Intramembranous ossification in alveolar ridge defect repair using noninductive biomaterials: experimental study

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BACKGROUND

Demand for reconstructive and restorative surgery in the maxillofacial region remains consistently high [1–6]. Various methods for replacing bone defects and/or deficiencies with contemporary biomaterials are used in clinical practice with varying degrees of success [7–10]. Bone wound healing, as a fundamental biologic process, consists of a series of multidirectional and sequential stages characterized by distinct patterns of modeling and remodeling of mineralized tissues [11–14]. The highly dynamic and complex interplay of cellular events—including migration, proliferation, and differentiation, as well as their timely coordination and regulation during bone formation and healing—remains insufficiently understood [15]. To date, numerous studies have reported outcomes of bone augmentation procedures in the alveolar processes of the jaws, presenting clinical and radiographic findings, morphologic and morphometric data, and assessments of vascular perfusion within regions of interest to verify the quality and volume of regenerated tissues. Indicators of successful outcomes typically include the extent of newly formed structures, the degree of mineralization of the regenerated supporting bone, and characteristics of the degradation of bone substitute biomaterials with differing properties [5, 10, 16–19].

The maxilla and mandible, along with certain other bones such as the clavicles, are classified as flat bones, which form mineralized structures via a pattern of direct bone formation—intramembranous ossification—without an intermediate cartilaginous phase [20, 21]. The role of angiogenesis in endochondral ossification is considered well established [22], with direct bone formation a priori assigned to the same pattern. Experimental investigation of reparative osteogenesis of the parietal bone of the skull, which is most frequently used as a model for comparative evaluation with the outcomes of jaw bone defect reconstruction, cannot be considered sufficiently convincing, at least due to differences in the osteogenic activity of the periosteum and the dura mater, as well as metabolic and morphogenetic differences [20, 22–25].

Although progress has been achieved in understanding the biocompatibility characteristics, composition and chemical properties, resorption behavior of biomaterials, and their interactions with the host organism, the events related to initiation and early progression of the osteogenic process in the alveolar ridge of the jaws remain insufficiently elucidated and undescribed. The existing gap in fundamental knowledge regarding the vascular network formation contributes to an incomplete understanding of reparative osteogenesis in the jawbones. Accordingly, we considered it both relevant and timely to investigate early cellular processes during reparative osteogenesis in artificially created alveolar ridge defects reconstructed with biomaterials commonly used in contemporary clinical practice.

AIM

The work aimed to investigate early events in the replacement of volumetric defects in the alveolar bone of the jaws in a large-animal model, within the context of intramembranous bone formation, using noninductive biomaterials.

METHODS

Study Design

A non-randomized experimental study was conducted.

Study Setting

The study was performed in the research and production unit of the Central Research Laboratory at Kuban State Medical University. Biomaterials were prepared at the same facility. Adult minipigs were obtained from the laboratory animal breeding facility of the B.P. Konstantinov Petersburg Nuclear Physics Institute, National Research Center Kurchatov Institute.

Intervention

Three adult minipigs of both sexes, each weighing 2.5 kg and approximately 6 months old, were included in the experiment. An additional animal served as a skin donor for the preparation of dermal matrices. Each minipig underwent a clinical examination to rule out overt pathology or signs of illness. During a 14-day adaptation period, the animals were quarantined in individual housing units and were subsequently transferred to standard cages, where they were kept under standard conditions with ad libitum access to feed and water. Baseline weights were recorded before initiation of the experiment. Throughout the study, the animals’ clinical status was documented in detail. Surgical procedures were performed in the operating suite of the vivarium under aseptic conditions, with general and local anesthesia.

Acellular dermal matrices without the epidermal component were prepared using a detergent–enzymatic method with 1% Triton X-100 and 4% sodium deoxycholate (Sigma-Aldrich, USA). The acellular dermal matrix (ADM) developed at the Central Research Laboratory of Kuban State Medical University was used in the study [26].

Experimental workflow and surgical protocol. In this phase of the animal experiment, which presents an analysis of experimental outcomes, ADM without the epidermal layer was used. The ADM was transplanted either onto artificially created bone defects and covered with a nonresorbable barrier membrane (group 1 [3 alveolar ridge sites]) or combined with an osteoconductive, naturally derived granular biomaterial without a barrier membrane (group 2 [3 alveolar ridge sites]).

General anesthesia was induced intravenously using dexmedetomidine and an injectable formulation containing tiletamine hydrochloride and zolazepam hydrochloride¹; local anesthesia was achieved with an injectable solution containing articaine plus epinephrine. The operative field was disinfected with povidone-iodine. After extraction of the first permanent molars with widely divergent roots, bilateral alveolar ridge defects were created by removing the outer cortical plate to half its height using a surgical round bur and excising the wide interradicular bone. Biomaterials were transplanted into the prepared defects according to the study design, mucoperiosteal flaps were mobilized, and the wounds were closed with VICRYL 910 synthetic suture material, size 5-0 (Ethicon, Johnson & Johnson, USA).

Postoperative antibiotics were not administered because (1) the study was designed and conducted under pathogen-free conditions and (2) exclusion of additional pharmacologic agents was necessary to avoid influencing the animals’ immune response. During the first postoperative week, the oral cavity—including tooth surfaces adjacent to the defects—was treated with a 0.05% chlorhexidine solution. During this period, animals were restricted from consuming hard feed while maintaining a balanced diet. Animals were sacrificed on day 120 by administering lethal doses of the anesthetic agents used for induction.

Morphologic analysis. For the laboratory phase of the study, the mandible was completely harvested from each minipig and immersed in 10% neutral formalin for fixation. Decalcification was performed in two changes of SoftiDec solution (BioVitrum, Russia) over six days. Tissue processing was carried out using a TP1020 tissue processor (Leica Biosystems, Germany). Paraffin blocks were sectioned at 4 µm thickness with an RM223 rotary microtome (Leica Biosystems, Germany), and the sections were mounted on charged adhesive slides for evaluation of newly formed structures in the mesiodistal direction. The specimens were stained with hematoxylin and eosin, Van Gieson’s picrofuchsin, and Masson trichrome (BioVitrum, Russia).

Statistical Analysis

The experiment was non-randomized. No statistical method was used to determine the number of animals included in the study or the volume of alveolar bone autopsy material.

RESULTS

The time required for primary wound healing was comparable between subgroups and totaled 120 days. Subgroup 2 demonstrated greater vertical loss of tissue structures, both within the defect area and on the contact surfaces of adjacent teeth; no exposure of biomaterial surfaces into the oral cavity was observed. At the time of euthanasia, the overlying tissues at the reconstruction sites were assessed as healthy.

In specimens from subgroup 1, the surface tissue overlying the defect was characterized by a reduced number of epithelial germ layers, uneven keratinization, absence of cellular anisomorphism, and lack of papillary layer formation, as well as discontinuity of the basal layer (see Fig. 1, a)². The dermal graft preserved its original thickness by the end of the experiment, with qualitative remodeling of its surface serving as a foundation for new structures. Small sinusoidal capillaries were observed beneath the barrier membrane (see Fig. 1, a, 1), containing maturing hematopoietic cells forming cellular islands. Oxyphilic unattached cells were visualized emerging from these sinusoids into the surrounding tissue (see Fig. 1, a, 2): via cytoplasmic bridging extensions, cells migrated transvascularly—often in groups—through gaps (pores) in the walls of the sinusoidal capillaries. A rapid increase in the quantity and morphological variability of cellular forms within the tissue was identified, represented by numerous basophilic segmented neutrophils, fewer band neutrophils, and larger transitional intermediate cells exhibiting variable staining intensity. Fusion events among some granulocytic elements were clearly visualized, resulting in cells that contained several dark nuclei and elongated, flattened cytoplasm (see Fig. 1, a, 3). Appearing as elongated purple cords or thinner cytoplasmic processes, they formed the walls of newly developed sinusoidal capillaries and nutritive vessels and lined the pores and irregularities of ADM, aligning along preexisting collagen fibers of the dermal graft matrix. Such cellular aggregates interacted with large fibroblast-like cells within the branching matrix network (see Fig. 1, a, 4), increasing the thickness of the developing tissue through the production of an organic substrate that served as a binding framework. In regions of developing reticular bone marrow tissue, lining cells were involved in the formation of more intensely stained, triangular-shaped cells, which were clearly identified in deeper intertrabecular spaces as well as in superficial submembranous areas, appearing as focal cellular clusters.

 

Fig. 1. Morphological pattern of tissue formation in an alveolar defect reconstructed with an acellular dermal matrix (ADM) and a barrier membrane:

a, superficial region, ×100, hematoxylin–eosin stain; I, epithelium; II, nonresorbable barrier membrane; III, acellular dermal matrix; 1, sinusoidal capillaries; 2, transvascular migration of cells; 3, fusion of granulocytes forming elongated cellular aggregates; 4, development of reticular connective tissue.

b, deep region, ×200, hematoxylin–eosin stain; 1, sinusoidal capillaries; 2, formation of reticular stroma within intertrabecular spaces; 3, lining cells; 4, cellular differentiation into osteoblasts and fusion-related events; 5, primary bone trabeculae.

c, Masson trichrome stain, ×200; 1, sinusoidal capillaries; 2, lining of primary bone trabeculae; 3, zone of granulocyte differentiation into lining and other active cells; 4, osteoblastic differentiation and cellular fusion events.

 

A large segment of the sinusoidal network and several smaller hemocapillaries were formed within the interpositional zone between the ADM and the region of primary bone trabeculae (see Fig. 1, b, 1). After migration of myeloid precursor cells from the hemocapillaries, cellular proliferation and differentiation were initiated, following the sequence of events described above, with the only difference being the absence of dermal matrix in this region. The resulting reticular connective tissue formed the stroma of hematopoietic bone marrow within the defect area (see Fig. 1, b, 2, 3, 5). Reticular cells interacted with one another and with other cells of the microenvironment—specifically transitional intermediate forms—via broad cytoplasmic filopodia that created a network-like architecture. Within this network, direct interactions with lining cells were evident, as were active cell fusion processes that preceded differentiation into osteoblasts—large polygonal cells with intensely basophilic nuclei containing multiple nucleoli (see Fig. 1, b, 4).

Subsequent cellular activation within the marrow intertrabecular compartment occurred during the thickening of newly formed bone through appositional growth. Basophilic cells of the endosteal layer—including prominent matrix-producing osteoblasts and granulocytic cellular forms joined by elongated cytoplasmic processes—migrated into the structure of reticulofibrous trabeculae. A portion of the osteoblastic population acquired the characteristics of osteocytes (see Fig. 1, b; Fig. 1, c). These phenomena remained visible later in mature lamellar bone of the alveolar ridge as nonmineralized or poorly mineralized interfaced intervals along which apatite crystals were deposited in a stencil-like pattern [23].

It should also be noted that all de novo tissue layers contained solitary large cells with intensely basophilic nuclei and a thin rim of pale-blue cytoplasm, likely derived from a pool of locally differentiated cells. Although in direct contact with lining cells, they exhibited no apparent functional activity.

In subgroup 2, almost complete biodegradation of the acellular dermal matrix was observed; a multilayered keratinized stratified squamous epithelium had formed (see Fig. 2, a). In most specimens from this subgroup, epithelial cords extended downward through the dense fibrous connective tissue, nearly reaching the level of the remaining nonresorbed osteoconductive granules (see Fig. 2, b, 1). Beneath the residual ADM layer, coarse fibrous trabecular structures were observed, displaying hypercellularity due to numerous intermediate cellular forms. Localized along the borders of intertrabecular spaces, these cells interacted with one another, forming small interwoven networks of cytoplasmic processes. Their contacts with regressing endosteal surfaces of trabeculae were evident; in some regions, endosteum was absent, and a “blurred” transition into the surrounding marrow spaces was observed. The architecture of the reticular tissue within the intertrabecular spaces was altered, showing increased density and focal zones of fibrosis. In previously formed bone trabeculae, the osteocytic component was replaced by avascular dense fibrous connective tissue, and residual nonresorbed granules of the biomaterial were identified within the trabeculae.

 

Fig. 2. Morphological pattern of tissue formation in an alveolar defect reconstructed with an acellular dermal matrix and osteoconductive granules:

a, hematoxylin–eosin stain, ×400; I, epithelium; II, zone of residual dermal graft; 1, reticulofibrous bone trabeculae; 2, intertrabecular spaces; 3, sinusoidal capillaries; 4, osteoid formation.

b, Masson trichrome stain, ×400; 1, epithelial cords; 2, osteoid formation; 3, death of hematopoietic cells within a sinusoid; 4, thickening of the walls of the sinusoid and of a trophic arterial capillary; 5, numerous residual biomaterial granules.

c, general view, hematoxylin–eosin stain, ×200; 1, fibrotic changes within intertrabecular tissue; 2, residual granules of the osteoconductive material; oval-marked areas, small sinusoidal capillaries and early primary bone trabeculae.

 

Findings from Masson trichrome staining clarified several adverse effects (see Fig. 2, b). Distinct morphologic alterations were identified in the sinusoidal capillaries: along the vessel wall, production of organic osteoid matrix was evident, with hematopoietic cells adhering to its surface. This was associated with impaired directed migration of these cells into the surrounding microenvironment and disruption of intravascular orientation within hematopoietic islands (see Fig. 2, b, 2). Marked changes in the walls of hemocapillaries were also noted, characterized by thick, acellular collagen fibers; within a large sinusoidal capillary, apatite crystals were observed growing from both poles, leading to occlusion of the vascular lumen and destruction of hematopoietic cell islands (see Fig. 2, b, 3). In deeper regions, large accumulations of residual osteoconductive granules were visualized.

Within the marrow spaces with fibrotically altered structures, large sinusoidal capillaries were absent; the remaining small vascular sinuses were nonfunctional (see Fig. 2, c). Dense fibrous connective tissue predominated in the intertrabecular spaces, where megakaryocytes exhibiting platelet shedding were observed, releasing small cytoplasmic fragments into the surrounding tissue. Irregularly arranged reticular tissue deviating from normal architecture was replaced by fibrous structures with avascular foci of bone marrow stromal condensation. Surfaces of previously formed reticulofibrous trabeculae demonstrated a thin endosteal layer.

In isolated loci of biomaterial degradation within the dense intertrabecular connective tissue, small hemocapillaries containing a limited number of hematopoietic cells, capable of migrating into surrounding tissue, were identified. Rare morphologic events observed at this time point (see Fig. 2, c, oval-marked regions) were characterized by the initiation of “delayed” osteogenic processes with reduced activity: granulocytic elements were present, together with bone lining cells and newly formed segments of reticular tissue, with an osteoblastic response manifested by the formation of small osteocytic-type bone trabeculae.

Regarding the direct effects of the biomaterials used, several observations should be emphasized. The acellular dermal matrix placed in a supra-alveolar position beneath a semipermeable barrier did not interfere with the formation of normal bone structures during reconstruction of volumetric alveolar ridge defects. Its additional positive role in stabilizing the overlying soft tissues—an attribute valuable in intraoral plastic surgery—was also verified in this work. In the absence of a barrier membrane during alveolar ridge reconstruction, the epithelium exhibited a marked tendency to proliferate downward into the defects. As a consequence, the ADM underwent rapid resorption, and the lost volume was replaced by dense, disorganized fibrous connective tissue with reduced vascularization.

DISCUSSION

This experimental study was conducted in adult minipigs, whose physiology, pathology, and immune responses closely resemble those of humans [24]. The work focused on early events of reparative osteogenesis occurring within the alveolar ridge. The pattern of direct bone formation, or intramembranous osteogenesis, characteristic of these structures is also observed in selected regions of the craniofacial skeleton and is less common than bone formation occurring via an intermediate cartilaginous callus [25]. We traced the sequential early events leading to the development of hybrid structures within surgically reconstructed volumetric alveolar ridge defects in the presence of noninductive biomaterials: acellular dermal matrix and naturally derived osteoconductive granules.

According to our findings, sinusoidal capillaries of the venous component of the regional vascular system formed actively within the reconstruction zones and initiated hematopoiesis. The development of the alveolar ridge was closely linked to vasculogenic events accompanying intramembranous bone growth. Hematopoietic stem cells of the bone marrow are known to differentiate into progenitors of the myeloid lineage, ultimately giving rise to erythrocytes, granulocytes, and platelets [25, 28]. As they mature, these cells enter the systemic circulation to maintain blood homeostasis, together with mature elements produced by the red marrow of other bones.

However, the functional scope of hematopoiesis appears to be broader than previously appreciated. We observed the initiation of sinusoidal capillary formation during the replacement of alveolar ridge defects: a single large myeloid precursor cell or several such cells adhered to lining cells, thereby triggering hematopoiesis within a newly formed hemocapillary. The discontinuous endothelial lining of these sinusoidal capillaries facilitated the migration of immature cells into the surrounding tissue, where proliferation and differentiation proceeded. As a result, numerous granulocytic cells accumulated within the tissues, the majority being small segmented neutrophils and, less frequently, band neutrophils.

Previous studies have demonstrated that neutrophils possess significant protein synthetic capacity, with mature neutrophilic granulocytes capable of producing more than 700 proteins [29, 30]. Morphologic alterations of neutrophilic granulocytes during surgical treatment of periodontitis using microporous barrier membranes have been reported earlier [31, 32]. In our earlier cytologic preparations—imprint smears of the superficial layer of the regenerate granulation tissue—the plasticity of granulocytes was visualized in the following forms: loss of nuclear connections with migration of individual nuclear segments beyond the cell boundary; cellular forms with elongated cytoplasm and multiple nuclei; various cellular fusion phenomena; formation of aggregates of extracellular protein structures derived from neutrophil granules with chromatin components; formation of syncytia; and other features. Taken together, these observations reflect a rather heterogeneous spectrum of transitional intermediate cellular forms at early stages of tissue formation.

In the present work, we focused on differentiation effects involving fusion of granulocytic elements and the emergence of basophilic, elongated cells with three or more flattened, dark nuclei. These cells formed long fibrillar extensions of varying thickness with numerous intercellular and cell–matrix contacts. Once formed, these lining cells functioned similarly to endothelial cells, contributing to the walls of sinusoidal capillaries and trophic vessels (arterioles and venules), and lining the surfaces and pores of residual nonresorbed dermal matrix. Similar phenomena were noted by other investigators who observed that endothelial cells, under reactive conditions, may demonstrate characteristics of connective tissue cells [33].

We additionally noted the participation of endothelial cells in the formation of reticular connective tissue within intertrabecular spaces—the stromal component of bone marrow that sustains hematopoietic homeostasis [34]. Local production of osteoid matrix within de novo reticular tissue enriched with functional blood capillaries was observed in association with a granulocytic response accompanying the differentiation of cells into osteoblasts responsible for the formation of primary bone trabeculae. These observations support the hypothesis that tissue granulocytes, lining/endothelial cells, and reticular structures of the marrow spaces may share a common lineage. Notably, these cell populations also exhibit several overlapping molecular differentiation markers [32, 35].

When excessive quantities of residual osteoconductive granules were present, marked disorganization of the sinusoidal capillaries occurred, resulting in loss of hematopoietic function and impaired transvascular migration of stem cells into the tissue. This resulted in morphofunctional disturbances in the intertrabecular spaces, including fibrotic changes within the bone marrow niche and the absence of observable intercellular interactions.

The mechanisms responsible for the initiation and restoration of hematopoiesis following disruption of the bone marrow niche (as the microenvironment regulating hematopoietic stem cell activity) remain to be elucidated when such disruption occurs in healthy animals under the influence of extrinsic factors.

CONCLUSION

Findings of this experiment indicate that successful replacement of alveolar ridge bone defects—structures characterized by direct bone formation via intramembranous osteogenesis—requires active vasculogenic processes (not arising from preexisting blood vessels, as in angiogenesis), together with a vascular supply sufficient to meet trophic demands at the surgical site. We found that the initiating event of direct bone formation within the defect areas was the development of a network of sinusoidal capillaries in the venous segment of the regional vasculature, where hematopoiesis was activated. In addition to the release of mature blood elements into the circulation, immature myeloid progenitor cells migrated into the bone defect, proliferated, and differentiated into granulocytic cells (primarily segmented neutrophils). Subsequent dynamic stages of cellular differentiation included widespread formation of lining/endothelial cells directly involved in generating reticular connective tissue, primary bone trabeculae, and parallel intertrabecular marrow spaces needed to support hematopoietic stem cell function.

It is also hypothesized that solitary progenitor cells, derived from a pool of locally differentiated cells and anchored to lining cells while being dispersed throughout de novo tissues, remain inactive until required.

The transplanted ADM did not affect intramembranous osteogenesis and had an additional beneficial role relevant to intraoral plastic surgery by stabilizing the developing soft tissues. The microporous polytetrafluoroethylene barrier membrane prevented early epithelial downgrowth into the bone defect, thereby increasing the volume of newly formed alveolar bone.

Disruption of synergistic interactions underlying direct bone formation in healthy experimental animals, observed in response to “external” factors (in this specific case, osteoconductive granules), led to distortion of signaling pathways regulating cellular behavior, abrogation of myeloid progenitor proliferation and differentiation, and morphofunctional insufficiency of bone marrow structures with fibrotic changes. In essence, this resulted in blockade of the niche responsible for sustaining stem cell viability within the region.

The morphologic findings documented here, reflecting the pattern of direct bone formation during reconstruction of volumetric alveolar ridge defects, should be taken into account when evaluating outcomes of reconstructive interventions involving the alveolar bone in dentistry and maxillofacial surgery. Moreover, these results may serve as valuable guidance for biotechnology specialists developing new bone grafting materials, particularly in the context of factors governing vasculogenesis and the condition of the bone marrow niche for hematopoietic stem cells. .

ADDITIONAL INFORMATION

Author contributions: M.D. Perova: conceptualization, data curation, investigation, data interpretation, writing—original draft, writing—review & editing; A.Yu. Ananich: resources, investigation; A.A. Verevkin: investigation, methodology, writing—review & editing; I.A. Sevostyanov: investigation; K.I. Melkonyan: project administration, investigation; I.D. Samokhvalova, I. Alayoub: resources, data curation. All the authors approved the version of the manuscript to be published 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: The animal experiment was approved by the local Ethics Committee of Kuban State Medical University (protocol No. 91, September 29, 2020). All procedures were conducted in accordance with bioethical principles based on the recommendations of the World Society for the Protection of Animals (WSPA), the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, March 18, 1986), Directive 2010/63/EU of the European Parliament and of the Council of September 22, 2010 (compliant with European Economic Area requirements), and Order No. 199n of the Ministry of Health of the Russian Federation dated April 1, 2016, “On the Approval of Good Laboratory Practice Rules”.

Funding sources: The authors declare no external funding was received for the study.

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: This article is an original research work by the author.

Data availability statement: Data are available in open access.

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.

 

¹ The drug is not registered in the Russian Federation.

² When the semipermeable nonresorbable barrier material remains stable within the wound, an epithelium with reduced structural organization forms; following membrane removal, the epithelial layer is restored within a short period to the normal state of stratified squamous keratinized epithelium [27].

About the authors

Marina D. Perova

Kuban State Medical University; LLC “Stomatological Сenter ‘Intelligent’”

Email: mperova2013@yandex.ru
ORCID iD: 0000-0001-6974-6407
SPIN-code: 5552-7988

MD, Dr. Sci. (Medicine), Associate Professor; Scientific & Clinical Base of Kuban State Medical University

Russian Federation, Krasnodar; Krasnodar

Artem Yu. Ananich

Kuban State Medical University

Email: ananicha.ksma@mail.ru
ORCID iD: 0000-0002-5166-2894
SPIN-code: 7324-7491
Russian Federation, 4 Mitrofan Sedin st, Krasnodar, 350063

Aleksandr A. Verevkin

Kuban State Medical University

Email: vilehand@bk.ru
ORCID iD: 0000-0002-4159-2618
SPIN-code: 8264-4990

MD, Cand. Sci. (Medicine)

Russian Federation, 4 Mitrofan Sedin st, Krasnodar, 350063

Igor A. Sevostyanov

LLC “Stomatological Сenter ‘Intelligent’”

Email: drsevostyanovia@gmail.com
ORCID iD: 0000-0002-8472-7279
SPIN-code: 9174-3102

MD, Cand. Sci. (Medicine); Scientific & Clinical Base of Kuban State Medical University

Russian Federation, Krasnodar

Karina I. Melkonian

Kuban State Medical University

Email: kimelkonian@gmail.com
ORCID iD: 0000-0003-2451-6813
SPIN-code: 2461-8365
Russian Federation, 4 Mitrofan Sedin st, Krasnodar, 350063

Inna D. Samoxvalova

Kuban State Medical University; LLC “Stomatological Сenter ‘Intelligent’”

Author for correspondence.
Email: samoxvalovai@mail.ru
ORCID iD: 0000-0003-0360-8882
SPIN-code: 9091-1041

Scientific & Clinical Base of Kuban State Medical University

Russian Federation, Krasnodar; Krasnodar

Iyad Alayoub

Kuban State Medical University

Email: iyadalayoub@yahoo.com
ORCID iD: 0009-0007-3888-8024
Russian Federation, 4 Mitrofan Sedin st, Krasnodar, 350063

References

Supplementary files