hsu T T, Huang T H and Shie M Y 2015 Human dental pulp cells responses to apatite precipitation from dicalcium silicates Materials 8 4491–504
[40] Huang M H, Kao C T, Chen Y W, Hsu T T, Shieh D E, Huang T H and Shie M Y 2015 The synergistic effects of chinese herb and injectable calcium silicate/b- tricalcium phosphate composite on an osteogenic accelerator in vitro J Mater Sci: Mater Med 26 161
[41] Mizuno M and Kuboki Y 2001 Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen J Biochem 129 133–8
[42] Hung C J, Hsu H I, Lin C C, Huang T H, Wu B C, Kao C T and Shie M Y 2014 The role of integrin αv in proliferation and differentiation of human dental pulp cell response to calcium silicate cement J Endod 40 1802–9
Figure Legends
Figure 1. The hMSCs/HUVEC co-cultured system. (A) The top view and side view of 3D printed substrates. (B) The photograph of 3D printed substrates. (C) The cytotoxicity of hMSCs and HUVEC cells cultured on tissue culture plate (TCP) and 3D printed substrates (3DP).
Figure 2. The PrestoBlue® assay performed for viability HUVEC cells treated with diode laser for different times. *p < 0.05, compared with Ctl.
Figure 3. (A) TNF-α, (B) IL-1, and (C) IL-10 secreted form HUVEC cells after treatment with diode laser for different times. *p < 0.05, compared with Ctl.
Figure 4. (A) OPG, (B) MMP-3, and (C) VEGF secreted form HUVEC cells after treatment with diode laser for different times. *p < 0.05, compared with Ctl.
Figure 5. The PrestoBlue® assay performed for viability of hMSCs cultured with HUVEC cells-treated diode laser. *p < 0.05, compared with Ctl.
Figure 6. Relative mRNA expressions of (A) ALP, (B) BSP, and (C) OC by comparing hMSCs cultured with HUVEC cells-treated diode laser. *p < 0.05, compared with Ctl.
Figure 7. (A) ALP and (B) OC secretion from hMSCs cultured with HUVEC cells- treated diode laser. *p < 0.05, compared with Ctl.
Figure 8. (A) Photograph and (B) quantification of calcium mineral deposits by Alizarin Red S assay of hMSCs cultured HUVEC cells-treated diode laser for 7 and 14 days. The scale bar was 100 μm. *p < 0.05, compared with Ctl.
Figure 9. Summary of possible effects of HUVEC cells in the process of the diode laser- treatment inducing osteogenic differentiation of hMSCs.
