CURRENT POSSIBILITIES AND PROSPECTS OF ALVEOLAR BONE DEFECT REPLACEMENT AND COVERING TISSUES: NARRATIVE LITERATURE REVIEW

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Background

In surgical dentistry and maxillofacial surgery, the number of patients in need of reconstructive and reconstructive bone surgeries using osteosubstituting materials is about 330 thousand per year, the absolute majority of whom are of working age [1]. According to the circulation data, the proportion of those in need of an increase in the volume of the alveolar jaw bone varies widely − from 43% to 77% [2]. Significant bone defects in terms of volume, where the need for regeneration exceeds the normal self-healing potential, can be replaced by no more than 10% of the lost volume during a person's lifetime [3, 4]. It is proved that in conditions of a large loss of supporting tissues in the human body, a morphologically verified decrease in the activity of inducing factors of systemic and local levels, which largely determines the quality of the final result of reconstructive treatment [5].

Replacement of intraoral integumentary tissue defects resulting from chronic dental diseases, injuries, congenital malformations or tumor eradication with autografts is hampered by a limited number of healthy donor sites, poorly predicted survival of full-layer flaps, the formation of scar contractures and deformities [6, 7]. Regeneration of the integumentary structures can potentially be achieved with the help of cell structures and biocompatible frame materials for insertion into the area of damage. The currently actively developing direction, called tissue engineering, already offers a number of biological structures that can help eliminate the deficit of integumentary intraoral structures.

The aim is to characterize the modern achievements of intraoral reconstruction of volumetric defects, including bioengineering for the regeneration of the alveolar jaw bone and integumentary tissues.

Methods

The search strategy included an electronic search of literature sources performed in the PubMed database using the keywords mentioned in the PubMed and MeSH headings: post-traumatic osteogenesis, neovascularization, endesmal regeneration, targeted bone regeneration, osteosubstituting biomaterials, mesenchymal stem cells, tissue engineering structures. The formats "Review", "Systematic Review" and "Clinical Trial" were requested.

 Inclusion criteria: 1) original scientific articles with controlled research design, reviews, systematic reviews and meta-analyses, 2) studies that evaluated the effect on vascularization processes when using biomaterials to replace bone defects; 3) studies that used various inducing agents - mesenchymal stem cells and (or) growth factors.

Non-inclusion criteria: 1) studies published more than 25 years ago; 2) studies that did not evaluate the vasculogenic effects of biomaterials in the replacement of bone defects.

Of the 378 articles, 44 met the inclusion criteria established for this review. The search depth is 20 years.

Results

1. Prerequisites for intraoral bone reconstruction

Acquired alveolar ridge bone deficiency in patients is most often caused by the loss of teeth of natural bite. After tooth extraction, the normal healing process takes about 40 days, starting with the formation of a clot and ending with filling the hole with bone covered with connective tissue and epithelium. However, resorption of the alveolar bone is inevitable, which leads to significant changes in its size and deformations. These changes are manifested by a loss of crest height by 1.5 mm - 2 mm and width - 40% - 50% in the period from six to twelve months [8]. Size reduction is especially active during the first 3 months and progressively continues with the loss of bone structures by more than 10% over the next 5 years. According to A. Ashman, post-extraction loss of bone height and width is 40% and 60% over 2-3 years, respectively [9].

Endesmal and enchondral ossification were previously considered as different types of regenerative process for different bones of the skeleton. Nevertheless, osteoblasts are cells that form bone of both types, and the main difference between them is on which basis osteoblasts deposit the bone matrix: in a pre-formed model of cartilage or perivascularly [10]. Osteoblasts form a bone matrix in a certain direction, since they lack different domains on the plasma membrane. In addition, osteoblasts require a spatial framework that serves for migration and deposition of functionally structured bone tissue, so that the type of scaffolds (or extracellular matrix) largely determines the "behavior" of cells [11]. The formation of the bone of the alveolar maxillary ridge after reconstructive operations occurs both on the skeletons of non-mineralized tissues or biological substances (direct bone formation de novo), and on the preserved mineralized bone or its tissue-engineered analogues. In addition, it has been reported that the combination of both endesmal and enchondral bone formation occurs simultaneously during the development of craniofacial structures [12], healing of mandibular fractures [13] and bone elongation due to distraction corns [14, 15].

The bones of the skull and jaws have a sandwich structure with a dense cortical plate with a longitudinal arrangement of osteons, which surrounds the medullary space from the outer and inner sides (16). The spongy bone contains skeletal internal partitions and rods of trabeculae (thickness from 50 to 300 microns) in the bone marrow mesh network [17], located in the direction of mechanical loads.  In the spongy bone, resistance to external forces (chewing pressure, for example) occurs differently than in the cortical bone: a feature of the submicrostructure of this bone is the presence of mineralized collagen fibers, inside which hydroxyapatite crystals (3 x 25 x 50 nm in size) and collagen molecules are located [18]. Therefore, the mineralized extracellular matrix of bone tissue is identified as a specialized connective tissue consisting of an organic phase (90% of type I collagen and about 5% of non−collagen proteins) and an inorganic one, mainly of hydroxyapatite and 10% - 20% water [19]. These two types of bones differ in the level of porosity of tissues and the activity of physiological processes. The trabecular bone with a porosity of 40% to 95% is less dense than the cortical bone, and has a flexible structure. Moreover, spongy bone is metabolically more active compared to compact bone [20, 21]. This is how the structure provides the function. Accordingly, achieving the "restoration ad integrum" outcome as a result of surgical reconstruction of the reduced alveolar ridge is of paramount importance for resisting masticatory loads, including the presence of osteointegrated dental implants.

2. Transplants and bioengineering products for intraoral bone reconstruction

Two variants of bone grafts are available for replacement of bone defects: natural (auto-, allo- and xenogenic) and artificial synthetic (alloplastic). In intraoral reconstructive surgery to increase the bone volume of the alveolar bone in the interests of dental implants, cortical and cortical-spongy autografts extracted from the outer oblique line of the lower jaw, less often the chin symphysis, are more often used; in maxillofacial surgery, they often resort to the selection of material from the iliac crest. Autografts remain the "gold standard" due to their inherent osteogenic, osteoinductive and osteoconductive characteristics due to the preservation of properties for the formation and maturation (remodeling) of tissues similar to the living host bone without rejection [22, 23]. These materials have the greatest potential for induction of microcirculation due to the presence of viable cells, such as multipotent bone marrow stem cells. Allografts are osteoconductive, have little or no osteoinductive properties, but are not osteogenic. Integration in the recipient zone occurs more slowly and to a lesser extent [24], there are risks of rejection of allografts in oral surgery. Xenogenic grafts (the basis is the inorganic bone matrix of pigs, horses, cattle) are well embedded in the recipient bone, but their low rate of resorption can negatively affect the healing and maturation of tissue structures, jeopardizing the mechanical and biological properties of the replaced bone. There are observations that particles of bone substitutes of animal origin are detected 10 or more years after surgery [25]. Both allo- and xenografts undergo a decellularization process to eliminate antigenic properties, as a result of which they were proposed to be classified as bioengineering products [26]. Alloplastic biomaterials (calcium carbonate, calcium sulfate, bioactive glass and ceramics, including synthetic hydroxyapatite, tricalcium phosphate) developed for interaction with living structures have osteoconductive properties, being a biocompatible matrix with imitation of the mineral phase of bone [27, 28]. Despite the fact that ceramics have a more pronounced mechanical strength in comparison with human cortical bone, it demonstrates a higher Young's modulus [29] and is considered undesirable when used in intraoral reconstructive surgery, for example, for the potential placement of dental implants. Hydroxyapatite and tricalcium phosphate, as well as their combination - two-phase calcium phosphate due to a composition similar to natural bone, acceptable biocompatibility, osteoconductivity and the ability to directly integrate with the surrounding bone, have become the most widespread among calcium-phosphate ceramics in bone bioengineering [30]. Tricalcium phosphate exists in two main different phases of crystals (α and β), similar in chemical composition, but differing in crystallographic characteristics that give them different resorption properties [31]. While hydroxyapatite has a relatively high crystallinity, it is hardly resorbed in vivo; tricalcium phosphate biodegrades more actively than hydroxyapatite [32]. Bioactive glass is a silicate-based material, is osteoconductive, although it may have osteoinductive properties in certain formulations − as a result of changes in the composition and proportions of the biomaterial (45S5, 58S or 1393); interest in bioactive glass is due to the ability to bind to both bone and soft tissues [33]. By changing the proportion of sodium oxide, calcium oxide and silicon dioxide, resorbable and non-absorbable forms of artificial bone substitutes are obtained [23]. It was revealed that after implantation of scaffold scaffolds at the level of interfacial bonds, the process of formation of a dense layer of hydroxyapatite carbonate, similar to the mineral phase of bone, is quickly started, providing, at the same time, unhindered cell adhesion [29]. Zhang J. Research Group et al. (2015) demonstrated the best characteristics of bioactive borate glass in comparison with tricalcium phosphate [34]. According to the results of our own clinical and histological study, granules of bioactive glass with a size of 250-350 microns implanted into the submembrane space during surgical treatment of periodontitis after 3 months were in the stage of active resorption - the cracking of granules into smaller particles (in the form of a "bicycle wheel") with the formation of reticulofibrous bone tissue directly on the surface of the biomaterial [35].

The success or failure of bone regeneration in reconstructive surgery depends on the key role of the vascularization process. Blood vessels perform the function of maintaining cell viability through perfusion of the healing zone during physiological development or bone regeneration. In particular, the critical point is the possibility of vascularization of volumetric bone grafts to provide the regeneration zone with oxygen, nutrients, growth factors, minerals, as well as for the transfer of metabolic products from the operating wound. Bone regeneration can be defined as a complex mechanism based on the interaction of osteogenic and vasculogenic processes that can stimulate bone growth and restore tissue. The formation of blood vessels is a necessary part of bone formation, the process of osseointegration of dental implants, the transport of growth factors that ensure the viability and interaction of cells. In addition to observing the basic surgical principles, in order to achieve an acceptable volume of the alveolar jaw bone, an adequate response of the host is necessary, due to which the restoration of microcirculatory blood flow is ensured [36]. The insufficient number of microcirculation vessels in the regeneration zone is directly related to the reduced formation of the supporting bone [37]. The growth of blood vessels is necessary both during the formation of bones in the embryonic period and in adults during post-traumatic regeneration; bone recovery and maturation presuppose activation and complex interactions between vasculogenesis and osteohistogenesis [38, 39].

Ambiguous results have been obtained from the use of biomaterials with growth factors - a combination of osteogenic factors with angiogenic signals [40] and their effect on bone healing [41, 42]. Several strategies of immobilization of growth factors on biomaterials have been implemented, as a result of which specific profiles of their release in the regeneration site have been obtained [43]. In relation to bone tissue, the most studied are bone morphogenetic proteins (BMPs) belonging to the family of transforming growth factors (TGFß), vascular endothelial growth factors (VEGF) with its receptors, placental growth factor (PIGF) – a representative of the VEGFs family, platelet growth factor (PDGF), fibroblast growth factor (FGF). BMPs play a role in the processes of mitogenesis and osteogenic differentiation of mesenchymal stem cells, as well as in the induction of angiogenesis [44-46]. BMP-9 was used to improve the functions of the composite skeleton, revealing its ability to differentiate bone marrow MSCs into osteoblasts in vitro and enhancing bone formation in vivo [47]. Vascular endothelial growth factor-A (VEGF-A) plays an important role in angiogenesis, it is produced by many types of cells. In addition, its activity includes not only bone formation [48-50]. VEGF stimulates migration, proliferation and differentiation of endothelial cells, inhibits their apoptosis, promotes the formation of capillary-like structures; indirectly stimulates osteogenesis by regulating the secretion of osteogenic growth factors through paracrine signals [51, 52]. However, overexpression of VEGF causes bone resorption due to the excessive presence of osteoclasts in tissues [53].

Explanations for the accumulated contradictions when using such combinations were given in his dissertation by I.Ya. Bozo (2017). Protein molecules rapidly biodegrade under operating wound conditions (exudation, high activity of proteolytic enzymes), which does not allow the material to show osteoinductive effect in full. The amount of protein in the biomaterial is limited, and the time of their "work" is short-term and obligate. A smaller part of the protein molecules that have separated from the carrier, while maintaining their biological activity, reaches the target cells, where they interact with specific receptors on their surface and realize a biological effect. In this case, the receptors are rapidly inactivated together with the ligand as a compensatory-adaptive mechanism that protects cells from excessive stimulation. The biological effect of the growth factor ceases, and its amount runs out [1].

Gene-activated materials are devoid of these disadvantages. Their main active component, determining the effect on the reparative regeneration of bone tissue, are gene constructs. Unlike products containing growth factors, the main component of gene-activated osteoplastic materials is able to act facultatively: the ingress of a transgene (growth factor) into the nucleus of a target cell does not force it to obligate protein expression. The cell retains its normal functional state and response to microenvironment stimuli. Thus, in the absence of a need for a therapeutic protein (in a specific period of time), due to intracellular post-transcriptional mechanisms of regulation of mRNA half-life, it is able to reduce the level of transgene mRNA and, thereby, prevent protein production [54]. In our country, as part of the creation of a technological platform, the world's first gene-activated material for starting osteohistogenesis has been registered, which contains the gene for vascular endothelial growth factor VEGFA [55]. Nucleotide sequences encoding the main osteoinducing and osteoblast-specific transcription factors are used as transgenes. Plasmid DNA with the VEGFA gene (pl-VEGFA), which has angiogenic activity critically important for reparative osteohistogenesis, was selected as a biologically active component of the gene-activated materials being developed [56, 57].

3. Possibilities of intraoral tissue reconstruction with the introduction of mesenchymal stem cells into the defect zone

The introduction of bioengineering technologies with the use of mechenchymal stem cells (MSCs) is considered to be one of the promising directions for the development of reconstructive surgery today [58, 59].

Natural bone formation during reparative regeneration of the cranio-maxillofacial region is carried out by recruiting MSCs from the bone marrow into the surgical intervention zone. These cells undergo osteoblastic differentiation and initiate the formation of a new bone, replacing the defect. In other words, this method is aimed at triggering bone tissue regeneration by simulating biological processes occurring during embryogenesis [60, 61]. The mechanism by which MSCs promote bone regeneration is aimed at differentiation into the osteogenic line – into the precursors of osteoblasts, which is associated with the beginning of the synthesis of bone matrix (deposits of organic substance) with subsequent mineralization. At this stage, mature osteoblasts undergo apoptosis or turn into osteocytes [62].

As a rule, the source of cellular material for the implementation of such technologies is the adult's own tissues, including bone marrow and adipose tissue. The most accessible, abundant and minimally invasive source of multipotent stem cells, without the risk of rejection for the patient, is adipose tissue from which 100-500 times more cells are isolated than from bone marrow. They have significant plasticity and, under the influence of microenvironment factors, are able to differentiate into cells of mesenchymal origin, as well as, under special conditions in vitro, into cells of ectodermal and endodermal phenotypes [63, 64]. Unlike other sources (bone marrow, embryos, intraoral tissues), adipose tissue stem cells are much less sensitive to aging and, very importantly, the material is easily scaled if necessary [65-67].

In 2001, P.A. Zuk et al. isolation of multipotent cells from lipoaspirates of different localization of the organism was described [68]. Mesenchymal stem cells of adipose tissue have much in common with respect to the profiles of surface markers of bone marrow cells, multipotent potential and growth characteristics [69]. The stromal-vascular fraction of adipose tissue (VSF-AT) is a heterogeneous population of stem/stromal cells isolated from the perivascular and connective tissue matrix of adipose tissue by enzymatic (or mechanical) disaggregation and subsequent centrifugation. Mature adipocytes and their precursors, fibroblasts, macrophages, blood cells, endothelial progenitors and mesenchymal stromal cells were found in the composition of the vascular-cellular fraction of adipose tissue. Many translational studies show that VSF-AT promotes tissue healing and regeneration through a combination of cell-mediated repair and, most importantly, paracrine effects [70-71]. This opened up the prospect of using VSF-AT as a source of cells suitable for various applications in regenerative medicine, including wound healing, soft tissue regeneration, skeletal tissue repair and other conditions. The spectrum of angiogenic, anti-inflammatory, immunomodulatory cytokines and growth factors secreted by VSF-AT cells is described [72].

Our first work to determine the characteristics of the VSF-AT was an experimental study on guinea pigs, which simulated large bilateral through defects of the mandible. 12 weeks after the introduction of autogenic HCF-VT into the defect zone, they were replaced by vascularized bone with the formation of cortical plates; histological and immunohistochemical studies confirmed the presence of mature mineralized bone de novo with an osteonic structure of lamellar bone. On the control side, the presence of fibrous and muscular tissue without bone formation was morphologically confirmed in the defect zone at the same time [58]. In a comparative clinical study, the possibility of using VSF-AT for the replacement of large bone defects of the jaws after cystectomy was studied. In the tested group, it was possible to significantly reduce the frequency of postoperative complications, to increase the frequency of positive clinical outcomes without relapses of the inflammatory-destructive process in the long-term follow-up to 94% (versus 21% in the control). The structural and functional restoration of a well-vascularized trabecular bone and cortical structure was noted, as well as the preservation of causal teeth that had been in contact with the environment of cystic formations for a long time [73]. The results of another controlled study showed the high efficiency of bone plastic surgery with the use of VSF containing osteoconductive materials in the regression transformation of the alveolar ridge of the upper and lower jaws. In patients of the test group, an acceptable degree of bone augmentation was achieved without the risk of complications and reoperations (8% and 21% of cases in the test and control groups, respectively), with optimal morphological characteristics of the regenerate (40.14 ± 3.36 and 24.23± 2.63% of viable mineralized bone tissue on sections of trepan biopsies from the test and control groups, respectively). This ensured reliable osseointegration of artificial supports in the reconstructed alveolar ridge and high efficiency of orthopedic treatment based on osteointegrated dental implants for up to 10 years (97% and 88% in the tested and control groups, respectively) [74].

To date, the results of the replacement of bone defects of the craniofacial-maxillofacial region with tissue-engineered structures, including VSF-AT, have been presented [75-77].

The results of the use of MSCs have shown that this is simply a feasible, safe and more predictable option for increasing the insufficient volume of the alveolar ridge of the jaw bones [78]. It was also demonstrated that patients who underwent lifting of the maxillary sinus floor to install dental implants benefited from the use of these cells. The percentage of osteoid substance and mineralized bone in the regeneration zone was significantly higher in the examined biopsies than in the control samples [66]. The additive effect of the VSF-AT supplement did not depend on the bone substitute − β-tricalcium phosphate or biphasic calcium phosphate.

4. Development of tissue engineering in the replacement of the deficiency of the gingival structures of the alveolar ridge

The first tissue-engineered structures in the form of layers of epithelial cells of the oral mucosa (IOM), transplanted into the zone of reconstructive intervention, turned out to be inconvenient in operation, and most importantly, had low rates of engraftment [79]. However, the idea of using epithelial layers remains relevant as before, and quality control of the cell graft according to a detailed developed technique presented in the work of Kasai Y. is recognized as a decisive success factor. et al (2020) in the form of a step-by-step standardized protocol for the production of epithelial cell sheets for the oral mucosa [80].

Another approach is known, using natural polymers such as fibrin gel, hyaluronic acid, chitosan and their combinations [81-85]. The disadvantages of using such materials were the lack of necessary stability, weak mechanical properties, rapid degradation in the wound, a tendency to compression and loss of volume [86-87].

The research group K. Izumi et al. (2000) proposed to overcome these disadvantages by developing an ex vivo produced equivalent of the oral mucosa (EVPOME) in a serum-free culture system that does not contain a nutrient layer [88]. The resulting biomaterial consists of primary autogenic human gingival keratinocytes seeded on a cell-free cadaveric dermis. It was the use of such a framework that, according to the authors, provided the best opportunities for manipulating the equivalent of a IOM as a graft. It was shown that the transplanted EVPOME had advantages over the use of an alloderm without an epithelium, which served as a control in this study: early revascularization was observed, cytological evidence of the presence of intact squamous epithelial cells in the EVPOME transplant center a week after surgery was obtained, as well as a decrease in the inflammatory response and a faster appearance of mature epithelium in the transplantation zone.

Cell-free allogeneic or heterogeneous dermis is by far the most used matrix in tissue engineering, in particular, for modeling three-dimensional structures of integumentary intraoral purposes. The advantages of 3D models were that they were easier to handle and could be used with significant tissue defects. They looked more like native tissue due to a more complex structure with an epithelium and its own plate of connective tissue, separated, as in normal, by a basement membrane. They also made it possible to incorporate different types of cells, had a high degree of differentiation and strength for histological evaluation [89-91]. Using these 3D models, it is possible to control tissue growth, protein or microRNA expression in situ [92].

There have been published reports on the use of in vitro reconstructions of intraoral mucose, which are divided into those consisting only of differentiated oral epithelium (epithelial substitutes) and containing their own plate of connective tissue with oral fibroblasts seeded on a framework. Thus, the unceratinized mucous membrane of the mouth was constructed using epithelial cells isolated from the cheek area covered with non-keratinizing epithelium [93], since early attempts to create a tissue-engineered construct of IOM using epithelial cells of non-intraoral origin led to keratinization of structures.

Various methods of manufacturing three-dimensional structures, including multilayer frames made using 3D bioprinting technology, have also been reported [94-96].

Currently, the use of matrices containing elastin, collagen, bioactive proteins has been described for periodontal plastic surgery, dental implantology, and reconstructive oral surgery [90, 91]. However, the dominant outcome of healing using this material is the formation of fibrous tissue, which leads to scar contractures of the recipient site. There are no publications on long-term studies of Alloderm and the possibilities of its use in thin gingival phenotype. In addition, the cost of this material is high, and for the purpose of sealing an operating wound during reconstructive surgery of the oral cavity, when using it, it is necessary to additionally use their own epithelial tissues, which, most often, are not enough. It was also noted that the post-traumatic inflammatory reaction in the regeneration site with Alloderm is more pronounced than when working with own donor flaps, and the time of superficial keratinization of the wound in periodontal plastic surgery is significantly increased, which negatively affected the results of treatment. It has been shown that Alloderm acts as a framework for host fibroblast migration and preserves the basement membrane complex to facilitate attachment of the surface epithelium [97]. At the same time, suboptimal ingrowth and migration of cells using this material was observed when attempts were made to use it as a framework for the regeneration of mucous membranes [98].

A large amount of research is focused on the creation of sophisticated tissue engineering constructs to improve functionality, biological properties, viability and survival when replacing damaged or lost structures [99-102]. In the work of Japanese authors K. Nishiyama et al. Information is provided on the creation of 3D-epithelized equivalents of IOM using cell layering technology. Histological analysis of the intraoral mucosa equivalents revealed distinct layers characteristic of human IOM, including the horn. Blood capillaries have been successfully incorporated into the connective tissue region, increasing the possibilities of medical use of such models as a graft [103].

Conclusion

The regeneration of intraoral tissues is a complex cascade of healing processes, which is the result of coordinated interactions of stem cells, applied biomaterials and the patient's immune system. The presented literature review includes known and new approaches to tissue regeneration based on the reconstruction of suitable microenvironment conditions in areas of lost or damaged structures. The first experimental and clinical effects of intraoral surgery using adipose tissue stem cells  without preliminary in vitro expansion, as well as the active development of the bioengineering direction suggest promising results in the search for an ideal paradigm of the regenerative process to create conditions for early cell differentiation followed by tissue replacement in areas of interest.

About the authors

MARINA PEROVA

Author for correspondence.
Email: mperova2013@yandex.ru
ORCID iD: 0000-0001-6974-6407
SPIN-code: 5552-7988
ResearcherId: ABG-8805-2021

Associate Professor, Professor of the Department of Surgical Dentistry and Maxillofacial Surgery

References

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