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Basic and Clinical Research

Concentrate Growth Factors Regulate Osteogenic Dysfunction of MC3T3-E1 Cells Induced by High Glucose Through PI3K/Akt Signaling Pathway

Dong, Kai DDS, MSC*; Hao, Pengjie DDS, PhD; Zhou, Wenjuan DDS, PhD; Liu, Zhonghao DDS, PhD

Author Information
doi: 10.1097/ID.0000000000000921
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Abstract

The implant therapy in dentistry is an attractive substitute to the traditional fixed/removable prosthesis.1,2 The osseointegration of titanium implants is primarily dependent on how quickly and completely the surrounding bone tissues grow in close apposition to the implant. Furthermore, the process is directly related to the proliferation and differentiation of osteoblasts.3 However, in patients with diabetes mellitus, such implants are associated with a high failure rate and poor bone-implant integration.4,5 One of the main causes is the lower levels of growth factors such as insulin-like growth factor-1 (IGF-1) and insulin growth factor–binding protein-3 (IGFBP-3) in patients with diabetes.6 Furthermore, a positive correlation has been found between IGF-1, IGFBP-3, and bone mineral density in both type 1 diabetes mellitus and type 2 diabetes mellitus.7

Growth factors are polypeptides that can either stimulate or inhibit cellular proliferation, differentiation, migration, adhesion, gene expression, and tissue angiogenesis.8 An appropriate source for delivering growth factors is through platelet concentrates. Platelets are known to release a variety of autologous growth factors, such as IGF, vascular endothelial growth factor, and transforming growth factor–β1 (TGF-β1).9 Concentrate growth factor (CGF) is a new generation of autologous platelet-rich fibrin biomaterial, which is obtained by single centrifugation of venous blood by use of a specially programmed centrifuge. Researches have demonstrated that CGF can promote bone formation through growth factor release10,11

To the best of our limited knowledge, there have been few studies indicating the effects of the soluble growth factors contained in the CGF on the osteogenic differentiation of osteoblasts under high glucose condition. In addition, MC3T3-E1 osteoblast-like cell line culture system represents a very useful model for studying the process of osteoblast function.12 Therefore, the aim of our study was to demonstrate in vitro whether the soluble growth factors contained in the CGF clot influence the proliferation and osteogenic differentiation of MC3T3-E1 cells under high glucose condition. In addition, we also explore the regulatory mechanism of PI3K/AKT signaling pathway, thus providing theoretical basis for the clinical applications of CGF.

Materials and Methods

Preparation of CGF and CGF Extract (CGF-e)

All surgical procedures involving animals were conducted after recommended procedures approved by the Institutional Animal Care and Use Committee of the Yantai Stomatological Hospital. CGF was prepared according to Sacco's protocol.13 Briefly, 5-mL venous blood was drawn from Sprague–Dawley rat (purchased from Shandong LVYE Pharmaceutical Co., Ltd., Yantai, Shandong, China), into a sterile glass tube without any anticoagulant solution. The tube was subsequently immediately centrifuged in a special centrifuge device (Medifuge; Slifadent Srl, Sofia, Italy) at a fixed temperature, and the rotor turned at alternating, controlled speeds (2700 rpm/2 min, 2400 rpm/4 min, 2700 rpm/4 min, and 3000 rpm/3 min). After centrifugation, the blood in the tube was separated into 4 layers, the second or buffy coat layer and the third layer, also called growth factor layer, were made up of the CGF. Then, the CGF was frozen at −80°C for 1 hour to separate trapped growth factors and cytokines from the fibrin meshes. After thawing, the leaked juice was harvested without fibrin fibers by centrifugation at 230g for 10 minutes and then filtrated (0.22 μm), named the CGF extract (CGF-e). According to the previous study, we chose 0.5% CGF-e for the following experiments.14

Cell Culture and Grouping

MC3T3-E1 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were maintained and differentiated as described in our previous study.15 According to the glucose level of α-MEM and CGF-e presented or not, 4 groups were divided including (1) the control group (C group): normal glucose (5.5-mM) α-MEM alone; (2) the high glucose group (H group): high glucose (25.5-mM) α-MEM alone; (3) the C + CGF group: 5.5-mM glucose + CGF-e; (4) the H + CGF group: 25.5-mM glucose + CGF-e.

Cell Survival Assay

Cell survival was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay through the protocol described.15 The absorbance values were read at 570 nm using an automated microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA).

Actin Cytoskeleton

MC3T3-E1 cells (1 × 104) were cultured for 24 hours. Then, cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. After washing with phosphate-buffered saline (PBS), cells were permeabilized with 0.5% Triton X-100 (10 minutes, RT; Solarbio Beijing, China), incubated with rhodamine phalloidin (10 nm, 200 μL; CytosKeleton, Denver, Colorado) for 30 minutes in the dark at RT, washed again, embedded with a coverslip using Fluoroshield mounting medium (Sigma-Aldrich, St. Louis, MO; Art. No. F6182) and examined on the laser scanning confocal microscopy (LSCM) (Leica TCS SPE).

Alkaline Phosphatase Assay

As described,15 alkaline phosphatase (ALP) activities in the culture supernatants were determined by measuring hydrolysis of p-nitrophenyl phosphate according to the manufacturer's instructions (Beyotime Institute of Biotechnology, China).

Mineralization Assay

As reported,15 the mineralized nodule formation was detected using Alizarin Red staining. Briefly, cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature. Then cells were stained with 2% Alizarin Red-S (pH 4.2; Sigma) for 20 minutes at room temperature. For quantification, the bound stain was eluted with 10% (wt/vol) cetylpyridinium chloride, and the absorbance of supernatants was measured by the automated microplate reader at 570 nm.

Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction PCR

The qRT-PCR was performed using the SYBR green kit (TaKaRa Bio Group, Dalian, China) and Rotor-Gene 3000 real-time PCR system (Corbett Research, Sydney, Australia). The detailed protocol was described in our previous studies.15 The specific primers were synthesized commercially from Sangon Biotech, Inc., (Shanghai Shi, China) and shown in Table 1. The mRNA-expression level was calculated based on the comparative 2−ΔΔCt method.

Table 1
Table 1:
The Primer Sequences for Quantitative Real-Time Polymerase Chain Reaction

Western Blot Analysis

As described,15 total intracellular proteins were isolated from cultured cells using a protein extraction kit (Cell signaling technology, Danvers, MA). Protein concentrations were assayed by using a BCA bicinchoninic acid protein assay (Pierce, Waltham, Massachusetts). Samples containing an equal amount of protein (10 mg) were separated by 15% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes (Millipore, Burlington, MA). These blots were then blocked (in 10% milk) and incubated sequentially with primary antibody and specific secondary antibodies. Blots were visualized with an enhanced ECL reagent (Santa Cruz Biotechnology, Dallas, TX). The intensity of the protein bands was quantified by densitometric scanning using Image J software and normalized to GAPDH (Zhong Shan-Golden Bridge Technology Inc., Beijing, China).

Statistical Analysis

All statistical analyses were performed using the SPSS 17.0 statistical software. Data were expressed as the mean ± SD. Statistical analyses were performed using one-way analysis of variance and Student's t test. Differences between groups were considered significant at P < 0.05.

Results

Effects of CGF-e on Cell Viability and Actin Cytoskeleton

High glucose inhibits osteoblast proliferation, but this effect is reversed by treatment with CGF-e. As shown in Figure 1, A, the number of cells in the control (C) group was increased gradually from days 3 to 14. The H group exhibited significantly lower growing cells than the control group (P < 0.01). However, CGF-e significantly increased the survival of MC3T3-E1 cells in both C + CGF group (vs C group, P < 0.05) and H + CGF group (vs H group, P < 0.01).

Fig. 1
Fig. 1:
Effects of high glucose and CGF-e on cell viability and actin cytoskeleton. (A) MTT assays. (B) Actin cytoskeleton of MC3T3-E1 cells after 24 hours. C group: Cells were well spread with numerous, pronounced actin stress fibers (inset arrow); C + CGF group: Actin filaments were obviously denser and longer than C group (inset arrow); H group: High-glucose result in cluster-like, shortened actin fragment throughout the retracted cell body (inset arrow); H + CGF group; with the present of CGF, cells maintain their morphology and actin filaments were reorganized and visible (inset arrow). (LSCM, 532 nm, scale bars 50 μm). *P < 0.05 and **P < 0.01 compared with the C group; #P < 0.05 and ##P < 0.01 compared with the H group.

As shown in Figure 1, B, LSCM exhibited typically long and straight actin stress fibers in C-group cells. In C + CGF group, the actin filaments had more density, whereas in H group, the actin cytoskeleton appeared to be not only significantly shorter compared with the C group but was also irregularly distributed. Interestingly, the actin filaments of H + CGF group were significantly longer and more regular than that of H group, indicated that the CGF seems to reverse the adverse effects of high glucose.

CGF-e Stimulates Osteoblast Differentiation

As shown in Figure 2, the level of ALP and osteogenic markers in H group was significantly lower than that in C group (P < 0.05; P < 0.01). However, the expression of ALP and osteogenic markers were markedly increased in C + CGF group and H + CGF group compared with C group and H group, respectively (P < 0.05; P < 0.01).

Fig. 2
Fig. 2:
Effects of high glucose and CGF-e on the osteoblastic genes expression of MC3T3-E1 cells. (A) ALP; (B) COL-I: collagen type I; (C) BMP-2: bone morphogenetic protein 2; (D) BSP: bone sialoprotein; (E) Runx2: runt-related transcription factor 2; (F) OCN: osteocalcin. *P < 0.05 and **P < 0.01 compared with the C group; #P < 0.05 and ##P < 0.01 compared with the H group. &P < 0.05 and &&P < 0.01 compared with the H + CGF group.

CGF-e–Enhanced Mineralization

High glucose inhibits mineralization, but this effect is reversed by treatment with CGF-e. As shown in Figure 3, both the images and the quantification of the Alizarin red staining demonstrated that high glucose significantly inhibited the formation of mineralized nodules in MC3T3-E1 cells (H vs C group, P < 0.01). However, the H + CGF group exhibited an increase in nodules after exposure to CGF-e (vs H group, P < 0.05).

Fig. 3
Fig. 3:
Effects of high glucose and CGF-e on the mineralization of MC3T3-E1 cells. *P < 0.05 and **P < 0.01 compared with the C group; #P < 0.05 compared with the H group.

CGF-e Induced p-Akt Promoted Osteoblast Differentiation in MC3T3-E1 Cells

As shown in Figure 4, A, the level of P-Akt expression in H group was significantly inhibited compared with the C group (P < 0.01). A significantly enhancement of P-Akt expression (C + CGF vs C group, P < 0.05; H + CGF vs H group, P < 0.01) was observed after treatment with CGF-e. To assay the association between PI3K/Akt pathway and CGF-e–induced osteogenic differentiation of MC3T3-E1 cells, cells were pretreated with 10 mM of PI3K/Akt inhibitor (LY294002; Sigma) before CGF-e. Result showed that the increased expression of ALP mRNA induced by CGF-e was significantly attenuated by addition of LY294002 (Fig. 4, B, P < 0.05).

Fig. 4
Fig. 4:
Effects of CGF-e on PI3K/Akt pathway. (A) Protein levels of p-Akt were determined. (B) Cells were pretreated with 10 mM of PI3K/Akt inhibitor (LY294002) for 24 hours, and the mRNA level of ALP was determined at 10 days. *P < 0.05 and **P < 0.01 compared with the C group; #P < 0.05 and ##P < 0.01 compared with the H group. &P < 0.05 compared with the H + CGF group.

Discussion

Dental implants have a high clinical success rate in generally healthy individuals, where the jawbone possesses a well-formed cortex and densely trabeculated medullary spaces with a good blood supply, providing for a high healing capacity. On placement of the implant into the prepared alveolar socket, the undifferentiated mesenchymal cells migrate into the site, attach, proliferate, and differentiate down osteogenic lineages, leading to contact osteogenesis (de novo bone formation on the implant surface) and distance osteogenesis (bone repair on pre-existing bone surfaces).16 By contrast, the use of dental implants in patients with uncontrolled diabetes mellitus is a debatable issue due to the adverse effects of hyperglycemia on osseointegration.17–22 Hyperglycemia associated with diabetes has been proved to be responsible for delayed bone healing, and it has the potential to affect different stages in the bone healing process, such as vascularization, clot formation, and bone matrix synthesis.23 Researches24,25 have reported that the osteogenic differentiation and mineralization process of osteoblasts were inhibited by high glucose. Our researches also have demonstrated that high-glucose significantly inhibited the proliferation, osteogenic differentiation, and mineralization of MC3T3-E1 cells.

CGF was first produced by Sacco in 2006 (unpublished data). It is an autologous leukocyte- and platelet-rich fibrin biomaterial containing many growth factors and has gained considerable popularity owing to its autologous nature, easy collection, simple, and cost-effective preparation, and safe clinical application, without the risks associated with immunological rejection.26 To the best of our knowledge, this study is the first to evaluate the effects of CGF-e on osteogenic differentiation of MC3T3-E1 cells under high glucose environment. Previous study has reported that beagle periodontal ligament cells cultured with CGF membrane showed increases in osteogenic markers such as collagen-I, bone sialoprotein, and osteocalcin.27 In addition, Honda et al reported that an hMSC line cultured with CGF-e promoted osteogenic gene markers, such as Runx2, Osterix, and Osteopontin.28 In line with above researches, our experiments showed that the proliferation, osteogenic differentiation, and mineralization of MC3T3-E1 cells in H + CGF group were significantly improved. It probably means that the growth factors released by CGF could probably prevent the adverse effects of high glucose on cell proliferation activity and osteoblastic differentiation. However, Kim JM et al13 reported that a highly concentrated CGF-e (20%) inhibited osteoblastic differentiation and mineralization, whereas a less-concentrated CGF-e (1% to 10%) promoted osteoblastic differentiation and mineralization of a human mesenchymal stem cell line in a dose-dependent manner. Therefore, we chose 0.5% CGF-e in this study.

Importantly, optimal cell adhesion is the prerequisite for proliferation and osteogenic differentiation.29 Moreover, the actin cytoskeleton is critical for cell adhesion, migration, and function.30 In this study, we found that the actin filaments were disorganized, and the cell spreading was limited in H group; however, the cytoskeleton was obviously promoted in H + CGF group. It probably means that the CGF-e could effectively promote the adhesion of MC3T3-E1 cells.

The osteogenic differentiation of osteoblasts is closely connection with the PI3K/Akt signaling pathway.31 PI3K is a heterodimeric enzyme important for proliferation and apoptosis. Akt is a downstream serine–threonine kinase that transmits survival signals from growth factors.32 PI3K/Akt pathway and its downstream signaling molecules are critical regulators of bone resorption and bone formation. Our research shows that high glucose significantly inhibited the expression level of P-Akt; however, CGF-e reversed the negative effect of high glucose. In addition, we found that the expression level of ALP mRNA in the H + CGF group was obviously higher than that in the H group; however, pretreatment with LY294002 could significantly eliminate the positive effects of CGF-e. This means that CGF-e promotes osteoblastic differentiation of MC3T3-E1 cells under high-glucose condition partly through the PI3K/Akt regulatory pathway.

Conclusion

The findings of our study demonstrated that the soluble factors released by CGF could significantly stimulate proliferation, cytoskeleton extension, differentiation, and mineralization of MC3T3-E1 cells through the modulation of PI3K/Akt pathway. However, the in vitro studies cannot mimic in vivo conditions. Therefore, further researches are required to clarify the in vivo actions and mechanisms.

Disclosure

The authors claim to have no financial interest, either directly or indirectly, in the products or information listed in the article.

Approval

All surgical procedures involving animals were conducted following recommended procedures approved by the Institutional Animal Care and Use Committee of the Yantai Stomatological Hospital (Approval number: 20170720018).

Roles/Contributions by Authors

K. Dong: experimental implementation and paper writing. P. Hao: data statistics. W. Zhou: language polishing. Z. Liu: experimental consultant.

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Keywords:

CGF; osteoblasts; hyperglycemia; osseointegration

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