Background
Stem cell-based technologies are an ideal source for regenerative medicine, immunological studies, and cell therapy because they induce tissue repair and regeneration [
1,
2]. Mesenchymal stem cells (MSCs) play a key role in tissue regeneration treatment. They are rapidly adherent, clonogenic, and capable of extended proliferation in vitro [
3]. In addition, they maintain stem cell properties such as self-renewal, long-term viability, and differentiation potential into mesodermal origin osteocytes, chondrocytes, and adipocytes [
4,
5]. As a result of their capacity to differentiate into various cell types, MSCs play a key role in tissue and organ regeneration and have recently attracted great interest in tissue engineering [
6,
7].
Even though MSCs can be isolated from many sources, such as cord blood, bone marrow, or adipose tissue [
8], a very promising source is the relatively easily obtainable dental tissue. There are five types of human dental stem cells: dental pulp stem cells (DPSCs) [
9], stem cells from exfoliated deciduous teeth (SHED) [
10], periodontal ligament stem cells (PDLSCs) [
11], dental follicle stem cells [
12], and stem cells from apical papilla [
13]. These MSCs express specific MSC markers, such as CD29, CD73, CD90, CD105, and CD166, and can differentiate into odontoblasts, chondrocytes, and adipocytes under appropriate circumstances [
9,
10]. DPSCs can easily be isolated from the dental pulp tissue of newly extracted teeth, making the procedure relatively more straightforward and avoiding ethical dilemmas [
14]. DPSCs are commonly used in the regeneration and reconstruction of dental structures in addition to bone tissue engineering after undergoing osteogenic differentiation [
15,
16].
Tissue engineering techniques, including the use of MSCs, often require scaffolds and cytokines serving as inductive factors [
17]. Some inflammatory cytokines alter stem cell functions as well as immune or inflammatory cells [
18]. In vitro studies have revealed that cytokines can affect the differentiation process of mesenchymal progenitor cells during tissue formation. Most of these in vitro studies have used MSCs by isolating them because of their ability to adhere to plastic [
19,
20].
Cytokines are commonly used superior markers of inflammation, modulating immune and inflammatory responses [
21]. Tumour necrosis factor-α (TNF-α) is defined as a proinflammatory cytokine expressed in injured tissues as well as in ischaemic situations [
22]. TNF-α also plays a major role in the repair process of injured tissues and promotes MSC recruitment [
23‐
25]. Similar to TNF-α, interleukin (IL)-6 can be detected in injured tissues and stimulates osteoblast differentiation. Both TNF-α and IL-6 are released from T-cells and macrophages [
26]. IL-1β plays an essential role in tissue damage and inflammation as well as cell proliferation and differentiation [
27]. It also induces various metalloproteinases (MMPs), causing extracellular matrix degradation and cell migration [
28,
29]. Thus, cytokines can affect MSC differentiation in addition to their role as an immune response started by injury. Kang et al. [
30] and Ries et al. [
31] have indicated that MSCs respond to various growth factors and cytokines. Studies have reported negative and positive effects of cytokines on the osteogenic differentiation potential of MSCs [
32,
33].
Dental pulp can also express many inflammatory mediators that can combat irritants [
34,
35]. Pulpal inflammation (pulpitis) increases with the progression of carious lesions [
36]. Caries bacterial antigens evoke proinflammatory cytokines in various amounts [
37]. Lipoteichoic acid (LTA), an amphiphilic molecule produced in large amounts by cariogenic bacteria, activates the innate immune system and induces proinflammatory cytokines such as TNF-α, IL-1, IL-8, and IL-12 [
38]. IL-6 and IL-1β are also secreted when dental pulp cells are challenged with Gram-positive bacteria. In the later stages of pulpitis, IL-6 becomes a critical component due to the increase of B cells [
39]. Releasing these mediators in the dental pulp triggers a series of inflammatory events, resulting in innate repair with the help of immune cells, protease inhibitors and other molecules [
40]. The present study also supports that the application of DPMSCs in the inflammatory niche may transform the MSCs into a phenotype of suppression of inflammation rather than tissue regeneration.
Many in vitro studies [
41‐
44] have evaluated the roles of inflammatory cytokines in osteogenic and chondrogenic differentiation of MSCs. However, no study has reported the role of inflammatory cytokines in the adipogenic, chondrogenic or osteogenic differentiation of DPSCs. The present study examined the effect of TNF-α, IL-1β, and IL-6 on the osteogenic, chondrogenic, and adipogenic differentiation of DPSCs in vitro. The findings can provide a better understanding of in vitro differentiation of cytokine-stimulated DPSCs and may develop the possible usage of autologous transplantation of DPSCs.
Materials and methods
Isolation of stem cells and DPSC culture
DPSCs were isolated from the impacted third molar teeth of three 22–30-year-old patients in the Marmara University Faculty of Dentistry Oral and Maxillofacial Surgery Department, Istanbul, Turkey. All patients provided informed consent, and the Ethics Committee of the Marmara University Clinical Researches in Istanbul, Turkey approved the study protocol (22.05.15–1). The extraction procedure was performed atraumatically and under sterile conditions. Extracted teeth were transported in Dulbecco’s phosphate-buffered saline (DPBS, Gibco, Grand Island, NY, USA) with 1% penicillin/streptomycin (Gibco, USA) within ice cubes within 4 h to the laboratory in the Department of Pediatric Allergy-Immunology, Marmara University Research Hospital, where all laboratory work was performed. The pulp was separated from the tooth by cracking the crown under sterile conditions. First, the pulp was broken down to 0.1–0.5-mm pieces mechanically with a sterile scalpel and then enzymatically treated with 2 mL of collagenase type I solution (3 mg/mL) (Gibco, USA); then, incubation for 45 min at 37 °C was carried out to digest the pulp tissue enzymatically. The enzymatic activation stopped with 2 mL of 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) containing Dulbecco’s modified Eagle medium (DMEM, Gibco, USA), followed by centrifugation at 1500 rpm for 5 min. The supernatant was aspirated, and cell pellets were obtained and then suspended with 5 mL of DMEM and cultivated in T-75 flasks with a 5% CO2 atmosphere under 37 °C for 7 days. The culture medium was changed every 2–3 days until the cells became confluent at 80%. Thereafter, adherent cells were cultured until the third passage to characterize and analyse specific surface markers. The third passage cells are used in the culture studies. These specific cellular determinations and analyses were performed using flow cytometry.
Flow cytometry analysis
The cells from the third passage were used to analyse cell surface antigen expressions. Approximately 1 × 106 cells were counted and homogenised in PBS and incubated with antibodies at room temperature in the dark for 15 min. After incubation, 0.1% sodium azide containing PBS was added and procedure followed by centrifugation at 1200 rpm for 5 min and the cell suspension analysed with FACSCalibur Flow Cytometry device with BD Cell Quest TM software (BD Biosciences, San Jose, CA, USA). CD29, CD105, CD146, CD73, and CD90 were determined as positive antibodies, whereas CD3, CD4, CD20, CD34, CD45, and HLA-DR were determined as negative antibodies.
Stimulation of DPSCs with inflammatory cytokines
After determining DPSCs with flow cytometry analysis for specific surface markers, DPSCs from three impacted third molar teeth were randomly divided into 5 main groups with 3 subdivisions for each group making a total of 15 groups. One group of unstimulated control and three groups treated with TNF-α (100 ng/mL) (R&D Systems, UK), IL-1β (100 ng/mL) (R&D Systems UK), or IL-6 (100 ng/mL) (R&D Systems UK) for 48 h [
45].
Differentiation of DPSCs
After culturing DPSCs with inflammatory cytokines, each group was divided into three subgroups with approximately 100,000 cells to induce osteogenic, adipogenic, and chondrogenic differentiation, leading to 12 separate differentiation groups in total. The cells were incubated in a 5% CO2 atmosphere under 37 °C for 7 days until they became 80%–90% confluent. For osteogenic (MesenCult, Stemcell Technologies, North America), adipogenic, and chondrogenic (Gibco, Grand Island, USA) differentiation, human MSC functional identification kits were used. For the differentiation procedure, the cells were plated in 6-well plates, and the differentiation medium was prepared following the manufacturer’s instructions. The differentiation medium changed every 3 days, and at the end of 21–28 days, the formed tissues were determined using different staining procedures.
Staining protocols and determination of differentiation
After 28 days, osteogenic, chondrogenic, and adipogenic differentiations were determined by staining with Alizarin red, Alcian blue, and oil red, respectively. Staining solutions were prepared according to the manufacturer’s instructions, and after all groups were examined by the biological light microscope under 20 × objective. Histological analysis of osteogenic, chondrogenic, and adipogenic differentiated pellets was assessed following staining protocols using a modified version of the Bern Score proposed by Grogan et al. [
46]. In brief, cell pellets were assessed using the following criteria: uniformity and intensity of staining and distance between cells/amount of matrix produced and cell morphology. Each of these three categories was scored from 0 to 3 (Table
1). The evaluation was performed by calculating the arithmetical means of the scores within three criteria, as given in Table
1.
Table 1
Scoring categories for osteogenic, chondrogenic and adipogenic stimulated pellets in the monolayer culture system
A. Uniformity and darkness |
No stain | | 0 |
Weak staining of poorly formed matrix | | 1 |
Moderately even staining | | 2 |
Even dark stain | | 3 |
B.Distance between cells/Amount of matrix accumulated |
Osteogenic | No osteogenic colonies/No calcium deposit | 0 |
No osteogenic colonies/Weak staining of calcium deposits | 1 |
Osteogenic colonies/Waek staining of calcium deposits | 2 |
Osteogenic colonies/High amount of calcium deposits | 3 |
Chondrogenic | Low cell densities with no matrix between cells | 0 |
Low cell densities with little matrix between cells | 1 |
Moderate cell density with matrix | 2 |
High cell density with moderate distance between cells | 3 |
Adipogenic | No adipocytes/No oil droplets | 0 |
No adipocytes/Weak staining of oil droplets | 1 |
Adipocytes with weak staining of oil droplets | 2 |
Adipocytes with high amount of oil droplets | 3 |
C. Cell morphologies represented |
Osteogenic | Condensed/necrotic/pycnotic bodies | 0 |
Spindle/fibrous | 1 |
Mixed spindle/fibrous with calcium deposits | 2 |
Majority calcium deposits/osteogenic | 3 |
Chondrogenic | Condensed/necrotic/pycnotic bodies | 0 |
Spindle/fibrous | 1 |
Mixed spindle/fibrous with cartilage forming | 2 |
Majority cartilage forming/chondrogenic | 3 |
Adipogenic | Condensed/necrotic/pycnotic bodies | 0 |
Spindle/fibrous | 1 |
Mixed spindle/fibrous with oil droplets | 2 |
Majority oil droplets/adipogenic | 3 |
Statistical analyses
Statistical analyses were performed using IBM SPSS v22 (IBM SPSS, Turkey). Statistical significance was determined using one-way analysis of variance. Correlations were assessed using Pearson’s test (two-tailed). P < 0.05 was set as statistically significant.
Conclusion
Our results indicated that DPSCs are highly proliferative MSCs in terms of osteogenic, chondrogenic, and adipogenic differentiation. In the present in vitro study, TNF-α, IL-1β, and IL-6 were demonstrated to inhibit DPSC differentiation and tissue formation. Further studies, including in vivo applications with different dental MSCs origins and diverse amount, type and appliance durations are required to more comprehensively understand the underlying molecular mechanisms for application in stem cell therapies.
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