Radiological and Microbiological Evaluation of the Efficacy of Alveolar Bone Repair Using Autogenous Dentin Matrix—Preliminary Study

Abstract

Dental procedures for alveolar bone augmentation may be carried out using autologous bone graft material derived from the patient’s own tooth. The material obtained is subjected to strict procedures aimed at reducing the amount of bacteria in the autograft. The aim of this study was the evaluation of the efficacy of the autogenous dentine matrix produced by grinding the patient’s own tooth for the augmentation of maxillary bone defects and the evaluation of the microbiological status of the material obtained. Alveolar bone repair was performed with an autogenous dentin matrix in four patients. In each case, an autogenous bone graft substitute obtained by grinding the patient’s own tooth was used. The tooth-derived material was then used for alveolar augmentation. The obtained material was tested to assess its microbiological profile. For the purpose of comparison, other materials and tissues were also subjected to microbiological testing. Bone healing was assessed by CBCT (cone beam computed tomography) scanning before and 6 months after surgery using the Hounsfield scale and the ImageJ software. Analysis of the bone regeneration process based on the bone density score in Hounsfield units showed significant differences in measurements on CBCT scans carried out on the treatment site, before surgery, and 6 months after it, using ImageJ software. All bacteria detected in the bone augmentation material constituted the patient’s bacterial flora. The microorganisms present in the augmentation material were also present in the patient’s bone and soft tissues. The use of an autogenous dentin matrix for alveolar bone repair ensures that the proper volume is obtained and that alveolar bone shape is preserved and does not introduce pathogenic microorganisms into the patient. The procedure for preparing and using an autogenous dentin matrix is described based on one clinical case.

1. Introduction

Dental surgeries often require tissue regeneration. Modern dentistry attaches great importance to preserving the adequate dimensions of bone tissue for both aesthetic and functional reasons. If the alveolar bone volume is insufficient, augmentation procedures are performed to rebuild the lost bone [1]. Alveolar bone resorption after tooth extraction leads to bone atrophy in the horizontal and vertical dimensions. Bone height decreases progressively by 25% during the first year after tooth loss, with a total of 4 mm height loss during this first year post-extraction [2]. This is often an obstacle to a correct implant placement [3]. Augmentation techniques include a wide range of surgical procedures using autologous, allogenic, xenogeneic, and alloplastic grafts. The most effective procedure in guided bone regeneration (GBR) is the transplantation of autogenous, living bone tissue [4]. Autogenous bone graft is regarded as a material of choice for bone regeneration because it is the only material with the capabilities of osteogenesis, osteoconduction, and osteoinduction [5,6]. The physical properties of the autogenous dentin matrix, such as density, roughness, and homogeneity, and its physiochemical characteristics, including the dissolution of calcium/phosphate ions, are similar to those of human cortical bone [7]. Clinical studies have also shown a negligible level of immune reaction and a low risk of infection in AutoBT (autogenous tooth bone graft material) graft sites [8]. Autografts do not trigger the body’s immune response; thus, they are not rejected, as occurs when foreign tissue is used [9]. Alveolar bone repair using bone grafts enables the placement of dental implants in sites previously considered impossible or in more functional and aesthetic positions. The survival of implants placed in the bone bed regenerated by bone graft was 93.2%, whereas, in the pristine bone, it was 94.6% [10]. One method to repair alveolar bone is through the use of autogenous graft material produced by grinding the patient’s own extracted tooth. Tooth-derived demineralized dentin matrix was first introduced in 1967. Several studies have shown that its chemical composition is similar to that of bone [11]. The obtained material undergoes strict procedures to reduce bacterial contamination of the bone graft. Tooth-derived demineralized dentin scaffold matrix is slowly and gradually replaced by bone [12]. An autogenous dentin graft (ADG) is osteoconductive and has osteoinductive characteristics. It has a feature similar to human bone. Therefore it can be used for guided bone regeneration [13]. Several studies have reported that extracted teeth from patients, which undergo a process of cleaning, grinding, demineralization, and sterilization, can be a very effective graft to fill alveolar bone defects in the same patient [14] and a viable option for alveolar bone augmentation following dental extraction [15]. In this study, we focused on the assessment of the bone tissue reconstruction process using an autogenous dentin matrix and on the microbiological assessment of the obtained material in one of the cases. However, this method has several drawbacks due to limited accessibility and donor site morbidity [16]. Improved wound healing is achieved with the use of platelet-rich fibrin, which accelerates soft tissue regeneration (Figure 1) [17].

Advanced platelet-rich fibrin (A-PRF) is an autogenous blood product with applications in dentoalveolar surgery. PRF is a platelet concentrate made of an autologous bioscaffold of a dense fibrin matrix with naturally integrated growth factors that are released from the scaffold over a sustained period to promote the healing of hard and soft tissues [18].

2. Aim of the Study

The null hypothesis is the use of an autogenous dentin matrix in bone regeneration procedures that was obtained from the patient’s own tooth. The first aim of this study is the evaluation of the efficacy of the use of autogenous dentine matrix produced by grinding the patient’s own tooth for the procedure of augmentation of maxillary bone defects. The second objective is the evaluation of the microbiological status of the material obtained on the basis of one case.

3. Materials and Methods

3.1. General Information

A total of four patients were enrolled in the study. Surgeries with the use of autogenous dentin matrix were performed at the Dental Surgery and Implantology Clinic in Bytom. At the start, each patient underwent radiological examination using GENDEX’s iCAT cone beam computed tomography (CBCT, Gendex Corp, Hatfield, PA, USA). Based on it, the size, nature of the lesion, and surgical feasibility, as well as the possibility of filling the cavity with autogenous dentin matrix, were evaluated. (Figure 2).

The inclusion criteria for the enrolment of a patient were the presence of an impacted third molar, which enabled obtaining an autogenous dentin matrix after grinding, age of at least 18 years, and the requirement of preservation of the alveolar process by GBR after tooth extraction before dental implant placement or after cystectomy. The exclusion criteria were as follows: smokers, immunosuppression, head-and-neck-irradiated patients in the past 5 years, regular intake of bisphosphonates, anticoagulants, chronic drug abuse or alcoholic habits, patients with poor oral hygiene (full-mouth plaque score and full-mouth bleeding score > 15%), lack of motivation, uncontrolled diabetes (reported levels of glycated hemoglobin exceeding 7%), and uncontrolled and/or untreated periodontal disease.

In total, 20 mL of venous blood was collected from each patient before the surgery to prepare an A-PRF membrane using a PRF Duo centrifuge (PRF process Choukroun, Nice, France), according to the current procedure for obtaining A-PRF, i.e., 1300 rpm for 8 min. A-PRF was used as a barrier membrane and mixed with autogenous tooth-derived bone graft material (AutoBT).

3.2. Surgery

The surgery was in two stages.

First, the impacted tooth was removed using classical methods for the extraction of impacted third molars and ground to be used as graft material. This is where stage one ended.

The extracted impacted tooth was prepared. To obtain the AutoBT from the impacted teeth, the Smart Dentin Grinder produced by KometaBio, (KometaBio, Fort Lee, NJ, USA) was used. The extracted tooth was processed according to the procedure’s protocol. The tooth was mechanically cleaned of soft tissue using a drill and sickle probes. These were some debris of tooth follicles or periodontal fiber. After drying, the tooth was put in the chamber of the KometaBio, where it was ground for 3 s, and then the material was sieved through the sieve system for 20 s to obtain a homogeneous grafting material with a particle size of 200–1000 um. The graft material prepared this way was immersed in cleanser (sodium hydroxide solution with 20% ethanol, (KometaBio, Fort Lee, NJ, USA), supplied by the manufacturer, for 10 min, and then after draining the cleanser for 3 min, the particulate was treated with saline (Dulbecco’s phosphate-buffered saline DPBS, without calcium, magnesium), (KometaBio, Fort Lee, NJ, USA), also provided by the manufacturer. Thus, prepared and drained particulate dentin was ready for grafting (Figure 3).

In the second stage, the actual treatment was performed. Within the group of patients enrolled in the study, 3 cystectomies were performed to remove a dentigerous cyst from the jawbone. The patients underwent total cyst excision using classical methods in dental surgery. The collected material was submitted for histopathological examination. One patient underwent root extraction of teeth not eligible for restorative treatment or prosthetic restoration. A decision was made to remove the patient’s roots, followed by augmentation with a demineralized autogenous dentin matrix to preserve the socket for subsequent implant insertion (Figure 4). The bone defect (after cystectomy) was carefully cleaned and prepared and then filled with a previously prepared autogenous dentine matrix with A-PRF and covered with a membrane produced from A-PRF. The wound was sutured with absorbable Dafilon sutures. The patient received systematic antibiotic therapy for 7 days. The use of a mouthwash containing chlorhexidine or propolis was recommended. (Figure 5, Figure 6 and Figure 7).

During the procedure, one patient was sampled for microbiological testing, and the sample was placed in tubes with a liquid transport medium for both aerobes and anaerobes. The tubes were as follows:

• Tooth after grinding;
• Tooth after 10 min immersion in cleanser (sodium hydroxide solution with 20% ethanol);
• Tooth after immersion in phosphate-buffered saline;
• Heterogenous bone substitute (comparative study);
• Patient’s own soft tissue (comparative study);
• Patient’s own bone (comparative study)-vestibular bone plate.

3.3. Microbiology

Microbiological tests of the above materials were performed in the Microbiological Laboratory of the Department of Microbiology and Immunology in Zabrze. In the first step, the samples were incubated at 37 °C until microbial growth as sediment was noticeable. In the next stage, for qualitative identification of the microorganisms, classical microbiological examination was carried out by spreading 20 microliters of the contents of each tube onto the surface of a solid medium (for aerobes, it was Columbia agar with 5% sheep blood, and for anaerobes, it was Schoedler K3, (Figure 8)). Then, after 24–48 h of incubation of the plates at 37 °C, the species of the cultured bacteria were identified using classical microbiological testing techniques.

The follow-up visits of the patients were after 2, 7, and 14 days from the surgery. Normal healing of postoperative wounds was observed in all patients during the check-ups. Sutures were removed on postoperative day 7. Follow-up CBCT scans were performed 6 months following the surgery (Figure 9).

3.4. Data Analysis

Data from preoperative and postoperative, performed 6 months after surgery, assessments were entered into ImageJ 1.52v. Average bone density was measured using the Hounsfield units. It was calculated as the mean pixel intensity of circled areas of interest in the figures, where T represents the treatment site and H is the healthy site (Figure 10, Figure 11, Figure 12, Figure 13, Figure 14, Figure 15 and Figure 16). Four measurements were taken in each patient, two before and two after the surgery. Measurements taken on the treatment site were considered as the study group, and those obtained on the other site as the control group. Prior to surgery, measurements were taken on the treatment site and on the control site. The same procedure was applied for CBCT scanning 6 months following the surgery. Bone regeneration was evaluated based on bone density measurements expressed in Hounsfield units. Statistical analysis was carried out using the Statistica 13.1 software (StatSoft Polska, Kraków, Poland). Data are expressed as average values ± standard deviation (SD). For normal distribution and homogeneity of variance, Student’s t-tests were applied. Otherwise, the Wilcoxon tests were conducted. The deeper analysis was performed using the Friedman ANOVA test. The significance level was set at p < 0.05. The significant results are marked in the graphs (Chart 1).

3.5. Limitations of Research

In our study, we present the results obtained from the analysis of 4 cases (from which only one followed microbiological analysis). Due to a small research group, these studies should be understood as preliminary studies.

4. Results

The analysis of the bone regeneration process based on the bone density score in Hounsfield units showed significant differences in measurements on CBCT scans carried out on the treatment site, before surgery, and 6 months after it, using the ImageJ software.(ImageJ 1.53k, Wayne Rasband and contributors National Institutes of Health, Kensington, MD, USA) The average bone density at the measurement points was 100 Hounsfield units before surgery and 1000 Hounsfield units after surgery. Chart 2 shows the measurements on the study site before and after surgery.

A clinical examination 6 months after surgery revealed a normal alveolar shape, with no apparent bone defects. Radiological examination using CBCT confirmed the complete filling of the bone defect after the healing period. The bacteria identified in each tube are presented in

Table 1. Caption. Species present in the microbiota identified by microbiological tests in selected materials.

Test-Tube	Microorganisms
1. Grounded tooth	Staphylococcus epidermidis MSCNS Streptococcus salivarius
2. Tooth after 10 min in NaOH + 20% ethanol	Staphylococcus epidermidis MSCNS Streptococcus sanguinis
3. Tooth after buffer	Blautia producta
4. Biomaterial	Sarcina spp.
5. Soft tissue	Streptococcus mitis Neisseria subflava
6. Bone	Staphylococcus epidermidis MSCNS Abiotrophia adiacens Streptococcus salivarius

.

More detailed analysis showed the presence of oral saprophytic bacteria such as Staphylococcus epidermidis, Streptococcus salivarius, and Streptococcus sanguinis, both in the tooth specimen just after grinding and in the cleaned material. A visual analysis of the samples showed that the amount of bacteria settled in tube 2 was significantly smaller than that in tube 1 after the same incubation time (Figure 17). The microorganisms found in the material from tube 3 were not pathogenic since Blautia producta is a typical bacterium of the normal oral microbiota. By visual analysis, we concluded that the number of microorganisms in the grafting material (tube 3) was lower than that in the starting material (tube 1). It is worth noting that saprophytic bacteria, similar to those present in the bone graft material, were also found in the patient’s own tissues (tubes 5 and 6). We would like to point out that Sarcina, present in the biomaterial from the sterile package that was tested for comparison, is an airborne opportunistic bacterium; thus, also the heterogeneous material was not completely sterile at the time of use.

All bacteria detected in the bone augmentation material constituted the patient’s bacterial flora. The microorganisms present in the augmentation material were also present in the patient’s bone and soft tissues.

5. Discussion

Currently, autografts, allografts, and heterogeneous materials are used for bone regeneration, yet autogenous materials are still considered the substitute of choice for bone augmentation procedures.

Some authors have suggested that processed dentin may have osteoinductive potential in addition to serving as an osteoconductive medium [19].

According to Kang-Mi Pang, autogenous demineralized dentin matrix from an extracted tooth grafted to extraction sockets for the augmentation of the vertical dimension was as effective as augmentation using an organic bovine bone graft [20]. Our study confirms that the autogenous dentin matrix can be used with great success in patients to repair bone defects and create conditions for restoring missing teeth.

Autogenous tooth-derived demineralized dentin matrix (AutoBT) grafted into extraction sockets for the vertical augmentation of an alveolar defect exhibited minimal tissue response and bone regenerative potential similar to that of the much-tested and widely used organic bovine bone [20]. In our study, we demonstrated that the volume of autogenous dentin matrix obtained from grinding a patient’s own tooth is suitable for filling post-cystectomy defects in the case of one-and two-wall bone defects.

The microbiological tests that were carried out showed that the method employed did not introduce pathogenic bacteria into the patient’s body.

The addition of A-PRF to the grafting material facilitates obtaining a normal bone density and has a positive effect on soft tissue healing [21]. Therefore, in our study, we used A-PRF as an additive to the material derived from each patient’s own extracted tooth. An interesting extension of previous studies in the field seems to be the use of a thermal imaging camera to assess the healing process. According to the authors, the analysis of temperature distribution dynamics indicates a correlation between changes in the temperature and repair processes occurring in the body [22]. This aspect could be further studied in future works.

It might also be a good idea to apply the “socket-shield technique’’. This method involves maintaining the vestibular root portion and immediate insertion of the dental implant in close proximity to the root [23].

It is a good idea to use post-treatment mouthwashes. Studies show a positive biological effect of oral rinses containing propolis against the oral microbiota [24].

The analysis of bone repair showed statistically significant differences in bone density measured by CBCT scanning before and 6 months after surgery.

Other authors also indicated that bone density assessed by CBCT scans six months after surgery in Hounsfield units was greater in the study group (in which autogenous dentin matrix was used to fill the bone socket after extraction of the impacted third molar) compared to the control group, where the surgical site was left to heal spontaneously [25].

6. Conclusions

• The use of an autogenous dentin matrix for alveolar bone repair ensures that the proper volume is obtained and that alveolar bone shape is preserved;
• Two surgical sites do not increase pain in patients and do not cause postoperative healing complications. For minor bone losses, this is one of several solutions that can be proposed to a patient;
• The use of an autogenous dentin matrix, prepared according to the manufacturer’s instructions, does not introduce pathogenic microorganisms into the patient.