Biphasic Calcium Phosphate Versus Demineralized Freeze-Dried Bone Allograft in the Treatment of Periodontal Disease: A Clinical and Radiographical Evaluation

Aim The study aimed to clinically and radiographically evaluate the effect of biphasic calcium phosphate (BCP) versus demineralized freeze-dried bone allograft (DFDBA) in treating periodontal disease. Method The study consisted of 44 patients. The sites were randomly assigned to receive one of two treatment modalities (BCP at site 1 and demineralized freeze-dried bone at site 2) by a computerized method. All the clinical data were measured with the help of a University of North Carolina-15 (UNC-15) probe at the baseline, three months, and six months postoperatively. Radiovisiographs were taken using a Rinn XCP® (Dentsply/Rinn Corp, Elgin, IL) system and an oral grid using the paralleling technique. A manual calculation of the defect area was undertaken at the end of six months and was compared with the other groups. Result The linear bone growth recorded for site 1 at the end of six months was 3.8 ± 1.14 mm, and site 2 was 4.6 ± 1.07 mm. The intergroup comparison showed more remarkable linear bone growth in site 2, which was statistically insignificant, with a mean difference of 0.8 ± 1.23 mm and a p-value of 0.07. Conclusion Improvements were observed on all the documented parameters. However, the sites treated with DFDBA showed better periodontal regeneration.


Introduction
Periodontitis is an inflammatory disease of the supporting tissues of the teeth caused by specific microorganisms or a group of specific micro-organisms, resulting in progressive destruction of the periodontal ligament and alveolar bone with pocket formation recession or both [1]. The pathological hallmark of periodontitis is the destruction of the supporting structures of the involved teeth with loss of attachment.
A decrease in the alveolar bone assistance is the typical symptom of degenerative periodontal diseases, which occurs as the anatomical continuation of the spread of periodontitis in the apical region [2]. Different forms of bone abnormalities result from periodontal diseases [3]. The crucial goal of periodontal management is the preservation of natural dentition in healthy function. Once the attachment apparatus is lost, regenerating the periodontium to its pre-diseased state requires optimal care [4].

Sample Size
The study sample consisted of 44 patients initially, but two patients failed to follow up. Patients who participated in this study were of both sexes. These cases were systemically healthy and diagnosed with generalized chronic periodontitis with deep intrabony bilateral/contralateral defects with a probing depth of ≥5 mm and at least 3 mm as detected on the radiograph. The following formula was used to determine the sample size: n = (Zα/2 + Zβ )2 *2*σ2/d2, where Z α/2 is the critical value of the normal distribution at α/2. Patients who failed to maintain adequate oral hygiene (plaque index >1) after phase. The total calculated sample size was calculated at 40 patients. Still, taking into consideration that few patients will fail to follow up, we included a larger number in our study therapy; pregnant and lactating women, smokers, habitual tobacco consumers, those who did not give consent, and those who failed to follow up were excluded from the study. Additionally, the study sample age range was 30-60 years. The plaque index assesses the amount of dental plaque visible on all teeth' vestibular and lingual surfaces, except the third molars. The sites were randomly assigned to receive either treatment modality by a computerized method (Figure 1).

Site 1: Biphasic Calcium Phosphate
The BCP used here was Meta Bonemedik-DM bone™ (Union Dental, New Delhi, India) in size 0.5-1.0 μm. It contains 60% HA and 40% β-TCP. This fully synthetic bone graft substance is a medically pure BCP composite. This ceramic might be classified as resorbable because of the β-TCP content. The ratio of 60:40 is ideal for new bone formation and osteoconduction. This ratio shows a high uptake of calcium when compared with other combinations of HA/βTCP [24].

Site 2: DFDBA
The DFDBA (TATA, Mumbai, India) particle size 500-1040 μm was used in the present study. It stimulates the immigration, attachment, and osteogenesis of the mesenchymal cells when implanted in highly vascularized bone. It also highly boosts endochondral bone regeneration when inserted into tissues that would otherwise not form bone. DFDBA encompasses bone morphogenic proteins (BMPs) such as BMP 7, 4, and 2, which assist in stimulating osteoinduction. The significant advantage of the DFDBA used here was its affordable cost [26].
The patients who fulfilled the inclusion criteria underwent phase I therapies and were given oral hygiene instructions. At baseline, personal history, dental history, and medical history were recorded. All the clinical records were taken, and an occlusal stent was fabricated for the treatment-targeted tooth. This offered a fixed reference point and angulation for the measurements for any future references at each site during the study period.

Clinical and radiographic parameters
All the scientific parameters like plaque index (PI) (Quigley-Hein plaque index updated by Turesky et al.) [27], modified gingival index (GI) [28], and probing pocket depth (PPD) were recorded. The Turesky modified version of the dental plaque index is as follows: 0 = no plaque, 1 = separate flecks of plaque at the cervical margin of the tooth, 2 = a thin continuous band of plaque (up to 1 mm) at the cervical margin of the tooth, 3 = a band of plaque wider than 1 mm but covering less than one-third of the crown of the tooth, 4 = plaque covering at least one-third but less than two-thirds of the crown of the tooth, 5 = plaque covering two-thirds or more of the crown of the tooth. A University of North Carolina-15 (UNC-15) probe recorded relative attachment level (RAL) with the help of a customized acrylic stent at the baseline, three months, and six months postoperatively.
Radiovisiographs (RVG) were obtained by the Rinn XCP® (Dentsply/Rinn Corp, Elgin, IL) system (paralleling technique) with a standard intraoral grid attached. The total zone of the bony defect was analyzed. The radiographic parameters were documented at the baseline and six months postoperatively. To avoid interexaminer bias, a single operator (dental surgeon 1) blinded to the study recorded all parameters.
BD is the distance from the cementoenamel junction (CEJ) to the most profound extension of the bony defect [30]. The alveolar crest (AC) is the distance from the cementoenamel junction (CEJ) to the alveolar ridge [30]. AUX I is an auxiliary line drawn in the direction of the tooth axis ( Figure 2). AUX II is the second auxiliary line perpendicular to the tooth axis, drawn through the most coronal extension of the lateral wall of the intrabony defect. INFRA 1 is the distance from CEJ to BD minus the distance from CEJ to AC (the difference between the distances from CEJ to BD and CEJ to AC). INFRA 2 is the distance from the point where AUX II crosses the contour of the root to BD. Bone defect width (BDW) is the measurement from the defect's lateral margin to the intersection of the point where AUX II crosses the root's surface. Linear bone growth is the BD to CEJ at the baseline minus the BD to CEJ after six months. The defect area equals ½ (INFRA 1 × BDW) of the bone fill % (linear bone growth/defect depth) × 100.

Surgical Procedure
The operative area was anesthetized with a 2% lignocaine hydrochloride solution (1:80,000). After achieving adequate anesthesia, infraclavicular incisions were placed using a 15-number blade. Full-thickness mucoperiosteal flaps were elevated on lingual and buccal sides for attaining access to the defect zone. The granulation tissues were detached using Gracey curettes, and accurate scaling and root planning were undertaken. After complete debridement, the osseous defect was measured by a UNC-15 probe. The bone defect width, vertical bone depth, and existing bony walls were recorded.
Pre-suturing and filling with BCP bone grafts occurred on site 1 and DFDBA grafts on site 2. The surgeries were performed on the respective sites at an interval of two weeks. The time difference was calculated from the day the first surgery was performed. Patients were advised not to take any other medicines than prescribed unless an emergency arose. The mucoperiosteal flaps that were pre-sutured were relocated and secured with interrupted sutures. The operating area was shielded with a non-eugenol (Coe-pack). A different single operator (dental surgeon 2) performed all surgeries.
The patients were advised to avoid chewing and brushing in the surgical area for two weeks. They were advised to avoid rinsing, spitting, and consuming hard or hot food for 24 hours. Patients were instructed to use 10 ml of 0.2% chlorhexidine gluconate solution twice daily for mouth rinsing for effective plaque control after the day of surgery.
They were prescribed medications, including 500 mg three times a day (TDS) of amoxicillin for three days and 400 mg TDS of diclofenac sodium for three days. After ten days, patients were recalled for sutures and periodontal dressing removal. The area was carefully inspected for healing. A single physician was allotted for this study, who questioned all the patients for signs and symptoms and also physically examined the patients if required. All the data regarding this was recorded in black and white.

Statistical Analysis
The data gathered were organized and subjected to statistical analysis using SPSS Software (IBM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. IBM Corp, Armonk, NY). The intragroup comparisons in site 1 and site 2 from baseline to three months and from baseline to six months were made using a student's paired t-test. Similarly, the intergroup comparison between site 1 and site 2 was made using an independent t-test and analysis of variance (ANOVA). The p-value <0.05 was considered statistically significant. 2022  The study initially consisted of 44 patients, but two patients failed to follow up. The attrition rate was adjusted to 4%. The remaining 42 patients completed the study. The mean age of the study subjects was 43 years. No post-surgical complications or allergic reactions were documented after the surgery, and all the sites displayed flawless healing. Although study respondents had flawless healing, a wound healing index was calculated.

Results
Regarding intragroup comparison, the baseline plaque index score, three-month score, and six-month score were 0.67 ± 0.2, 0.61 ± 0.16, and 0.56 ± 0.2, respectively, with a p-value of 0.122, 0.01, and 0.173, respectively. The plaque score was reduced considerably from baseline to six months. The baseline modified gingival index scores, three-month score, and six-month score were 0.88 ± 0.29, 0.75 ± 0.13, and 0.6 ± 0.16 with p-values of 0.109, 0.008, and 0.015, respectively ( Table 1). The gingival score dwindled considerably from baseline to six months and from three months to six months.   The pocket probing depth (in mm) of the intragroup comparison for site 1 was 8.4 ± 1.35 at baseline. At six months, it reduced to 4.8 ± 0.79, showing a substantial reduction in probing depth with a mean difference of 3.6 ± 1.07 and a p-value of <0.001, which is statistically significant. For site 2, the PPD (in mm) documented at baseline and six months was 8.8 ± 1.32 and 3.7 ± 0.48, respectively, showing a mean difference of 5.1 ± 1.29 from baseline to six months and a p-value of <0.001, which is statistically significant (   Regarding the intergroup comparison between sites 1 and 2 after the t-test, the PD was statistically nonsignificant at baseline and three months, given that the p-values were 0.479 and 0.089, respectively. However, at six months, the PD reduction was statistically significant in site 2 than at site 1, with a p-value of 0.003 (Table 3).  On intergroup comparison between Site 1 and Site 2 after the t-test, the relative attachment level was statistically non-significant at baseline, as the p-value was 0.853. However, at 3 months and 6 months, the RAL reduction was statistically significant in Site 2 than in Site 1, with p-values of 0.009 and <0.001, respectively (  The linear bone growth recorded for site 1 at the end of six months was 3.8 ± 1.14 and 4.6 ± 1.07 for site 2.

Groups
The intergroup comparison showed more remarkable linear bone growth in site 2, which was statistically non-significant with a mean difference of 0.8 ± 1.23 and a p-value of 0.07.
The percentage of bone fill recorded for site 1 at the end of six months (Figures 3A, 3B) was 56.29 ± 11.17 and 63.38 ± 8.78 for site 2 ( Figures 4A, 4B). The intergroup comparison showed a statistically significant greater percentage of bone fill in site 2, with a mean difference of 7.09 ± 8.37 and a p-value of 0.025 ( Table  5).

Discussion
Deep periodontal pockets coupled with osseous lesions characterize the specific risk factors for or indicators of disease progression [31]. Thus, treating them to halt the progression of periodontal disease is of prime importance to the tooth. Bony lesions often require access to flap surgery unaided or in combination with bone resective or regenerative techniques; they may not be available for periodontal debridement through non-surgical therapy [32].
After periodontal flap surgery, the unpredictability of osseous defect fill has investigated various bone grafting materials. The autogenous bone graft is considered the gold standard. Still, the limitation of the number of bone grafts available and the morbidity associated with the second surgical site for graft procurement are drawbacks [12]. This material is recognized in the 1996 World Workshop in Periodontics consensus report to fulfill all criteria for the promotion of periodontal regeneration [33]. The disadvantages of DFDBA are cost, lack of patient acceptance due to fear of disease transfer from the donor, and radiolucency [34].
Alloplasty overcomes some of the disadvantages of autogenous and allogenic bone grafts [35]. BCP has osteoconductive properties and may be appropriate as a bone graft material [13]. The capability of BCP to be bio-absorbed and to form a direct tough bond with the host bone also makes it a better choice as a bone graft. In the histological sections, it was observed that it completely integrated into a secondarily formed spongy bone. This resulted in complete osseous regeneration of the former defect area [36].
The present study was a split-mouth, triple-blinded, randomized crossover controlled clinical trial. The randomized controlled trial (RCT) is often considered the gold standard for a clinical trial. It offers the advantage of minimizing allocation bias and matching both known and unknown prognostic factors in the assignment of treatments. A split-mouth design was applied in this study to minimize the bias theoretically determined by differences in individual elements, avoid natural variation between individuals, and limit patient-based and defect-based factors. In our research, plaque and gingival index showed improvement from baseline to six months. This could be attributed to an improvement in the home care and reinforcement of oral hygiene instructions at each recall visit.
Our study's PI at baseline, three months, and six months was 0.67 ± 0.2, 0.61 ± 0.16, and 0.56 ± 0.2, respectively. The plaque score was reduced from baseline to three months and three months to six months, but it was not statistically significant. The plaque score decreased significantly from baseline to six months. The insignificant quantity of plaque did not hamper the regeneration, and all patients maintained appropriate oral hygiene during the study stage. The deterioration in PI scores throughout the study could be attributed to an improvement in home care and an emphasis on oral hygiene instructions at each recall visit.
A comparable tendency of a declining plaque index was observed in a study by Franco and Rubia [37] and Agarwal et al. [38]. The modified gingival index scores at baseline in the study were 0.88 ± 0.29, which reduced to 0.75 ± 0.13 at three months and, significantly, to 0.6 ± 0.16 at six months. This is in agreement with an investigation by Stein et al. [39].
The pocket probing depth at sites treated with BCP showed a significant decrease at the end of six months. Previous research observed similar results [7,[39][40][41]. Similarly, the pocket probing depth at sites treated with DFDBA also showed a significant decrease at the end of six months. The current study results align with earlier studies [42][43][44][45][46]. But the sites treated with DFDBA showed a better improvement when compared to the sites treated with BCP.
One of the most important parameters for assessing periodontal destruction is the loss of connective tissue attachment to the tooth root surface. The "gold standard" for recording changes in periodontal status is a longitudinal measurement of CAL from the CEJ or a RAL from a fixed reference point. The assessment of attachment levels using fixed reference points provides better information relating to gain or loss of attachment to the root surface and in assessing disease progression compared with pocket depth measurement.
The RAL at the sites treated with BCP showed a significant gain at six months. The present study results follow the findings of earlier studies [7,[39][40][41]47]. The RAL at the sites treated with DFDBA also showed a significant gain at six months. These results are similar to previous studies [45,46,[48][49][50]. The DFDBA grafted sites showed better gains in RAL than the BCP grafted sites.
The linear bone growth recorded for site 1 at the end of six months was 3.8 ± 1.14 and 4.6 ± 1.07 for site 2.
The intergroup comparison of site 2 showed comparatively higher linear bone growth, which is statistically non-significant. The results are analogous to those in the studies of Aspriello et al. [51] and Jayakumar et al. [52], which examined enamel matrix derivative (EMD) and demineralized freeze-dried bone allografts (DFDBA) with DFDBA alone for the treatment of defects. The results are also similar to an animal study conducted by El-Dien et al. [53], in which bilateral defects were arbitrarily treated with DFDBA + plateletrich plasma (PRP) (test) or DFDBA alone (control). The bone fill at site 2 (DFDBA) was 4.6 ± 1.07 mm at six months, which is in line with previous studies [45,46,[48][49][50]54].
The percentage of bone fill recorded for site 1 at the end of six months was 56.29 ± 11.17 and 63.38 ± 8.78 for site 2. The intergroup comparison showed a more significant percentage of bone fill in site 2, which was statistically significant. Similar results were observed in a study done by El-Dien et al. [53].
DFDBA contains BMPs that help stimulate osteoinduction by: (1) acting as mitogens on undifferentiated mesenchymal cells and osteoblast precursors; (2) inducing the expression of the osteoblast phenotype (e.g., increasing alkaline phosphatase activity in bone cells); and (3) acting as chemo-attractants for mesenchymal cells and monocytes as well as binding to extracellular matrix type IV collagen [55]. This may be the reason behind the more significant percentage of bone fill in site 2 in the present study.
The current study was a cross-sectional study with its inherent limitations. Thereby incapable of determining the incidence, studying uncommon health disorders, and making causal inferences. This single-center study was conducted at the Department of Periodontology, School of Dentistry, Karnavati University, India. Thereafter, it is difficult to generalize the findings all over the country.

Conclusions
With the extensive array of grafting biomaterials currently available, clinicians often find themselves lost in selecting the best option. This study is intended to make the selection procedure easier for dental clinicians. This study's findings had the potential to conclude the following: (I) The plaque and gingival index decreased in both groups, which indicates an overall improvement in the oral hygiene status of the patients after treatment. (II) Healing was uneventful, with absolutely no allergic reactions, which shows that both the graft materials were safe for human use. (III) Reduction in pocket depth was more in the sites treated by DFDBA, which offers a significant regenerative potential of DFDBA. (IV) Radiographic parameters, i.e., area of defects, linear bone growth, and percentage of bone fill, were more in the sites treated with DFDBA. This signifies the better regenerative potential of DFDBA. Although this study has certain limitations, it can be concluded that the use of DFDBA for treating intrabony defects is more beneficial when compared to BCP.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Karnavati University (KU) issued approval KU/KSD/2019/675, Dated: Dec 2019. This study obtained the Institutional Review Board's approval from Karnavati School of Dentistry, Gandhinagar, India. This was conducted in accordance with the declaration of Helsinki. The patients had been explained the study protocol and procedure, and informed written consent was obtained from them before the commencement of the study. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.