Cytokines secretion from human mesenchymal stem cells induced by bovine bone matrix

Abstract

BACKGROUND:

Bovine bone matrix is a natural material that has been used in the treatment of bone lesions. In this study, bovine bone matrix Nukbone^® (NKB) was investigated due its osteoconductive and osteoinductive properties. This biomaterial induces CBFA-1 activation and osteogenic differentiation, although the cytokines involved in these processes is still unknown.

OBJECTIVE:

The aim of this work was to determine the influence of NKB on the pro-osteoblastic and anti-osteoblastic cytokines secretion from human mesenchymal stem cells (hMSCs).

METHODS:

The hMSCs were cultured onto NKB and cytokines IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ and TNF-α were analized at 0-14 days by immunoassay. In addition, hemocompatibility of NKB and characterization of hMSCs were evaluated.

RESULTS:

NKB induces an increase on pro-osteoblastic cytokine secretion IL-4 and a decrease on anti-osteoblastic cytokine IL-6 secretion, at days 7 and 14 of cell culture. Interestingly, there was no statistical difference between secretion profiles of others cytokines analized.

CONCLUSIONS:

The up-regulation of IL-4 and down-regulation of IL-6, and the secretion profiles of other cytokines examined in this work, are findings that will contribute to the understanding of the role of NKB, and similar biomaterials, in bone homeostasis and in the osteoblastic differentiation of hMSCs.

Keywords

Bovine bone matrix mesenchymal stem cell bone regeneration cytokine nukbone

1. Introduction

Extracellular matrix (ECM) is a three-dimensional network composed of collagens, proteoglycans/glycosaminoglycans, and several other proteins. This biological network regulates diverse cellular functions, such as survival, growth, migration, differentiation, and apoptosis, all of these for maintaining of normal homeostasis. ECM is a highly dynamic structural network whose components vary in concentration, type and three-dimensional arrangement in according to the tissue of which they are part [1]. In native bone tissue, the ECM not only provides a platform for cell migration and adhesion, but also regulates cell behavior in response to external stimuli, which allows balancing the resorption and bone formation processes [2,3]. It is difficult to replicate the native bone ECM in vitro, however, decellularization of bone matrix from natural sources has become popular in tissue engineering applications due to the natural ECM can provide an appropriate cellular microenvironment for some kinds of cells [4,5].

In this context, bovine bone matrix has been widely used in medical applications for the treatment of bone lesions and has been demonstrated its participation in bone consolidation and also in process related with bone regeneration [2–5]. However, the mechanism involved in these process induced by bovine bone matrix are still not thoroughly understood and need additional investigations. In this study, the bovine bone matrix Nukbone^® (NKB) was analized. NKB is a decellularized bone from bovine origin, the main component is hydroxyapatite (HA) however, has a component of organic matter approximately 20% by weight corresponding to the collagen, in addition to HA, NKB also presents pure calcium carbonates and calcium phosphates with a calcium/phosphorus (Ca/P) ratio of 2.08 [6,7]. This xenoimplant has been used as bone filler in several medical areas such as orthopedics, odontology and maxillofacial surgery, among others. It even has shown positive effects in pre-clinical and clinical studies; where it has been proven to be an excellent option in the treatment of osseous pathologies [6–11]. Nowadays, more than 66,000 patients have received NKB grafts.

In a previous work, it was found that NKB has osteoconductive and osteoinductive properties [8–10]. Also, it was demonstrated, by means of Reverse Transcription Polymerase Chain Reaction (RT-PCR), that the gene expression of two key markers is involved in the osteoblastic differentiation of mesenchymal stem cells from the human amniotic membrane (hMSCs). The two markers in question are the Core Binding Factor A-1 (CBFA-1) and Osteocalcin (OC), although the mechanism of action by which NKB induces this gene expression was not determined. The effect of NKB on osteogenic differentiation has been attributed to topographic characteristics and the chemical composition of this biomaterial, because it has been shown that the main constituents of NKB, collagen and hydroxyapatite, have shown positive effects in the bone consolidation [12,13]. Also, the nano, micro and macroporosity of biomaterials like NKB, are involved in cell adhesion and proliferation, and other biological processes [14]. However, we consider that the study of the participation of chemical mediators involved in the bone resorption and bone formation, such as cytokines, would contribute to elucidate the mechanisms involved in bone consolidation induced by NKB. In this regard, there are reports that show the participation of cytokines in the consolidation and bone regeneration [15–20]. Particularly, it has been reported that cytokines such as IL-4, IL-10, IL-12 and IFN-γ, possess the ability to induce bone formation, i.e. they are pro-osteoblastic cytokines. This is achieved by suppressing osteoclast formation and by increasing the gene expression of osteoprotegerin (OPG) [16,21–25]. In contrast, there are other cytokines, like IL-6 and TNF-α, that are related to osteoclast formation (differentiation and/or activation), and consequently with the bone resorption phenomenon; these cytokines are named anti-osteoblastic [22,23,26,27].

Keeping in mind these considerations, the aim of this work was to measure the secretion of pro-osteoblastic and anti-osteoblastic cytokines in hMSCs cultures in presence of NKB, in order to gain fundamental knowledge on the mechanism of action of role of NKB, and similar biomaterials, used in bone tissue engineering applications. In addition, hemocompatibility properties of NKB were assessed, through hemolysis assay.

2. Materials and methods

2.1. Reagents

Bovine bone matrix NKB discs were provided by Biocriss S.A. C.V. (Mexico City, Mexico). Dulbecco’s Modified Eagle Medium (DMEM), antibiotic–antimycotic solution, and trypsin–EDTA solution were obtained from Life technologies (Thermo Fisher, MA, USA) whereas type II collagenase, ascorbic acid, dexamethasone and β-glycerol phosphate were supplied by Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) and Alamar Blue Cell Viability Reagent were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA) and Invitrogen Life Technologies (Carlsbad, CA, USA), respectively. The antibodies for flow cytometry were purchased from BD Pharmigen (California, USA).

2.2. Isolation and characterization of human mesenchymal stem cells

hMSCs were obtained from amniotic membranes from five healthy women, with prior informed consent for participation in the experiment. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Facultad de Medicina, UNAM (project 101-2012). Samples of amniotic membranes (5 cm²) were fragmented with a scalpel into fragments of approximately 2 mm. The fragments obtained were incubated with an enzymatic solution, containing 0.01% type II collagenase in DMEM without SFB, and were incubated for 1 h at 37 °C at 5% CO₂ with constant agitation. After this time, the cell suspension resulting from the enzymatic digestion was centrifuged at 1200 rpm, at 25 °C, for 15 minutes. Finally, the cell button was resuspended in DMEM supplemented with 10% SFB and 1% antibiotic–antimycotic. The cultures were maintained at 37 °C at 5% CO₂ in DMEM until they reached 80% confluence for subsequent experiments, with medium change every third day.

The cells obtained were characterized in according to the International Society for Cellular Therapy standards [28]. Briefly, the Colony Forming Unit (CFU) assay was evaluated through crystal violet staining. For this, 0, 25, 50, 100 and 200 cells/cm² were cultured in a 6-well culture dish for triplicate, and cultured for 14 days at 37 °C at 5% CO₂ in DMEM. Then, the cells were washed twice with PBS and fixed in methanol for 5 min before staining with 0.5% (w/v) crystal violet for 5 min. The excess dye was removed with consecutive PBS washes and the CFU (violet points) were counted manually.

For immunophenotypic characterization, aliquots of 1 × 10⁶ cells (passage 3) were resuspended in PBS and incubated with monoclonal fluorescein isothiocyanate-conjugated antibody against CD90, phycoerythrin-conjugated antibody against CD73 and CD34, and PECy5 and PECy7-conjugated antibodies against CD105 and CD44 respectively for 30 min at 4 °C in the dark. After this time, the cells were washed with PBS and fixed in 0.5% (w/v) paraformaldehyde and analysed by flow cytometry in a BD FACSCalibur system (BDBIOSCIENCES, San Jose, CA, USA).

Finally, cells were subjected to osteogenic and adipogenic differentiation. Cells (2 × 10³) were seeded in triplicate on 24-well culture dish and incubated under osteogenic and adipogenic conditions. For osteogenic condition, the cells were cultured in DMEM containing 82 μg/mL ascorbic acid, 100 nM dexamethasone and 10 mM β-glycerophosphate. For adipogenic differentiation, the cells were incubated in DMEM containing 5 μg/mL insulin, 0.5 mM isobutyl-methyl-xanthine and 60 μM indomethacin. The cell cultures were maintained for 14 days under standard culture conditions with medium changes every 3 days. To show osteogenic differentiation, the cultured cells were washed with PBS and fixed in 10% (v/v) p-formaldehyde in PBS for 15 min at 25 °C. After washing, cells were staining with 2% (v/v) Alizarine Red S for 10 min at 25 °C, the visualization of calcium deposition was visualized under optical microscope (TCM-400, Labomed). For adipogenic differentiation, cells were washed with PBS and fixed in 10% (v/v) p-formaldehyde in PBS for 1 h at 25 °C. After this time, cells were stained with 0.5% (w/v) Oli Red O solution for 1 h at 25 °C, and then, the cells were washed twice with 60% isopropanol in PBS, once time with PBS and finally with distiller water. The lipid deposition was observed under optic microscopy.

2.3. Cell adhesion and hMSCs proliferation onto bovine bone matrix NKB

Cell proliferation was determined by Alamar blue (AB) reduction at 7 days, using a 3 × 10³ cell density in 96-well culture plates, according to the manufacturer’s instructions. The cell-NKB interaction was observed in a JEOL-JSM-6300 Scanning Electron Microscope; samples were treated as reported previously [29].

2.4. Hemolytic assay

The hemolytic assay was performed in according to the ISO 10993–4 [30]. Three discs of NKB from 3 different batches (n = 9) were placed in contact with 1 mL of 5% (v/v) human erythrocytes in saline solution, and incubated at 37 °C for 1 h. The negative control was a NKB sample containing erythrocytes in an isotonic solution, while erythrocytes in distilled water were the positive control. The hemolysis % was determined by absorbance at 415 nm in a Cytation3 Cell Imaging Multi-Mode Reader (BioTek^® Instruments, Inc) using the following formula: $\begin{eqnarray}\displaystyle \mathit{hemolysis}(\%):{\displaystyle \frac{Abs_{\mathit{sample}}-\overline{Abs}_{\mathit{negative}}}{\overline{Abs}_{\mathit{positive}}-\overline{Abs}_{\mathit{negative}}}}\ast 100. & & \displaystyle \nonumber\end{eqnarray}$

2.5. Cell culture and cytokine analysis

The hMSCs (5 × 10⁵) from passages 3–5 were cultured in DMEM supplemented with a 1% antibiotic-antimycotic solution and 10% heat-inactivated FBS. Samples were incubated for 14 days onto NKB discs (12 mm diameter and 2 mm thickness) (+NKB). Cell cultures were maintained at 37 °C under standard culture conditions. The cell culture supernatant was collected at 0, 3, 7 and 14 days and centrifuged for 30 min at 4 °C and 4000 g. Then, cytokines IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ and TNF-α were determined from 200 μL supernatant aliquots using the Bio-Plex Precision ProTM Human Cytokine immunoassay panel (Bio-Rad). The samples were processed according to the manufacturer’s manual and analysed using a Bio-Plex 200 System with the Bio-Plex Manager 6.1 software (Bio-Rad).

A cell culture without NKB was used as negative control (−NKB), whereas culture medium was employed as a blank to assess baseline signals (+NKB, −NKB). For this experiment, hMSCs from five amniotic membranes were used and the cytokine secretion was measured in triplicate for each one.

2.6. Statistical analysis

Data are reported as the mean and standard deviation of at least 3 repeats. The statistical significance between experimental conditions (−NKB, +NKB) was determined using an ANOVA test (p < 0.05 was considered significant) with the OriginPro 8.5.1 software.

3. Results

3.1. hMSCs characterization

The hMSCs obtained from amniotic membranes exhibited the typical morphologic evolution of mesenchymal cells (Fig. 1a). Immediately after the isolation process, the cells showed a spherical morphology at day 0; later, cell adhesion was observed at day 3, and some cells exhibited fibroblast-like morphology. At the seventh day, most of cells exhibited an elongated morphology, and finally at day 14, the culture reached confluence and all hMSCs exhibited fibroblast-like morphology and tended to align and orient.

The self-renewal capability of hMSCs was demonstrated by CFU assay. This experiment allowed us to observe the formation of violet cell colonies at different cell densities (Fig. 1b). Regarding the multipotency that hMSCs should exhibit, it was possible to demonstrate the ability of this cells to differentiate towards osteogenic and adipogenic phenotypes. Osteogenic differentiation was evidenced by the calcium deposits which were stained with alizarin red; while lipid deposition (adipogenic differentiation) was evidenced through Oil Red O staining on day 14 (Fig. 1c).

For immunophenotypic characterization, hMSCs from passage 3 were subjected to a flow cytometric analysis for the identification of mesenchymal markers. The 73.1% of the cell population were positive for CD90 marker, while 61.6% of the cells were positive for CD105 and 82.4% of the cells were positive for expression of CD73. In contrast, the cell population was slightly for hematopoietic markers CD34 (19.2%) and CD45 (9.29%) (Fig. 1d).

Fig. 1.

hMSCs characterization. (a) hMSCs morphology. The images show the morphologic changes of the hMSCs, from a spherical morphology at 0 day, until like-fibroblast morphology and confluence at 3, 7 and 14 day. Arrows indicated the like-fibroblast cellular morphology. (b) Clonogenic capability of cells. The graphic shows the UFCs manually measured at different cell densities, the image shows the formation of colonies staining with violet crystal n = 3. (c) Multipotency of hMSCs. The micrographs show the cells cultured without inductor culture medium and stained with violet crystal; cells cultured with osteogenic medium and stained by alizarin red staining and cells cultured with adipogenic culture medium and stained by Oil red O staining. Scale bar = 20 μm. (d) Immunophenotypic characteristics of hMSCs. Flow cytometry of hMSCs compared according to total number of cells analysed.

3.2. Cell adhesion and proliferation onto bovine bone matrix NKB

The electronic micrographs showed that NKB promotes cellular adhesion. hMSCs cultivated onto NKB during 24 h showed adhesion onto the biomaterial surface in a panoramic approach, and at higher magnifications it was possible to see the morphology of cells on the porous surface (Fig. 2).

Fig. 2.

SEM analysis of hMSCs cultured onto NKB. Micrographs showed the cell adaptation to NKB. (a) Panoramic vision of hMSCs around the NKB pore. (b) Magnification of cell on the surface of the biomaterial. Micrographs were registered to 24 h of cell culture. Red asterisk shown some cells adhered to NKB superficies.

Also, the cell proliferation in +NKB or −NKB conditions showed no significant statistical difference (Fig. 3).

Fig. 3.

hMSCs proliferation in contact with NKB. There is no significant difference between +NKB, and −NKB conditions. n = 3.

3.3. NKB hemocompatibility

In order to evaluate the interaction between NKB and biological fluids, the hemolytic assay was carried out. The integrity of red cells in contact with this biomaterial was preserved after 1 h of incubation. Hemolysis registered in contact with NKB (2.16 ± 0.04) was under the acceptable value (below 5%) in accordance with the ISO 10993-4 international standard (Fig. 4).

Fig. 4.

NKB hemocompatibility. (a) The hemolysis induced in contact with NKB (+NKB) was under 5%. (b) Visual grades of hemolysis were observed in the positive (erythrocytes in distiller water), and negative condition (erythrocytes in isotonic solution of NaCl). n = 9.

3.4. Cytokine secretion induced by bovine bone matrix NKB

Figure 5 shows that pro-osteoblastic cytokine induced by NKB, of the cytokine analysed, the bovine bone matrix NKB induced a higher secretion of the pro-osteoblastic cytokine IL-4 (1.33 ± 0.12 pg/mL) in comparison with no NKB containing experimental condition (0.05 ± 0.02 pg/mL) at day 14 of cell culture; while on days 0, 3 and 7, no significant changes were shown in the secretion profile of IL-4. Interestingly, results also indicated that there was no statistical difference between the secretion profiles of other pro-osteoblastic as IL-10, IL-12, IFN-γ in presence (+NKB) or absence of NKB (−NKB).

Fig. 5.

Pro-osteoblastic cytokines induced by NKB. IL-4, IL-10, IL-12 and IFNγ, the changes in IL-4 secretion were observed. n = 5, ^∗ p < 0.05.

In regards to anti-osteoblastic IL-6, showed lower secretion at days 7 (7166.97 ± 1240 pg/mL) and 14 (11628.60 ± 1000 pg/mL) than no NKB containing culture conditions, which showed values of 20633.5 ± 3198.46 pg/mL and 16102.62 ± 300 pg/mL at days 7 and 14, respectively (Fig. 6). In contrast, TNFα secretion remained unchanged in +NKB and −NKB experimental conditions in the times studied.

Fig. 6.

Anti-osteoblastic cytokine induced by NKB. IL-6 and TNFα. IL-6 secretion showed effects at 7 and 14 days of cell culture. n = 5, ^∗ p < 0.05.

The behaviour of IL-12 was also studied. This cytokine has multiple clinical applications because of its immune-regulatory properties as its participation in the activation of immune cells, however its participation in the process of bone remodelling has not yet been clarified. hMSCs cultured with or without NKB showed no changes in the secretion of IL-12 (Fig. 7).

Fig. 7.

Immunoregulatory cytokine IL-2 secretion induced by NKB. There is no influence from NKB. n = 5.

4. Discussion

Human amniotic membrane is a tissue easy to acquire. Cells obtained from this tissue exhibited adherence to the culture plastic and the like-fibroblast morphology, clonogenic capability, potential to differentiate towards the osteogenic and adipogenic lineage, and were positive for mesenchymal markers and negative for hematopoietic; as previously reported for hMSCs [28]. These kind of cells represent a valuable tool in the field of tissue engineering, because various aspects related to cells, scaffolds or biomolecules involved in the tissue regeneration processes can be explored, in particular for this work on bone regeneration.

Bone homeostasis has been the goal of many scientific efforts in order to understand the mechanisms involved in osseous remodelling [8,9,18,26,27]. These efforts, have contributed to demonstrate that cytokines and grow factors, play an important role during bone formation and bone resorption [9,15,21–27]. The cytokines involved in bone remodelling have been classified into pro-osteoblastic cytokines (IFNγ, TGF-β, IL-10, IL-4, IL-6, IL-12, OPG) and anti-osteoblastic cytokines (TNF-α, IFNγ, TGF-β, IL-17A, IL-6, IL-12, RANKL) due to their ability to induce bone formation or bone resorption, respectively [25,32–35]. Interestingly, there are also data about the stimulatory and inhibitory effects of IL-6 and IL-12 on osteoblast activity [21,22,27,31,34–36]. In addition, there are other cytokines, such as IL-2, whose participation in the process of bone remodelling has not yet been clarified; however, this cytokine has multiple clinical applications, because of its immune-regulatory properties [37,38].

The secretion of certain cytokines is modulated by other cytokines and some drugs [38–41]. In this work, we evaluated the secretion of pro-osteoblastic cytokines IL-4, IL-10, IL-12, anti-osteoblastic cytokines IL-6, TNF-α, and the immune-regulatory cytokine, IL-2, on hMSCs cultures in presence of bovine bone matrix NKB.

Results indicate that NKB induces an increase in IL-4 at 14 days of growth. IL-4 is a pro-osteoblastic cytokine whose main activity in bone metabolism is to block pro-osteoclastogenic cytokines such as IL-1, IL-6 and TNFα, as well as collagenase secretion. Moreover, IL-4 has been demonstrated to prevent osteoclast differentiation and maturation through the activation of the activator of transcription STAT6 [39], and it has been associated with the increase of OPG and the suppression of RANK expression [39–41]. This is in agreement with the results obtained in this work, as IL-6 secretion decreased significantly at days 7 and 14 of hMSCs culture in presence of NKB. This effect was reported by Zhang et al. [41], who showed that an immune agent (FTY-720P) increases IL-4 levels and decreases those of IL-6, culminating with the suppression of osteoclastic formation.

However, it is important to note that IL-6 has been considered a stimulator of osteoclast differentiation, but some studies described an opposite effect [34,42], suppressing osteoclast differentiation. This pleiotropic function of IL-6 seems to depend on the microenvironment. The anti-osteoblastic role of IL-6 has been demonstrated by its participation as a potent stimulator of osteoclastogenesis in mieloma cells [43,44], and its positive activity as a regulator in osteoclast differentiation via the RANK signalling pathway, which involves NF-kappaB, JNK, and p 38 [44], as well as the IL-6 receptors expressed on osteoblastic cells [45]. In contrast, Yoshitake et al. [34] demonstrated a pro-osteoblastic effect of IL-6; they observed that co-stimulation with IL-6 and RANKL can also act directly on osteoclast progenitors to suppress their differentiation through regulation of the enzymes and transcription factors related to the ubiquitin pathway. Moreover, IL-6 has been involved in osteogenesis through the up-regulation of Runx2 and Dlx5, resulting in decreased multipotency and causing primitive multipotent cells to undergo osteogenic lineage commitment [42]. In contrast, IL-10, IFNγ, IL-12, TNFα, and IL-2 did not show changes in the presence of NKB. Additionally, hemocompatibility of NKB was validated through the hemolysis assay.

5. Conclusions

In conclusion, these findings indicate that bovine bone matrix NKB is able to induce the up-regulation of the pro-osteoblastic cytokine IL-4 and the down-regulation of IL-6, both cytokines related with bone homeostasis. However, more detailed studies are required to further understand the precise role and mechanism by which, IL-4 and IL-6 cytokines secretion is modulated by NKB, and possible participation of this cytokines in osteogenic differentiation induced by biomaterials like NKB. So that, the experiments using IL-4 blocking antibody or supplemented cell cultures with IL-6 should be performed.

Footnotes

Acknowledgements

The authors thank Carlos Flores Morales, Armando Zepeda Rodríguez and Alejandra Alvarado from UNAM for their technical assistance. This research was funded by CONACYT (214128, 248378), PAPIIT-UNAM (IN224316) and PAPIME-UNAM (PE211115).

Conflict of interest

The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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