Analysis of Reparative Osteogenesis in Augmented Zone Using Allogeneic Lyophilized Cancellous Bone Graft

Full Text

BACKGROUND

Significant atrophy of the maxillary alveolar ridge often necessitates augmentation to restore bone volume [1–3] and ensure optimal implant positioning. Sinus floor augmentation is a well-established surgical procedure that enables implant therapy in cases of posterior maxillary atrophy with insufficient bone height for implant placement [4]. The success of dental implantation largely depends on bone quality; however, no grafting material offers ideal properties [5]. According to Chatzopoulos et al. [6], simultaneous maxillary sinus grafting and implant placement result in inferior osseointegration outcomes and higher failure rates. Therefore, monitoring early osteogenesis within the augmented zone is critical to prevent potential complications, including maxillary sinus inflammation [7, 8].

Although radiographic methods remain the primary tools for postoperative monitoring [9, 10], it is essential to identify premorbid signs of inflammatory and destructive changes in the augmented zone before clinical manifestation [11, 12]. Minimizing postoperative complications during correction of bone deficiency in implant sites requires data that characterize reparative osteogenesis [13]. Salivary and gingival crevicular fluid biomarkers have emerged as promising diagnostic tools in implant dentistry [14]. These biomarkers can be assessed using parameters that reflect bone destruction, mineralization, and demineralization. Oral fluid, which maintains direct contact with the dentoalveolar system and supports its homeostasis, may serve as a biologic medium for identifying criteria of structural and functional impairment [15–17]. Direct in vivo assessment of metabolic processes in bone tissue is challenging; therefore, evaluating bone status through biomarkers of osteogenesis and bone resorption remains a relevant noninvasive approach for indirect assessment [18].

AIM:

The work aimed to evaluate reparative osteogenesis in the augmented zone using allogeneic lyophilized cancellous bone graft through the analysis of metabolic markers.

METHODS

An allogeneic lyophilized human cancellous bone graft was used as the osteoplastic material in the maxillary sinus augmentation zone (registration certificate No. ФСР2010/08120 of February 12, 2021, for the medical device “Allogeneic bone bioimplants mechanically and ultrasonically processed, lyophilized, sterile, dental, TU 9398-001-01963143-2010”). The graft was developed and manufactured at the Tissue Bank of the BioTech Biological Center, Samara State Medical University. This biomaterial is a bionic tissue-engineering product with a 3D porous, acellular architecture composed of a scaffold of collagen proteins and extracellular matrix proteins that provide a balanced set of osteogenic stimulators and inhibitors. The material is biocompatible, nontoxic, and undergoes controlled biodegradation in the recipient, gradually being replaced by native bone.

Oral fluid served as the biologic specimen. Biochemical assessment included markers of bone modeling—osteocalcin, alkaline phosphatase, and parathyroid hormone—and the bone-resorption marker C-terminal telopeptide of type I collagen (β-CrossLaps).

Osteocalcin is a noncollagenous bone matrix protein consisting of 49 amino-acid residues. It is synthesized by osteoblasts and subsequently participates in mineralization. Collagen degradation results in the formation of hydroxyproline and the specific peptide fragment β-CrossLaps. Parathyroid hormone is the principal regulator of calcium–phosphate homeostasis, which activates transcriptional processes in osteoclasts.

Alkaline phosphatase is an organ-specific enzyme whose activity reflects metabolic processes in bone. Its function is the hydrolytic cleavage of phosphate groups from organic compounds and the formation of a pool of mineral phosphates readily mobilized for various structure-forming processes. It also contributes to organic matrix formation.

Study Design

A prospective, interventional, non-randomized controlled study was conducted in 58 participants: 21 men (36.2%) and 37 women (63.8%) aged 35–60 years (mean age, 44.0 ± 2.31 years). Participants were assigned to 2 groups—the experimental group and the control group—with a follow-up period of 1–12 weeks. The augmented zone in the experimental group (41 participants) was in the osseointegration phase. The control group included 17 healthy individuals. The groups were comparable in age, sex, and baseline periodontal status.

Eligibility Criteria

Inclusion criteria: significant atrophy of the maxillary alveolar ridge in the region of the maxillary sinus; use of an allogeneic lyophilized cancellous bone graft as the biomaterial.

Exclusion criteria: refusal to participate; age younger than 35 or older than 60 years; use of grafting materials other than the allogeneic lyophilized cancellous bone graft.

Study Setting

The study was carried out at the Department of Prosthodontics and the Department of Fundamental and Clinical Biochemistry with Laboratory Diagnostics, Samara State Medical University.

Participants were recruited among patients seeking prosthodontic rehabilitation as part of routine outpatient care.

Study Duration

Patient recruitment, group allocation, and visit scheduling were performed prospectively. Analysis of reparative osteogenesis based on metabolic markers was conducted over 1–12 weeks. The total follow-up period for participants was up to 3 years. The final stage involved summarizing and interpreting the findings.

Intervention

To increase alveolar ridge volume for dental implantation, sinus floor augmentation was performed on the maxilla under local anesthesia using an allogeneic lyophilized cancellous bone graft.

Panoramic radiography and computed tomography were used to assess osteogenesis. Dental implants were placed 3–6 months after augmentation.

Main Study Outcome

To assess the reliability of the study outcome, metabolic markers of bone turnover were analyzed in both the experimental and control groups. All cases of impaired reparative osteogenesis were recorded in a summary table. A comparative assessment of metabolic marker values was performed to identify destructive and regenerative processes occurring in the augmented zone.

Treatment outcomes were evaluated using radiographic imaging during osteogenesis and after its completion.

Additional Study Outcomes

The additional outcome included the clinical appearance of the augmented zone in the oral cavity. Examination revealed no inflammatory or destructive changes, fistulas, or other complications.

Subgroup Analysis

Participants were divided into the experimental and control groups according to the inclusion criteria, age, and severity of maxillary alveolar ridge atrophy.

Outcomes Registration

To register main and additional outcomes, oral fluid collected 1–12 weeks after augmentation with the allogeneic lyophilized cancellous bone graft was analyzed. Participants collected oral fluid independently by expectorating into a sterile disposable plastic container in the morning before eating, after rinsing the mouth with warm water, and no earlier than 15 minutes after toothbrushing. One hour before sampling, the oral cavity was rinsed with boiled water. Physical and emotional exertion and smoking were avoided before sampling. Samples contaminated with blood were excluded [19].

Changes in metabolic markers—osteocalcin, alkaline phosphatase, parathyroid hormone, and β-CrossLaps—were evaluated. Their concentrations in oral fluid were determined using a solid-phase electrochemiluminescent assay based on streptavidin–biotin technology with a ruthenium label on the Elecsys 2010 immunoassay analyzer (Roche, Switzerland) [20].

Additionally, panoramic radiography and computed tomography were performed to assess the restored bone volume in the maxillary sinus region.

Statistical Analysis

Statistical processing was performed using SPSS Statistics v.25 (IBM, USA; license No. 5725-A54). Descriptive statistics are presented as the median and quartiles: Me [Q1; Q3]. Intergroup comparisons were conducted using the Mann–Whitney test, and comparisons across different follow-up periods were performed using the paired Wilcoxon test. Illustrative materials are shown as overlaid boxplots and violin plots generated with the ggplot2 module in R (http://r-project.org). Results were considered significant at p < 0.05.

RESULTS

The obtained data on bone metabolism are presented in Table 1.

 

Table 1. Metabolic markers of bone turnover in the experimental and control groups, Me [Q1; Q3]

Follow-up period, weeks	Experimental group (n = 41)	Control group (n = 17)	P-value
C-terminal telopeptide of type I collagen, ng/mL
1	0.006 [0.001; 0.022]	0.010 [0.002; 0.015]	0.710
2	0.006 [0.001; 0.025]		0.572
3	0.016 [0.009; 0.030]		0.050
4	0.020 [0.010; 0.036]		0.007
12	0.010 [0.001; 0.020]		0.466
Osteocalcin, ng/mL
1	0.55 [0.53; 0.58]	0.55 [0.54; 0.57]	0.953
2	0.56 [0.54; 0.60]		0.235
3	0.57 [0.55; 0.61]		0.024
4	0.59 [0.55; 0.63]		0.001
12	0.56 [0.54; 0.59]		0.170
Alkaline phosphatase, U/L
1	26.15 [22.0; 30.90]	31.30 [20.70; 34.30]	0.841
2	25.70 [21.08; 30.33]		0.323
3	24.60 [22.0; 28.65]		0.209
4	23.65 [19.75; 27.05]		0.046
12	24.15 [21.0; 28.43]		0.255
Parathyroid hormone, ng/mL
1	1.96 [1.83; 2.09]	1.87 [1.72; 2.01]	0.157
2	1.97 [1.81; 2.10]		0.110
3	2.05 [1.89; 2.15]		0.010
4	2.09 [1.90; 2.28]		0.003
12	2.04 [1.79; 2.17]		0.060

 

At baseline, the β-CrossLaps concentration (see Fig. 1) in the experimental group was 0.006 [0.001; 0.022] ng/mL and did not differ significantly from the control group, where the value was 0.010 [0.002; 0.015] ng/mL (p > 0.05). During weeks 3–4, significant differences were identified between the experimental group and the same participants at weeks 1–2 (both p < 0.001), as well as compared with the control group (p = 0.050 at week 3 and p = 0.007 at week 4).

 

Fig. 1. Concentration of C-terminal telopeptide of type I collagen (β-CrossLaps) in oral fluid in the experimental and control groups at different follow-up periods.

 

Outlier values were recorded in the experimental group at week 3 in three participants: 0.051, 0.055, and 0.075 ng/mL. The median and interquartile range at this time point were 0.016 [0.009; 0.030] ng/mL.

By week 4, high-end outliers were observed in four individuals: 0.066, 0.075, 0.079, and 0.090 ng/mL, with a median and interquartile range of 0.020 [0.010; 0.036] ng/mL.

By week 12, markedly elevated β-CrossLaps concentrations were not observed in the experimental group, and no significant differences were found relative to baseline or the control group.

Marker values returned to baseline and matched those of the control group. These findings indicate complete resorption (destruction) of the bioplastic material with synchronous replacement by native bone, which was supported by subsequent analyses of bone-modeling metabolic markers in oral fluid.

At baseline, osteocalcin levels (see Fig. 2) did not differ from those in the control group, with values of 0.55 [0.53; 0.58] ng/mL in the experimental group and 0.55 [0.54; 0.57] ng/mL in the control group (p > 0.05). At week 2, two individuals in the experimental group exhibited marked deviations from the group trend (0.74 and 0.78 ng/mL); however, no significant differences from the control group were identified.

 

Fig. 2. Osteocalcin concentration in oral fluid in the experimental and control groups at different follow-up periods.

 

At weeks 3–4, significant differences were observed in the same participants compared with week 1 (both p < 0.001). At week 3, osteocalcin concentration in the experimental group reached 0.57 [0.55; 0.61] ng/mL (p = 0.024 compared with the control group). Markedly elevated values were recorded in three participants: 0.84, 0.87, and 0.89 ng/mL.

One month after study initiation, four individuals in the experimental group demonstrated pronounced upward deviations in osteocalcin concentration, measuring 0.83, 0.91, 0.94, and 0.96 ng/mL. The median and interquartile range at this time point were 0.59 [0.55; 0.63] ng/mL (p = 0.001 vs. the control group and p < 0.001 vs. week 1).

By 3 months (week 12), osteocalcin levels in the experimental group nearly returned to baseline: 0.56 [0.54; 0.59] ng/mL, with no significant difference from the control group (p = 0.170). One participant demonstrated an elevated value (0.74 ng/mL) relative to the rest of the group.

At week 1 of follow-up, salivary alkaline phosphatase activity in the main group (see Fig. 3) was 26.15 [22.0; 30.90] U/L, which did not differ significantly from the values in the control group: 31.30 [20.70; 34.30] U/L (p > 0.05). At week 2, enzymatic activity in the experimental group remained essentially unchanged. At week 3, increased dispersion was observed due to upward and downward outliers (see Fig. 3).

 

Fig. 3. Alkaline phosphatase activity in oral fluid in the experimental and control groups at different follow-up periods.

 

At week 4, dispersion remained higher than in the control group, and significant differences in central tendencies were detected. The median and interquartile range in the experimental group were 23.65 [19.75; 27.05] U/L (p = 0.046 compared with the control group). Clinically, cases of reduced alkaline phosphatase activity were notable. Four participants exhibited values below 10 U/L: 5.0, 6.0, 8.0, and 9.0 U/L.

At week 12, alkaline phosphatase activity in oral fluid in the experimental group reached 24.15 [21.0; 28.43] U/L and again did not differ significantly from the control group (p > 0.05), although a small but significant difference persisted compared with week 1 (p = 0.011). One participant in the experimental group continued to show markedly reduced alkaline phosphatase activity (8.3 U/L).

In the experimental group, parathyroid hormone concentration in oral fluid (see Fig. 4) at week 1 was 1.96 [1.83; 2.09] ng/mL, which did not differ significantly from the control group: 1.87 [1.72; 2.01] ng/mL (p > 0.05). One week later, no substantial changes were observed.

 

Fig. 4. Parathyroid hormone concentration in oral fluid in the experimental and control groups at different follow-up periods.

 

At week 3, higher parathyroid hormone values were found in the experimental group: 2.05 [1.89; 2.15] ng/mL (p = 0.010 compared with the control group and p = 0.009 compared with week 1). Three participants exhibited pronounced elevations: 2.36, 2.45, and 2.47 ng/mL.

Increased concentration persisted into week 4, when parathyroid hormone in the experimental group reached 2.09 [1.90; 2.28] ng/mL (p = 0.003 vs. the control group and p = 0.001 vs. week 1). High-end outliers were identified in four patients: 2.51, 2.55, 2.60, and 2.68 ng/mL.

At week 12, no significant differences were found compared with the control group or baseline; values were 2.04 [1.79; 2.17] ng/mL.

DISCUSSION

The use of an allogeneic lyophilized cancellous bone graft in the augmented zone yielded the following results. At baseline (weeks 1–2), no significant differences were found between the studied markers and control values. However, by weeks 3–4, β-CrossLaps increased significantly, indicating a possible destructive process in which the telopeptide with collagen fragments enters oral fluid. Concurrently, osteocalcin levels exceeded control values, reflecting decreased mineralization in the augmented zone and, consequently, depletion of the plastic resources required for bone formation. Changes in alkaline phosphatase activity during this period further supported reduced phosphate availability for mineralization, creating unfavorable conditions for physiologic osteogenesis. Reduced parathyroid hormone concentration led to increased calcium and phosphate ion levels in oral fluid. The most pronounced deviations were seen in four individuals, possibly associated with previously undiagnosed systemic conditions.

Thus, during weeks 3–4, the secondary remodeling phase develops through the formation of osteoid matrix, accompanied by the formation of immature trabecular bone in areas of active osteoclastic resorption of undifferentiated bone tissue. Accordingly, weeks 3–4 represent a period of increased risk for complications. Preventive measures should include avoiding traumatic factors, maintaining mucosal integrity in the augmented zone, and ensuring meticulous oral hygiene.

Follow-up of the experimental group at 12 weeks or longer demonstrated restoration of baseline metabolic markers of bone turnover, resulting in replacement of immature bone with functionally adapted mature bone structures. No pathologic changes were observed on panoramic radiography.

Representative clinical cases include the panoramic radiography of patient Ch. and patient B. before and after augmentation with the lyophilized cancellous bone graft (see Figs. 5, 6).

 

Fig. 5. Panoramic radiography of patient Ch.: a, at baseline; b, augmentation with a lyophilized cancellous bone graft; c, dental implant placement, osseointegration phase; d, clinical presentation at 3-year follow-up.

 

Fig. 6. Panoramic radiography of patient B.: a, at baseline; b, augmentation with a lyophilized cancellous bone graft; c, dental implant placement, osseointegration phase.

 

CONCLUSION

A comprehensive assessment of bone turnover demonstrated that augmentation with a lyophilized cancellous bone graft supports optimal osteogenesis and can be effectively used to increase bone volume in dental implant sites.

ADDITIONAL INFORMATION

Author contributions: V.S. Tlustenko: supervision, investigation, data curation, formal analysis, conceptualization, writing—original draft, writing—review & editing; F.N. Gilmiarova: investigation, formal analysis, supervision; V.P. Tlustenko: writing—original draft, writing—review & editing, conceptualization; L.T. Volova: writing—original draft, writing—review & editing; V.A. Koshelev: resources, formal analysis; N.V. Nogina: resources. All 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: According to Protocol No. 303 dated April 9, 2022, the Bioethics Committee of Samara State Medical University approved the research study titled Analysis of Reparative Osteogenesis in the Augmented Zone Using Allogeneic Lyophilized Cancellous Bone Graft. All participants provided written informed consent prior to inclusion in the study.

Consent for publication: Written informed consent was obtained from the patient for the publication of personal data, including photographs (with faces obscured), in Russian Journal of Dentistry and its digital version (signed on April 23, 2022). The scope of published data was approved by the patients.

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.

Disclaimer: The authors declare that the views expressed in this article are solely their own and do not necessarily reflect the official positions of their affiliated institutions.

About the authors

Vladimir S. Tlustenko

Samara State Medical University

Author for correspondence.
Email: vlastt@yandex.ru
ORCID iD: 0000-0002-7429-9135
SPIN-code: 8537-5155

MD, Cand. Sci. (Medicine), Associate Professor

Russian Federation, Samara

Frida N. Gilmiyarova

Samara State Medical University

Email: kaf_biohim@samsmu.ru
ORCID iD: 0000-0001-5992-3609
SPIN-code: 7638-1812

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Samara

Valentina P. Tlustenko

Samara State Medical University

Email: stas-763@yandex.ru
ORCID iD: 0000-0002-2756-5277
SPIN-code: 2737-9657

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Samara

Larisa T. Volova

Samara State Medical University

Email: volovalt@yandex.ru
ORCID iD: 0000-0002-8510-3118
SPIN-code: 1499-7910

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Samara

Vladimir A. Koshelev

Samara State Medical University

Email: stomvk@yandex.ru
ORCID iD: 0000-0001-8479-0342
SPIN-code: 6886-6628
Russian Federation, Samara

Natalya V. Nogina

Samara State Medical University

Email: noginanatalya@mail.ru
ORCID iD: 0000-0001-8109-8713
SPIN-code: 1680-9834

MD, Cand. Sci. (Medicine), Associate Professor

Russian Federation, Samara

References

Supplementary files