|Year : 2015 | Volume
| Issue : 1 | Page : 33-39
A comparative study of bovine bone used alone and in combination with transforming growth factor-beta for the treatment of periodontal osseous defects in humans
Khalid S Hassan, Adel S Alagl
Department of Preventive Dental Sciences, Division of Periodontics, College of Dentistry, University of Dammam, Dammam, Saudi Arabia
|Date of Web Publication||20-Jan-2015|
Khalid S Hassan
Department of Preventive Dental Sciences, Division of Periodontics, College of Dentistry, University of Dammam, P.O. Box 1982, Dammam 31441
Objectives: Transforming growth factor-betas (TGFβs) are multifunctional growth factors with a broad range of biological activities in various cell types in many different tissues. The purpose of this study was to evaluate the treatment of intrabony defects with anorganic bovine bone mineral matrix combined with TGFβ-1 with the use of anorganic bovine bone alone.
Materials and Methods: Thirty-two sites from sixteen patients were selected using a split-mouth study design for each patient, determined randomly through a biased coin randomization. One site received a mucoperiosteal flap, and the osseous defect was filled with the combined therapy (Group 1). The other site treated was with anorganic bovine bone alone and served as a control (Group 2). All the treated sites were covered with a bioabsorbable collagen membrane. The clinical parameters and radiographic follow-up examinations were recorded after 3, 6, 9, and 12 months.
Results: Clinically, there was a statistically significant gain in the clinical attachment level (+5.03 0.14 mm) and a statistically significant reduction of pocket probing depth (−5.16 mm 0.13) for Group 1 sites compared to sites in Group 2 (P ≤ 0.01). In addition, there were significant differences in bone density and a significant decrease of marginal bone loss after the combined therapy compared with the use of anorganic bovine bone alone (P ≤ 0.01).
Conclusion: The use of anorganic bovine bone mineral matrix combined with TGFβ-1 seemed to be effective in the treatment of intrabony defects. This showed an improvement in the clinical outcome of periodontal therapy superior to the use of anorganic bovine bone on its own.
ةجسنلأل ةيمظعلا تاوجفلا جلاع يف اتيب ومنلا زفحمو نيتوربلا ةعوزنم ةيمظع تاوشح نم طيلخ مادختسا ةنراقم ةسارد ىلع ثحبلا اذه موقي ةعشلأا روصو ةيكينيلكلإا تلالادلا تاسايق ذخأب ناثحابلا ماق .ةنمزملا ةينسلا لوح ضارمأ نم نوناعي ىضرم رايتخا مت .ناسنلإا ىدل ةينسلا لوح لوح تاباهتللااو ةيديدصلا بويجلا نم ليلقتلا اضيأو .مظعلا ةفاثك ةدايزو ةيمظعلا تاوجفلا ءلاتما ةساردلا هذه تتبثأ .ةينسلا لوح ةجسنلأل ءلم يف اريثأت لضفأ اتيب ومنلا زفحمو نيتوربلا ةعوزنم ةيمظع تاوشح نم طيلخ مادختسا نأ ىلإ ةساردلا تصلخ دقو .ةينسلا لوح ةجسنلأا.نانسلأاب ةطيحملا ةجسنلأا بيصت يتلا ةيمظعلا تاوجفلا
Keywords: Anorganic bovine bone mineral matrix, osseous defects, periodontal regeneration, transforming growth factors beta
|How to cite this article:|
Hassan KS, Alagl AS. A comparative study of bovine bone used alone and in combination with transforming growth factor-beta for the treatment of periodontal osseous defects in humans. Saudi J Med Med Sci 2015;3:33-9
|How to cite this URL:|
Hassan KS, Alagl AS. A comparative study of bovine bone used alone and in combination with transforming growth factor-beta for the treatment of periodontal osseous defects in humans. Saudi J Med Med Sci [serial online] 2015 [cited 2019 Jun 19];3:33-9. Available from: http://www.sjmms.net/text.asp?2015/3/1/33/149671
| Introduction|| |
The ultimate goal of a regenerative periodontal surgery is not only to reduce pocket depths, but to regenerate a new attachment apparatus and reconstruct the periodontal unit within previously existing normal physiologic limits. The use of bone grafts and artificial membranes for periodontal surgeries are measures in this direction.
A wide range of bone graft materials that have been developed and were available include: Autografts, allografts, xenografts and alloplasts. Autografts, the gold standard in grafting procedures, are bone specimens taken from one area of a patient's body and implanted in osseous defects somewhere else in the body.  However, the lack of adequate donor material and the fear of the remote chance of disease transmission have limited the use of autogenous grafts and allografts on a routine basis. These factors have led to the development of xenografts and alloplasts or synthetic bone substitutes for periodontal applications.
A natural anorganic bovine-derived bone mineral matrix has recently been introduced as a bone graft material for regenerative periodontal surgery. Moreover, Bio-Oss (Geistlich Pharma AG, Wolhusen, Switzerland) is a bovine bone widely used as dental xenograft material that can result in pocket reduction, attachment gain and bone fill in periodontal defects to the same extent as that achieved with demineralized freeze-dried bone. 
In recent years, progress in regenerative medicine has made recombinant growth factors (GFs) available as an alternative treatment for periodontal tissue regeneration. These multifunctional biological mediators are able to regulate the proliferation and migration of various cell types. They were found to be beneficial within bony sites, including the periodontium. 
Transforming growth factors betas (TGFβs) are multifunctional GFs with a broad range of biological activities in various cell types in many different tissues. Three isoforms of TGFβ have been found in humans (TGFβ-1 to -β3), are synthesized by many different cell types and stored as an inactive complex with latency-associated peptide in the extracellular bone matrix. ,,, Therefore, their evaluation for use in bone regeneration in various experimental settings have shown both stimulating and inhibiting effects on bone formation. Overall, the stimulating effects on bone healing and bone formation predominate. 
A considerable emphasis has been placed in research on "combination therapy," that is, an approach aimed at combining the positive aspects of the different regenerative principles. Greco et al. also stated that synergy between two or more entities was usually considered a positive attribute of the combination, and that the observed effect was greater than the known effect of each agent working alone.
A combination of various biomaterials has recently been proposed for use in bone regeneration. ,,, Therefore, for the purposes of this study, the research question was whether anorganic bovine bone mineral matrix combined with TGFβ-1 would give a therapeutic effect in the periodontal regeneration. The aim of the study was to evaluate the treatment of intrabony periodontal defects with anorganic bovine bone mineral matrix combined with TGFβ-1 as opposed to the use of anorganic bovine bone mineral matrix alone.
| Materials and methods|| |
Sixteen patients (8 males and 8 females with a mean age of 37 ± 2.06 years) with chronic periodontitis were selected to participate in this split-mouth randomized single-blind and controlled clinical trial. The study population was composed entirely of patients who presented to the Outpatient Clinic, College of Dentistry, University of Dammam (Dammam, Saudi Arabia) for evaluation and management of intrabony defects between March 2010 and March 2011. To be included in the study sample, the following criteria were used for patient selection: Absence of any systemic diseases, non-smokers, not pregnant (female cases) and no history of taking systemic antibiotics or nonsteroidal anti-inflammatory drug within the 3 months prior to treatment. In addition, the bony defects had to have the following characteristics: Probing pocket depth (PPD) and clinical attachment level (CAL) more than 5 mm, a minimum of two osseous walls and the site should not have been treated surgically in the year before the start of this study.
Each patient was prepared for surgery with an initial phase of therapy that included oral hygiene instruction and a full-mouth scaling and root-planing. Approximately 4 weeks after the initial therapy, patients were re-evaluated to assess their clinical parameters and plaque control. All subjects were required to achieve a level of good oral hygiene, which was set at an O'Leary Plaque Index (PI)  of less than 20% in order to progress to the surgical phase of therapy.
Written informed consent was obtained from each patient prior to the surgical procedure, and the protocol was reviewed and approved by the Ethical Committee of the College of Dentistry, University of Dammam, Saudi Arabia. In addition, all patients presented for evaluation of the clinical and radiographic parameters between March 2010 and March 2011.
The following clinical parameters (PI  , gingival index (GI)  , PPD and CAL  ) were assessed at the baseline, 3, 6, 9 and 12 months postoperatively by the same calibrated examiner (AA), who was blind to the patient's group assignment. This examiner used a periodontal probe (NUC-15, Hu-Friedy, USA), and collected the data for each patient. The clinical and radiographic examinations were performed by the same examiner during follow-up appointments after 3, 6, 9, and 12 months.
The same surgeon performed all procedures (KH). Thirty-two sites were selected by use of a split-mouth design for each patient, which was randomly determined through a biased coin randomization. Intracrevicular incisions were made on the buccal and oral aspects of the teeth, of the jaw at each the defect site. Vertical releasing incisions were made only if necessary. For patients in Group 1 (test sites), mucoperiosteal flaps were then raised at both the buccal and oral aspect of the teeth [Figure 1]a. The defect was debrided, and the roots planed with hand instruments and ultrasonic scalers. A bioabsorbable collagen membrane (Bio-Gide; Geistlich Pharma AG, Wolhusen, Switzerland) was trimmed and adapted so that it covered the entire defect and extended at least 3.0 mm beyond its margins. Prior to the final placement of the Bio-Gide membrane, anorganic bovine bone mineral matrix [Bio-Oss; Geistlich Pharma AG, Wolhusen, Switzerland, Figure 1]c combined with TGFβ [(10 μg/mL rhTGFβ-1, Research and Development (R&D) Systems, United states of America, [Figure 1]c and d)] was packed into the defect [[Figure 1]b and d]. The membrane was fixed by means of a bioabsorbable ligature around the neck of the adjacent teeth, and the mucoperiosteal flaps were coronally displaced to fully cover the membrane and achieve primary closure. The flaps at the defect site were sutured by means of interrupted 4/0 Vicryl sutures (Ethicon, Johnson and Johnson, US).
|Figure 1: 2-wall osseous defect (a), The osseous defect filled with anorganic bovine bone mineral matrix combined with transforming growth factor beta-1 (b), materials which are used (c), and recombinant human transforming growth-beta-1 mixed with anorganic bovine bone mineral matrix (d).|
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For patients in Group 2 (controls), the same procedures were performed except that the defects were filled with anorganic bovine bone mineral matrix only. All the patients received systemic antibiotic therapy with a combination of Amoxicillin 500 mg and Metronidazole 250 mg/day for 5 days. They were instructed to rinse their mouths twice a day with 0.2% chlorhexidine digluconate for 4 weeks postoperatively to aid in plaque control.
Standardized radiographs were taken with the Rinn film holder before surgery and again at intervals of 3, 6, 9 and 12 months postoperatively.  Identical exposure parameters were used at all examinations and the films were processed automatically. All periapical radiographs digitized were saved in a tagged image file format and the bone density and marginal bone levels were measured using imagej software.  In this software, the area/areas to be measured which are called regions of interest (ROI) were selected (color density selection). A single pixel that represents a specific color (white pixels in radiographs) was selected, or a threshold allowing for automatic selection of all other pixels in the ROI threshold areas were traced and counted as a number of pixels that can be calculated as the ratio of the entire ROI. Average density was determined on a scale of 0-256. The number 256 (8 bits) stands for the whitest pixel on the screen while number 0 represents the areas of the darkest pixels on the screen. The ROI of these radiographs was a circle of a fixed size to contain the critical size defect precisely. The program calculates every pixel in the image and then performs the calculations necessary to get one number representing the average density of all the pixels which must be between the values of 0 and 256. The marginal bone level measurements were obtained by measuring to points from cement-enamel junction. This was also done to the defects in the preoperative radiographs, and compared with the postoperative radiographs.
Data were analyzed using the Statistical Package for Social Sciences version 13 (SPSS, Chicago, Ill). Descriptive statistics were produced for all variables. The baseline values to assess the homogeneity of the groups (excluding the relative values) were compared using the unpaired t-test. For the statistical evaluation of changes from baseline to 12 months in each treatment group, the paired t-test was used. For comparisons between the groups, the unpaired t-test was used. In the calculations, measurements of the deepest sites per defect were included. The level of significance was set at P < 0.05.
| Results|| |
All patients completed the study and were recalled for evaluation at 3, 6, 9 and 12 months. Baseline analysis did not display any statistically significant differences between the two groups for any of the assessed variables, suggesting that final differences between treatment modalities were not influenced by initial defect characteristics, thus allowing post-treatment results to be compared. With regards to the gingival and plaque indices, there were no statistically significant differences between test (Group 1) and control sites (Group 2) (PI was 0.19 ± 0.10 at baseline for Group 1, 0.19 ± 0.11 for Group 2, 0.20 ± 0.15, for Group 1, and 0.19 ± 0.13 for Group 2 postoperatively. GI was 0.19 ± 0.30 at baseline for Group 1, 0.22 ± 0.35 for Group 2, 0.20 ± 0.33 and 0.21 ± 0.38 postoperatively for Group 1 and 2, respectively). These data are shown in [Table 1].
|Table 1: Clinical measurements (mean±SD) at baseline and the different observation periods for Group 1 and 2|
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The present study represented a comparison between the two groups with regard to probing depth and CAL. There was a statistically significant gain in CAL of 5.03 ± 0.14 mm (71.4%) for patients who received the anorganic bovine bone mineral matrix combined with TGFβ compared with 4.25 ± 0.16 mm (60.02%) for sites treated with anorganic bovine bone mineral matrix alone (P ≤ 0.01). With respect to probing depth, there was also a significant reduction in probing depth of 5.16 ± 0.13 mm (70%) for sites treated with anorganic bovine bone mineral matrix with TGFβ (Group 1) compared with 4.62 mm ± 0.14 (63%) for Group 2 sites treated with anorganic bovine bone mineral matrix only (P ≤ 0.01).
The radiographic findings of the present study revealed that there were statistically significant differences in the bone density (in pixel) for sites treated with anorganic bovine bone mineral matrix. This was combined with TGFβ compared with sites treated with anorganic bovine bone mineral matrix only (P ≤ 0.01). Moreover, there was a significant decrease of marginal bone loss in sites treated with anorganic bovine bone mineral matrix combined with TGFβ, compared with sites treated with anorganic bovine bone mineral matrix alone (P ≤ 0.01) [[Table 2] and [Figure 2]a-d].
|Table 2: Bone density (in pixel) and marginal bone loss (in mm) in Group 1 and 2 during the different observation periods|
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| Discussion|| |
The present study was designed to address a specific question, whether the application of anorganic bovine bone mineral matrix combined with TGFβ-1 provides an additional clinical benefit to the use of anorganic bovine bone mineral matrix alone. This study did not intend to assess the ability of procedures or materials to produce periodontal regeneration in histological terms.
Regeneration of the lost attachment apparatus is the treatment of choice for intrabony osseous defects in contemporary clinical practice. The results of this study demonstrated that both treatment methods provided statistically significant improvements in the measurements of clinical parameters. In addition, the statistical analysis of data revealed a significant improvement after the combined therapy about the clinical and radiographic measurements compared to the use of anorganic bovine bone mineral matrix alone.
In our study, the randomized, split-mouth design was chosen because it was intended to exclude patient-specific characteristics. The healing results of a single site, however, were dependent on a number of factors at baseline that may differ from site to site.  In addition, at the time of each follow-up examination, measurements were made without consulting the randomization schedule to determine which side the treatment was or control or what treatment the subject received. Moreover, the use of standardized periapical radiographs was included in this study to avoid changes in the apparent size and shape of a tooth on the radiographs, and to standardize the relationship between the tooth, film and x-ray source.
Analysis of research published to date, suggests that the clinical success of GFs for periodontal regeneration appears to a large degree, to be dependent upon the characteristics of the specific carriers.  Supporting matrices for engineering bone and soft tissue have included processed bone allografts, synthetic and natural polymers, synthetic ceramics, bovine type I collagen and calcium sulfate. These were formulated into porous scaffolds, Nano-fibrous membranes, micro-particles and hydrogels. Moreover, there is an evidence that a combination of several materials may offer the best opportunity for beneficial clinical outcomes.
The rationale for the therapeutic use of GFs in the stimulation of bone healing is based on the hypothesis that through appropriate signaling, they may induce or accelerate the whole healing process.  GF may be administered either via a protein therapy, resulting in direct Recombinant Human Growth Factor (rhGF) delivery to the regeneration site or via gene therapy, delivering GF into cells through their encoding genes.
Transforming growth factors-beta are members of a large superfamily of GFs. They have the ability to promote or inhibit proliferation of many cell types in postnatal tissues and are modulators of cartilage and bone differentiation.  Nimni 1997,  hypothesized that the term 'GF' was probably a misnomer for these polypeptides because they did not always encourage growth, but acted rather as the modulators of cellular activities. Moreover, latent TGFβ activated by osteoclasts during bone resorption, stimulates osteoblastic function and bone formation, suggesting that TGFβs play a key role in bone remodeling. 
In this respect, very few periodontal regeneration studies using TGFβs have been performed. TGFβ-1 has been evaluated in dogs , and sheep, but with disappointing results. Until todate, few studies have been carried-out out using TGFβ-1 for periodontal tissue regeneration in humans.
In vivo study demonstrated that the continuous application of 1 and 10 micrograms natural TGFβ-1 to a plated tibial osteotomy in rabbits increased the mechanical bending strength and callus formation at 6 weeks. In addition, in a dog model with unloaded implants surrounded by a gap, 0.3 microgram recombinant human TGF beta-1 (rhTGFβ-1) adsorbed to gritblasted tricalcium phosphate coated implants, was able to enhance mechanical fixation, bone ingrowth and gap bone formation  . On the other hand, McCauley and Somerman in 1998  , demonstrated that TGFβ-1 inhibited the formation of mineralized nodules in vitro. Moreover, TGFβ-1 expressed by platelets in the fracture sites or by osteoclasts during bone remodeling may stimulate the formation of an osteoid matrix with no mineral phase  .
In this study, periodontal defects implanted with rhTGFβ-1 combined with anorganic bovine bone showed virtually complete healing [[Figure 2]c and d] at all sites. However, the amount of alveolar bone regenerated by rhTGFβ-1 on its own was significant when compared to that of anorganic bovine bone mineral matrix alone and was highly significant when compared to the preoperative defect. This may be a true reflection of the regenerative capabilities of TGFβ-1, in that the periodontal osseous defects implanted with rhTGFβ-1 performed well with a 54.04% bone density and 60% marginal bone regenerated [[Figure 2]b, d and [Table 2]].
|Figure 2: Periapical radiograph showing intrabony defect before treatment (a), 6 months postoperative radiograph (b), postoperative photograph showing normal gingiva without recession or inflammation (c), and postoperative radiograph after 12 months demonstrated bone fill the osseous defect (d).|
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The study by Palioto et al., 2011  , demonstrated that the exposure of human osteoblastic cells to TGFβ-1 showed early increased cell proliferation, reduced alkaline phosphatase activity and matrix mineralization. The results of the combination therapy in the present study are in agreement with the concept of this study, whereby there was an increase in bone density and marginally reduced bone loss. In addition, our results revealed that there was no significant difference in the periodontal tissue regeneration of mandibular and maxillary defects. There was also no significant difference in the periodontal tissue regeneration between defects of the first and second molars. Finally, the use of TGFβ-1 has been proposed as a strategy to support periodontal tissue regeneration.
| Conclusion|| |
From the results of the present study, it can be concluded that the use of anorganic bovine bone mineral matrix combined with TGFβ-1 seemed to be effective in the treatment of intrabony defects and showed a greater improvement in the clinical outcome of periodontal therapy than anorganic bovine bone mineral matrix used on its own.
| Limitations and recommendations|| |
This study was the first step in investigating the regenerative capabilities of rhTGFβ-1 in periodontal applications in humans. The results indicate that rhTGFβ-1 may be on the verge of being therapeutically applied. Additional studies involving rhTGFβ-1 are recommended. However, to determine if such a treatment should be uniformly used in regenerative periodontal procedures.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]