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 Table of Contents  
Year : 2020  |  Volume : 14  |  Issue : 2  |  Page : 59-65

Histomorphometric analysis of bone interphase

1 Indian Naval Dental Centre - Danteshwari, Colaba, Mumbai, Maharashtra, India
2 Department of Periodontics, R and R – Delhi Cantt, New Delhi, India
3 Military Dental Centre, Nasik, Maharashtra, India

Date of Submission06-Apr-2020
Date of Decision11-May-2020
Date of Acceptance20-May-2020
Date of Web Publication15-Jul-2020

Correspondence Address:
Reenesh Mechery
INDC, Mumbai, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JODD.JODD_19_20

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Background: Different surgical techniques have been advocated for esthetic rehabilitation of deficient alveolar ridges with implant placement: Guided bone regeneration (GBR), Alveolar distraction osteogenesis, Onlay block graft (autogenous and allograft); However, autogenous block graft still provides with predictable outcome since being the gold standard for regeneration and augmentation.
Objective: The aim of the present study was to compare histomorphometrically the bone interphase of augmented site with and without GTR (guided bone regeneration) membrane before implant placement.
Material and Methods: In this study, a total of 16 patients with missing incisor with Siberts Class 1 alveolar defects were selected, parasymphysial block graft were harvested and stabilized with miniscrews, DFDBA graft material around the block graft were placed, and 8 were covered with GTR resorbable membrane. Miniscrews were removed after 6 months. Bone core biopsy of interphase were retrieved by 2.9 mm diameter trephine bur and sent for histologic and histomorphometric analysis using histometry software.
Results: Data were evaluated using paired sample t-test and Shapiro-Wilk test. Histological evidence of fibrous tissue interphase was observes in specimen without GTR group. Though there were no statistically significant difference between the two groups in terms of amount of new bone 31.47 versus 30.6% (P = 0.5362), while there was a statistically significant difference in percentage of residual grafted material higher in non GTR augmented site (t value= 4.501 P = 0.0003) and marrow space in GTR group with statistical significance (t = 2.887 P = 0.0098).
Conclusion: Only few studies have tested histomorphometrically to see the exact uptake of the augmented site, comparing interpositioning of fibrous tissue, newly formed bone at site, residual graft material and marrow space. It is most appropriate to use an evidenced-based approach when a treatment plan is being developed for bone augmentation cases. This study will add to the evidence of use of autogenous block graft with GTR membrane thus providing a connective tissue interphase free augmentation.

Keywords: Block graft, guided tissue regeneration, histomorphometric analysis

How to cite this article:
Mechery R, Mukherjee M, Shreehari A K. Histomorphometric analysis of bone interphase. J Dent Def Sect. 2020;14:59-65

How to cite this URL:
Mechery R, Mukherjee M, Shreehari A K. Histomorphometric analysis of bone interphase. J Dent Def Sect. [serial online] 2020 [cited 2022 Jan 25];14:59-65. Available from: http://www.journaldds.org/text.asp?2020/14/2/59/289747

  Introduction Top

Resorption of alveolar bone is the common sequela of tooth loss. This may jeopardize the esthetic outcome and compromise the functional and structural aspect of treatment. Augmentation and regeneration of the lost bone often are thus necessary.[1] To satisfy the goals of implant dentistry hard and soft tissue needs to be present in adequate volume and quality. Regeneration refers to the reconstitution of lost or injured part by complete restoration of architecture and function. Various techniques are employed for ridge augmentation like particulate grafting, ridge splitting, block grafting and distraction osteogenesis, either alone or in combination.[2]

Today, implantologists oftenface challenge of implants in anatomically less favorable positions with regard to the quantity of the available bone. This has necessitated the development of technique and materials that promote predictable regenerative treatment. There are very few studies on exact type of bone integration and regeneration at augmented site and its uptake. The aim of the present study was to compare histomorphometrically the bone interphase of augmented site with autogenous parasymphyseal block graft with and without GTR membrane before implant placement so as to histologically and histomorphometrically observe the amount of newly formed bone, marrow spaces, and residual bone graft at site, thus adding valuable evidence for implant practice.

  Materials and Methods Top

Sixteen patients with Sieberts Class 2 (horizontal bone loss) of the upper anterior region were selected for the present study. They all showed a residual bone height ranging from 3 to 6 mm measured by computed tomographic scan. All patients required placement of single implant after augmentation of block graft after 6 months. The protocol of the study was approved by local Ethical Committee of AFMC, Pune, and all interested patients signed written informed consent.

Exclusion criteria

  1. General contraindication to implant surgery
  2. Irradiation, chemotherapy, or immunosuppressive therapy over the past 5 years
  3. Poor oral hygiene and motivation
  4. Active/recurrent periodontitis
  5. Uncontrolled diabetes
  6. Pregnancy or lactating
  7. Smokers
  8. Psychiatric patients
  9. Substance abuse
  10. Positive for HIV or hepatitis B/C
  11. Unrealistic expectations of patients
  12. Autoimmune diseases such as Rh arthritis, SLE, scleroderma, and Sjogren's syndrome
  13. Treated or under-treatment of bisphosphonates (tablets/intravenous [IV])
  14. Previous history of augmentation or failure
  15. Under chronic treatment with steroids/NSAIDs.

A total of 16 patients were considered eligible for the study with average mean age of 32 years. Of these candidates with alveolar defect, eight patients were augmented with block graft and eight were treated with block graft and GTR membrane. Incidentally, the patients who gave consent for the surgery were males since females did not opt for the block graft due to involvement of secondary site. The treatment plan formulated comprised Phase I therapy, re-evaluation, surgical Phase II of ridge augmentation with autogenous block graft from parasymphyseal site. This was followed by regular recall checkup, and after 6 months of uptake of block graft, implant was planned. During the implant placement, 2 mm of interphase bone was removed for removed for histomorphometric analysis.

Presurgical evaluation

Comprehensive pretreatment evaluation with routine blood and urine examination and conventional radiographic investigations using radiovisiography and cone-beam computed tomography were done. Mounted models were used to evaluate the interocclusal relationships and ridge shape providing model for template fabrication and implant angulations. All selected patients underwent rigorous oral hygiene maintenance with professional debridement when necessary 2 weeks before the bone augmentation procedure. All patients received antibiotics 1 day before the surgery (augmentin 1 gm) followed by continuation till 4 days postoperatively.

Surgical procedure

Augmentation procedure

All patients were treated under local anesthesia. Recipient maxillary region was prepared followed by block graft harvesting from the mandibular parasymphyseal region. A paracrestal incision was made through the palatal side of the recipient side with raising a full-thickness flap with release incision vertically exposing the site to be augmented.

In the donor site, vestibular/sulcular incision was given to expose the parasymphyseal bone depending on the case. A template was made for the recipient site using tinfoil and marked onto the donor site in the parasymphyseal region. Care was taken to harvest sufficient thickness and width of bone from desired site without encroaching into vital structures such as mental foramen, inferior alveolar nerve, apices of mandibular incisors, and canine and inferior border of the mandible. The “Rule of 5” of leaving 5 mm space from vital structures was followed for harvesting to provide a margin of safety to prevent morbidity during osteotomy.[3] Osteotomy was made using postage stamp method using surgical burr and osteotome and a corticocancellous block graft of desired template proportions. The harvested site was packed tightly with gauze to control bleeding. The block graft was screwed into the recipient site with titanium miniscrews after smoothening the edges and decortications of recipient site with 2 mm round burr for increasing vascularization [Figure 1]a and [Figure 1]b.
Figure 1: (a and b) Autogenous block graft Harvesting (common step for both group)

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The above mentioned procedure was same for both the groups wherein, non-GTR group, the DFDBA graft was packed around the block graft and the flap was sutured in position [Figure 2]. In GTR group, Pro Gide TM GTR (Bitextured resorbable barrier, Equinox Med Teq, Netherlands) was sutured with resorbable suture over the block graft packed with DFDBA bone graft (Dembone, Pacific coast tissue bank, USA) [Figure 3]a and [Figure 3]b. Donor site was also suture with silk sutures after hemostasis; a pressure bandage was applied to chin for 48 h to avoid pooling of blood from donor site. Postoperative instructions and antibiotics were given to all the patients (augmentin 1000 mg twice daily, combiflam twice daily, serratiopeptidase twice daily, and 0.2% chlorhexidine mouthwash to rinse twice daily). Sutures were removed after 10 days and the patient were recalled on regular recall checkup for the next 6 months. No prosthetic removable dentures were allowed.
Figure 2: Non-GTR group (surgical and post 6 months)

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Figure 3: (a and b) GTR group (surgical and post 6 months)

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Bone core biopsy and Implant placement

Clinical and radiographic evaluation of the augmented site was confirmed by measuring the postoperative width of edentulous augmented ridge after 6 months of surgery, comparing it to baseline. Again, a paracrestal full-thickness flap was raised for implant placement. The miniscrew used for stabilization of the block graft was removed. A bone core biopsy of the interphase of the recipient maxillary bone and augmented donor bone from the mandible was retrieved using 2-mm trephine bur. This also was used as the initial sequential drilling site for implant placement. Implants were inserted to the site using increasing diameter drill with 25 Ncm2 torque. Cover screw of the implant was placed, and flap was closed with 3-0 silk sutures. Postoperative instructions, antibiotics, and analgesics were prescribed and advised for oral hygiene maintenance.

Histological procedure

Bone core for histomorphometric analysis was retrieved from the interphase of the augmented site with help of 2 mm of trephine bur used as initial ditch and guide pin for implant. The retrieved bones were stored immediately in 10% buffered formalin and sent for histopathological ground sectioning to the Department of Pathology, AFMC, Pune. It was processed to obtain thin ground section. Slides were stained with acid fuchsin and toluidine blue for histologic slide and examined in normal transmitted light using light microscope (Dewinter, Italy) connected to high-resolution camera with PC interphase and monitor, connected to output digital pad (Dewinter software) along with a histomorphometry microscope measurement analysis software package (Biowizard 4.2) [Figure 4]. The image captures gave a software reading of the field of percentage of formed bone, residual grafted material, and marrow space details.
Figure 4: Dewinter light microscope with PC interphase for histomorphometric analysis

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  Statistical Analysis and Results Top

Statistical analysis was evaluated with MATRIX LABORATORY 7.1 version, developed by Mathwork, Natrick, USA. Paired sample t-test on pre and postsurgical cases was applied with P < 0.05. Bonferroni adjustment for P value was done as a part of post hoc analysis to reduce the outlier values and also to minimize the error emerging out of the smaller sample size [Figure 5]. Data were also evaluated by Shapiro–Wilk test wherein all data are presented as mean ± SD; statistical significant difference was accepted as P < 0.05.
Figure 5: Pre- and post-surgical clinical width gain

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The failures and complications that occurred during the entire study period were limited and listed as under:

  1. Exposure to titanium miniscrew after 5 month was observed in one case and early removal of miniscrew was done; thus, satisfactory healing and closure were achieved
  2. Infection of the harvested donor site was seen in one case with temporary paresthesia in the mandibular anterior region. It was treated with debridement and resuturing of the site under antibiotics coverage. No dropouts were reported or any major complications during entire period of the case study.

Of the 16 patients who gave consent for the study were mostly males of average mean with 32 years of age. Paired t-test on pre and postoperative clinical width gain of the alveolar ridge was also assessed with P < 0.05. Due to smaller sample size Boferroni adjustment was considered which also proved to be statistically significant with P < 0.01.

Histological results

Non-GTR group [Figure 6]
Figure 6: GTR GP-osteoid matrix with osteons in vicinity of grafted bone at ×10 and ×40

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A significant amount of grafted bone surrounded by newly formed bone was observed clinically at the augmented site. The autogenous grafted bone showed irregular-shaped margins due to remodeling process. On histological slide in almost all the eight cases of non-GTR group, interposition of fibrous tissue was evident, and a demarcation line separating the bones was clearly seen in the slides. No signs of inflammatory infiltrates were present. In some areas, bone remodeling was conceivable with a rim of osteoblasts depositing osteoid matrix.

Block graft with GTR group [Figure 7]
Figure 7: Non GTR GP-interposition of fibrous tissue was evident at ×10 and ×40

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In all the analyzed samples, a good amount of newly formed bone could be observed clinically without much remodeling and rough borders. In histologic slides, a tight contact between the grafted block graft and recipient bone without much fiberous tissues was found, indicating good uptake of the block graft to recipient site. Osteoid matrix with osteons in vicinity of grafted bone could be observed in histologic sections. The newly formed bone had a high affinity for dyes and was acid fuschin positive. Due to compartmentalization and selected cell repopulation due to GTR, some grafted materials of DFDBA were bridged by newly formed bone in outer and inner portions with no osteoclast around the block graft in the slide.

The histomorphometric results are summarized in [Table 1], [Table 2], [Table 3].
Table 1: Newly formed bone

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Table 2: Residual graft material

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Table 3: Marrow spaces

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There is no statistically significant difference in values on intergroup comparison in amount of new bone formation at site.

There is a statistically significant difference in values on intergroup comparison indicating more of residual bone in non-GTR group.

There is a statistically significant difference in values on intergroup comparison indicating more of well-developed marrow spaces in GTR group.

  Discussion Top

For patients with insufficient bone width in the anterior maxillary region, several procedures were introduced to compensate for a narrow edentulous ridge, among which the simultaneous or staged approach with guided bone regeneration (GBR) has been used extensively to create new bone.[2],[3] When the implant placement is combined with GBR, a two-stage approach of the implant surgery is imperative, and its results rely strongly on the technical skills of the surgeon. The healing of autogenous block grafts has been described as “creeping substitution” where viable bone replaces the necrotic bone within the graft and is highly dependent on graft angiogenesis and revascularization.[1],[3]

A classification for alveolar ridge defects has been described by Seibert in 1983, to standardize communication among clinicians in the selection and sequencing of reconstructive procedures designed to eliminate these defects. A Class I defect has buccolingual loss of the tissue with normal ridge height in an apicocoronal direction. A class II defect has apicocoronal loss of the tissue with normal ridge width in a buccolingual direction. A Class III defect has a combination buccolingual and apicocoronal loss of tissue, resulting in loss of height and width. Thus, the bone augmentation technique employed to reconstruct these different ridge defects is dependent on the horizontal and vertical extent of the defect.[4]

In 1992, Simion et al. introduced a “split-crest” technique to widen the edentulous ridge and place the implant simultaneously. Autogenous block graft has long been advocated to be a reliable and predictable approach for gaining ridge width. However, the disadvantages are the second surgical site morbidity and long delay in implant placement.[5],[6] In 2009, Felice et al. performed a randomized controlled clinical trial to evaluate two different kinds of graft materials: bone from iliac crest and bovine anorganic bone. There were ten selected partially edentulous patients having 5–7 mm of residual crystal height above the mandibular canal. Four months after bone grafting, a bone core was retrieved from each side using a 3-mm external diameter trephine for the histological evaluation. The histomorphometrically data showed the only statistically significant difference in the mean percentage of residual graft (between 10% and 13%, p values between 0.008 and 0.009) that was greater in the Bio-Oss group, while there were no statistically significant differences in the percentage of newly formed bone and in the marrow space between the two groups.[7]

More recently, in 2014, Laino et al. in their randomized control trial compared histomorphometrically the bone interphase in atrophic posterior mandible augmented with sandwich technique between block graft and corticocancellous bone allograft. Both procedures supported good results although the use of bone blocks allograft was less invasive and preferable than harvesting bone from the mental symphysis. No statistically significant differences between the two groups were observed regarding the percentage of newly formed bone (31.47 ± 2.2 vs, 30.6 ± 3.7%). The results of the study suggested slower integration of the grafted material, which was not clinically appreciable, and the use of autologous bone blocks does not seem to provide particular advantage.[8]

  Conclusion Top

Though there are many techniques for effective bone augmentation and are largely dependent on the extent of the defect and specific procedures to be performed for the implant reconstruction. The approach largely is dependent on the extent of the defect and specific procedures to be performed for the implant reconstruction. It is most appropriate to use an evidenced-based approach when a treatment plan is being developed for bone augmentation cases. This study will add to the evidence of use of autogenous block graft with GTR membrane, thus providing a connective tissue interphase-free augmentation. The result of the present preliminary investigation encourages further studies on the present topic, and it will be interesting in the future to compare the autogenous block graft and allograft at augmented site histomorphometrically.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Misch CM. Implant site development using ridge splitting techniques. Oral Maxillofac Surg Clin North Am 2004;16:65-74, vi.  Back to cited text no. 1
Misch CM. Ridge augmentation using mandibular ramus bone grafts for the placement of dental implants: Presentation of a technique. Pract Periodontics Aesthet Dent 1996;8:127-35.  Back to cited text no. 2
Pikos MA. Alveolar ridge augmentation using mandibular block grafts: Clinical update. Alpha Omegan 2000;93:14-21.  Back to cited text no. 3
Seibert JS. Reconstruction of deformed, partially edentulous ridges, using full thickness onlay grafts. Part I. Technique and wound healing. Compend Contin Educ Dent 1983;4:437-53.  Back to cited text no. 4
Simion M, Baldoni M, Zaffe D. Jawbone enlargement using immediate implant placement associated with a split-crest technique and guided tissue regeneration. Int J Periodontics Restorative Dent 1992;12:462-73.  Back to cited text no. 5
Simion M, Jovanovic SA, Tinti C, Benfenati SP. Long-term evaluation of osseointegrated implants inserted at the time or after vertical ridge augmentation. A retrospective study on 123 implants with 1-5 year follow-up. Clin Oral Implants Res 2001;12:35-45.  Back to cited text no. 6
Felice P, Cannizzaro G, Checchi V, Marchetti C, Pellegrino G, Censi P, et al. Vertical bone augmentation versus 7-mm-long implants in posterior atrophic mandibles. Results of a randomised controlled clinical trial of up to 4 months after loading. Eur J Oral Implantol 2009;2:7-20.  Back to cited text no. 7
Laino L, Iezzi G, Piattelli A, Lo Muzio L, Cicciù M. Vertical ridge augmentation of the atrophic posterior mandible with sandwich technique: Bone block from the chin area versus corticocancellous bone block allograft—clinical and histological prospective randomized controlled study. Biomed Res Int 2014;2014:982104.  Back to cited text no. 8


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]

  [Table 1], [Table 2], [Table 3]


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