Evaluation of the Cytotoxicity of BMP-2 in the Coating of Dental Implants: an  In Vitro  Study

Alexander G. Stepanov; Степанов Александр Геннадьевич; Samvel V. Apresyan; Апресян Самвел Владиславович; Eduard G. Nacharyan; Начарьян Эдуард Георгиевич; Maxim V. Kopylov; Копылов Максим Валерьевич; Genrikh G. Kazarian; Казарян Генрих Геворгович; Enar D. Jumaniazova; Джуманиязова Энар Денисовна; Victoria Е. Karyagina; Карягина Виктория Евгеньевна

doi:10.17816/dent654080

Evaluation of the Cytotoxicity of BMP-2 in the Coating of Dental Implants: an In Vitro Study

Authors: Stepanov A.G.¹, Apresyan S.V.¹, Nacharyan E.G.¹, Kopylov M.V.¹, Kazarian G.G.¹, Jumaniazova E.D.¹, Karyagina V.Е.¹
Affiliations:
1. Peoples' Friendship University of Russia
Issue: Vol 29, No 3 (2025)
Pages: 210-218
Section: Original Study Articles
Submitted: 12.02.2025
Accepted: 24.02.2025
Published: 27.06.2025
URL: https://rjdentistry.com/1728-2802/article/view/654080
DOI: https://doi.org/10.17816/dent654080
EDN: https://elibrary.ru/GACLGK
ID: 654080

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Abstract
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Abstract

BACKGROUND: Bone morphogenetic protein 2 (BMP-2) is increasingly incorporated into bone graft materials due to its positive effects on osseointegration and de novo bone formation. While its efficacy in bone regeneration is well established, concerns have been raised about adverse effects, including inflammation, ectopic bone formation, soft tissue swelling, and even oncogenesis. Understanding the basis of these effects requires investigation of BMP-2 impact on various cell types.

AIM: The study aimed to evaluate the biological effects of BMP-2–containing implant coatings on the human monocytic leukemia cell line THP-1.

METHODS: THP-1 cells were seeded in 12-well plates at 2 mL/well and a concentration of 250 × 10³ cells per well, with PMA (phorbol 12-myristate 13-acetate) added to a final concentration of 150 nM. The cells were incubated at 37 °C with 5% CO₂ for 4–6 h until fully adhered to the culture plastic. Experimental implants (one per well) were then added and incubated for 48 h. To detect apoptotic cells after 48-hour incubation with implants, they were stained with propidium iodide (Lumiprobe, Russia). For each sample, 1 × 10⁵ cells were analyzed. Immunophenotyping was performed using anti-CD45 monoclonal antibodies (130-113-681, clone 5B; Miltenyi Biotec, Germany). Samples were analyzed using a NovoCyte Advanteon flow cytometer (Agilent, USA), and data were processed with Flowing Software 2.

RESULTS: No significant differences in cytostatic effects were observed between implants with and without BMP-2 coating. However, culture medium alone differed significantly from the implant-containing groups, suggesting that the mere presence of an implant affects THP-1 cell behavior. After incubation with the test implant samples, the percentage of apoptotic THP-1 cells, detected by flow cytometry following propidium iodide staining (PI test), did not significantly change between groups, despite a slight increase in this parameter among coated samples. Notably, a significantly higher proportion of CD45⁺ cells was detected after incubation with coated implants.

CONCLUSION: The study showed that implants with and without coating do not differ in their cytostatic properties when incubated with THP-1 cells. When assessing the percentage of apoptotic THP-1 cells, no significant difference was observed between groups of implants with and without coating. However, the group of coated implants exhibited a significantly higher percentage of CD45⁺ cells.

Keywords

bone morphogenetic protein 2, THP-1 monocytic cell line, coated dental implants, CD45+ cells

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BACKGROUND

Bone morphogenetic protein 2 (BMP-2) belongs to the transforming growth factor beta (TGF-β) superfamily, which plays a key role in osteogenesis as well as bone growth and regeneration. BMP-2 was first isolated by Urist in 1965 [1], but it was not until 2007 that the US Food and Drug Administration (FDA) approved its clinical use in dentistry. Today, incorporation of BMP-2 into various bone graft materials has gained increasing popularity due to its beneficial effects on osseointegration and de novo bone formation. This is particularly relevant for patients with impaired bone healing, such as those with diabetes mellitus or a history of radiotherapy for maxillofacial neoplasms [2]. During implant placement surgery, BMP-2 is delivered as an osteoinductive growth factor to accelerate bone remodeling around the dental implant. Although BMP-2 itself has demonstrated efficacy in promoting bone regeneration, the use of carrier systems extends its biological activity and reduces the local concentration required to achieve osteoinductive effects at the graft site [3, 4]. Despite the well-established regenerative potential of BMP-2, concerns have been raised about its adverse effects, including inflammation, ectopic bone formation, soft-tissue edema, and even oncogenesis. Most of these complications are associated with supraphysiologic concentrations resulting from rapid release of BMP-2 from its delivery matrix [5, 6]. Understanding the mechanisms behind these effects requires investigation of BMP-2 impact on various cell types. In this study, we evaluated the effects of BMP-2–containing implant coatings on immune cells.

AIM

The work aimed to evaluate the biological effects of BMP-2–containing implant coatings on the human monocytic leukemia cell line THP-1.

METHODS

The human monocytic leukemia cell line THP-1 was cultured in RPMI-1640 medium supplemented with L-glutamine (PanEco, Russia), 10% fetal bovine serum (Capricorn Scientific, Germany), and 1% penicillin–streptomycin (PanEco, Russia). THP-1 cells were seeded into 12-well plates at a total medium volume of 2 mL per well and a concentration of 250 × 10³ cells per well. Phorbol 12-myristate 13-acetate (PMA) was added to a final concentration of 150 nM. The cells were incubated at 37 °C with 5% CO₂ for 4–6 hours until fully adhered to the culture plastic. Experimental implants—5 with BMP-2 coating and 5 without coating—were placed individually into separate wells. The cell cultures with implants were incubated for 48 hours under identical conditions (37 °C, 5% CO₂).

To detect apoptotic cells after 48-hour incubation with implants, they were stained with propidium iodide (Lumiprobe, Russia). For each sample, 1 × 10⁵ cells were analyzed in 300 µL of phosphate-buffered saline (PBS). Samples were incubated with the dye for 5 minutes at room temperature in the dark. For immunophenotyping, anti-CD45 monoclonal antibodies (catalog No. 130-113-681, clone 5B; Miltenyi Biotec, Germany) were used. For CD45 surface marker staining, 1 × 10⁵ cells per sample were resuspended in 100 µL of PBS containing 1% bovine serum albumin and incubated with the antibodies for 15 minutes at +4 °C. Flow cytometry was performed using a NovoCyte Advanteon flow cytometer (Agilent, USA). A minimum of 10,000 events per sample was recorded. Data were processed and analyzed using Flowing Software 2.

For assessment of cell viability, THP-1 cells were further cultured in 96-well plates at a total volume of 200 µL per well and a density of 15,000 cells per well, with PMA added to a final concentration of 150 nM to induce differentiation into macrophages. The cells were incubated with the test implants for 48 hours at 37 °C in a 5% CO₂ atmosphere. After incubation, the medium was removed, and MTT reagent (PanEco, Russia) dissolved in RPMI-1640 to a final concentration of 0.5 mg/mL was added to each well. The plates were incubated under the same conditions for 4 hours. Subsequently, 100 µL of dimethyl sulfoxide (DMSO) (PanEco, Russia) was added to each well to dissolve the formazan crystals. The plates were kept at room temperature for 15 minutes, and optical density was measured at 540 nm using a Multiskan GO multimode microplate reader (Thermo Fisher Scientific, USA).

Statistical Analysis

Statistical analyses were performed using Prism 8 (GraphPad, USA). The Shapiro–Wilk test was applied to assess the normality of distribution. For comparisons among more than two groups, one-way ANOVA followed by Tukey’s post hoc test, or the Kruskal–Wallis test followed by Dunn’s post hoc test, was used as appropriate. For pairwise comparisons, the Mann–Whitney U test or Student’s t test was applied. Differences were considered significant at p < 0.05.

RESULTS

A total of 10 implants were examined—5 with BMP-2 coating and 5 without coating. No significant differences in cytostatic effects were observed between implants with and without BMP-2 coating. However, the control culture medium without implants differed significantly from both experimental groups, indicating that the mere presence of an implant in the culture well influences THP-1 cell behavior (see Table 1, Fig. 1).

Table 1. Optical density at 540 nm in samples after the MTT assay

Replicate No.	Control (medium without implant)	Implant without coating (n = 5)	Implant with BMP-2 coating (n = 5)
1	2.005	0.991	1.245
2	1.881	0.869	1.011
3	1.072	1.043	1.5
4	2.225	1.269	0.832
5	2.452	1.808	0.603
M ± SD	1.92 ± 0.52*	1.19 ± 0.37	1.0382 ± 0.3400

Note: * p < 0.05 vs. the “implant without coating” and “implant with BMP-2 coating” groups.

Fig. 1. Viabilit y of THP-1 cells after incubation with coated and uncoated implant samples and in the control culture medium; p < 0.05 vs. “implant without coating” and “implant with BMP-2 coating” groups.

The obtained data demonstrated that, after incubation with the test implant samples, the percentage of apoptotic THP-1 cells detected by flow cytometry following propidium iodide (PI) staining did not differ significantly between groups, despite a slight increase observed in the coated samples (see Table 2, Fig. 2).

Table 2. Percentage of propidium iodide–positive (apoptotic) THP-1 cells after incubation with coated and uncoated implant samples

Replicate No.	Implant without coating	Implant with BMP-2 coating
1	3.56	3.11
2	2.84	3.44
3	2.81	4.89
4	1.96	3.75
5	2.82	6.92
M ± SD	2.79 ± 0.56	4.42 ± 1.54

Fig. 2. Percentage of propidium iodide–positive apoptotic cells after incubation with implant samples with and without coating.

Representative dot-plot diagrams after staining are shown in Figs. 3 and 4.

Fig. 3. Representative dot-plot of propidium iodide–positive (apoptotic) THP-1 cells after incubation with uncoated implants (right panel). Left panel shows the unstained control. SSC-H, side scatter height; PE-H, phycoerythrin height.

Fig. 4. Representative dot-plot of propidium iodide–positive (apoptotic) THP-1 cells after incubation with BMP-2–coated implants (right panel). Left panel shows the unstained control. SSC-H, side scatter height; PE-H, phycoerythrin height.

Analysis of CD45⁺ cells after incubation with coated and uncoated implants revealed a significant increase in CD45 expression in the coated implant group (see Table 3, Fig. 5). Representative staining is shown in Fig. 6.

Table 3. Percentage of CD45⁺ cells after incubation with coated and uncoated implant samples

Replicate No.	Implant without coating	Implant with BMP-2 coating
1	35.5	51.6
2	34.8	54.5
3	30.3	48.8
4	38.1	47.9
5	35.4	49.5
M ± SD	34.8 ± 2.8	50.46 ± 2.60*

Note: * p < 0.05 vs. the “implant without coating” and “implant with BMP-2 coating” groups.

Fig. 5. Percentage of CD45⁺ cells after incubation with coated and uncoated implant samples; p < 0.05 by Student’s t test.

Fig. 6. Representative staining of THP-1 cells with anti-CD45 antibodies (blue signal) after incubation with implant samples without coating (a) and with BMP-2 coating (b). Red signal indicates control (unstained) cells.

DISCUSSION

Placement of a biocoated dental implant inevitably triggers an immune response, which may adversely affect implant integration and bone regeneration. Despite advances in biomaterial engineering over recent decades, undesirable immune-mediated events—such as inflammation, fibrous encapsulation, tissue degradation, and implant isolation or rejection—remain among the most common complications associated with coated dental implants. In this study, we evaluated the effect of BMP-2 incorporated into the dental implant coating on the THP-1 cell line and on CD45⁺ cells.

The human acute myeloid leukemia cell line THP-1 is commonly used as a model of the monocyte–macrophage system and for evaluating its role in the immune response under both pathological and physiological conditions. Monocytes and macrophages are key components of the innate immune system. The THP-1 cell line is an appropriate in vitro model for studying the modulation of monocyte and macrophage functions. Macrophages derived from the myeloid lineage predominate during the inflammatory phase and play a stimulatory role in normal osteogenesis. It has been shown that macrophages secrete a broad spectrum of cytokines, including BMP-2, oncostatin M, and interleukin 23 (IL-23), which collectively regulate bone formation processes [7].

CD45, also known as protein tyrosine phosphatase, receptor type C or leukocyte common antigen, is a key transmembrane glycoprotein expressed on all nucleated hematopoietic cells. CD45 expression can increase in response to various stimuli and is closely associated with immune cell activation. External stimuli, particularly pathogen-associated molecular patterns, can upregulate CD45 expression. For instance, stimulation of whole-blood samples from patients with COVID-19 by lipopolysaccharides resulted in elevated CD45 expression in granulocytes and monocytes [8]. Inflammatory responses and cellular stress can also lead to increased CD45 expression. Activation of THP-1 cells may also result in increased CD45 expression, which can be interpreted as a marker of monocyte or macrophage activation in vivo. Moreover, CD45 is involved in the regulation of cell proliferation. Alterations in the cell cycle or in the proliferation rate of THP-1 cells may potentially lead to increased CD45 expression. The multifaceted role of CD45 in immune cell function—from regulating signal transduction to influencing cell proliferation and differentiation—makes it a dynamic marker whose expression can shift in response to various cues, both intrinsic and extrinsic.

CONCLUSION

Summarizing the findings of the present study, the authors found that implants with and without the coating did not differ in their cytostatic properties when incubated with THP-1 cells. When assessing the percentage of apoptotic THP-1 cells, no significant difference was observed between groups of implants with and without coating. However, the group of coated implants exhibited a significantly higher percentage of CD45⁺ cells.

ADDITIONAL INFORMATION

Author contributions: A.G. Stepanov: methodology, conceptualization, investigation, writing—original draft, writing—review & editing; S.V. Apresyan: investigation, writing—original draft, writing—review & editing; E.G. Nacharyan: investigation, writing—original draft, writing—review & editing; M.V. Kopylov: writing—original draft, writing—review & editing; G.G. Kazarian, E.D. Jumaniyazova, V.E. Karyagina: methodology, investigation, formal analysis. All authors approved the final version of the manuscript for publication and agreed to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval: Not applicable.

Funding sources: No funding.

Disclosure of interests: The authors have no relationships, activities, or interests for the last three years related to for-profit or not-for-profit third parties whose interests may be affected by the content of the article.

Statement of originality: No previously published material (text, images, or data) was used in this work.

Data availability statement: All data generated or analyzed during this study are included in this article.

Generative AI: No generative artificial intelligence technologies were used to prepare this article.

Provenance and peer review: This paper was submitted unsolicited and reviewed following the standard procedure. The peer review process involved two external reviewers, a member of the editorial board, and the in-house scientific editor.