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Basic and Clinical Research

Radiological and Histomorphometric Outcomes of Homologous Bone Graft in Postextractive Implant Sites

A 6-Year Retrospective Analysis

Baldi, Domenico MD, DDS*; Pesce, Paolo DDS, PhD; Musante, Bruno DDS, PhD; Pera, Francesco DDS, PhD§; Fulcheri, Ezio MD; Romano, Filomena MD; Menini, Maria DDS, PhD#

Author Information
doi: 10.1097/ID.0000000000000920
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Abstract

Guided bone regeneration (GBR) is increasingly used in dentistry when the positioning of dental implants is impossible due to reduced vertical and/or horizontal residual bone.1,2 Various methods of intervention have been described using different types of grafting biomaterials.3–7 The ideal characteristics of biomaterials for GBR are biocompatibility, mechanical strength, flexibility, osteoconductivity, osteoinductivity, osteogenicity, biodegradability, sterilizability, easy availability, and easy storage.8,9

Autologous cancellous bone graft remains the most effective grafting material because it provides all the elements required for bone regeneration.7 However, autogenous grafting could be associated with several shortcomings and complications, including limited quantity of bone for harvest, greater morbidity, etc. Intraoral bone harvesting is often associated with complications such as damage of the mental or alveolar nerve, devitalization of teeth in the adjacent operation field, deterioration of the facial esthetic, and increasing risk of mandibular fractures depending on the donor site.10–12

For these reasons, new techniques for bone grafting are being tested almost daily,13,14 and several substitutes have been proposed as follows: homologous or allogenic grafts (human bone bank), allografts or xenograft (animal bones: cattle, sheep, and horses), and grafts with alloplastic materials (synthetic biomaterials).15–19 These bone substitute materials have been clinically demonstrated to promote acceptable outcomes. Ideally, augmentation with bone substitute materials would include good bone tissue integration, osseoinduction, and long-term stability. However, an ideal bone substitute material does not exist yet.7

Mineralized human bone allogenic graft seems an excellent bone substitute material, and in some European countries, its use was recently authorized after years of restriction, whereas in the United States, it has been broadly applied.11,20

Therefore, the aims of this study were:

  1. To radiographically investigate the in vivo efficacy in regeneration and maintenance of postextraction bone before implant surgery using a homologous cancellous bone particulate graft (Puros, Zimmer Dental);
  2. To evaluate histologically and histomorphometrically the regenerated bone;
  3. To analyze the regenerated bone resorption after implant insertion over time (6 years).

Materials and Methods

The present research was conducted in accordance with the Declaration of Helsinki, and the local Ethical Committee of Genoa University (Genoa, Italy) approved the study (prot. 62549).

All patients were carefully informed about the study protocol and provided written informed consent before the start of the study. The research is reported according to the CONSORT Statement (http://www.consort-statement.org).

Inclusion and Exclusion Criteria

Between January and December 2010, patients referring to the Department of Implant and Prosthetic Dentistry of Genoa University were screened for possible inclusion in this study.

Inclusion criteria

Patients requiring extraction of compromised teeth, postextraction site implementation, and subsequent implant placement; age greater than 18 years; and nonsmokers or light smokers (<10 cigarettes/day).

Exclusion criteria

The presence of serious disease or condition such as autoimmune diseases, uncontrolled diabetes and endocrinopathies, severe osteoporosis, polyarthritis, psychiatric illness, treatment with immune suppressants, drug addiction, pregnancy, immune deficiencies, intravenous bisphosphonate medication, orofacial cancer, chemotherapy or head and neck radiotherapy, or heart attack during the preceding 6 months.

First-Stage Surgery—Tooth Extraction

Patients underwent pre-extraction initial professional oral hygiene sessions 1 week before extraction.

After anesthesia (mepivacaine + epinephrine 1:100,000), the first surgical phase consisted in the minimally invasive extraction of the tooth (T0) using a piezoelectric tool (EX1-Mectron).

Radiographic Evaluation—Presurgery

The following day (T1) patients underwent a CBCT investigation (GXCB-500; Gendex Dental System, Biberach, Germany) at the level of the postextraction site to check the integrity of the alveolus walls and to evaluate the implant sites using a radiological-surgical guide in resin. That was created on the diagnostic wax-up to insert the implant in the most correct position. A hollow metal cylinder with an internal diameter of 2 mm was embedded in the resin guide consenting both a radiographical evaluation of the site and the guided insertion of the first bur during surgery.

Second-Stage Surgery—GBR

Seven days after tooth extraction, patients underwent a second operation for bone regeneration. Preoperative antibiotic prophylaxis was administered with 2 g of amoxicillin + clavulanic acid 1 hour before and 1 g after surgery and every 12 hours for 6 days thereafter.

After elevation of a mucoperiosteal flap, all the residual granulation tissue was removed and the alveolus was curetted to have a good bleeding useful for the engraftment (T2). The material was inserted with the use of pluggers, and the postextraction site was filled up at least for 2/3 of its volume. For a proper healing and protection of the graft, a resorbable bovine CopiOs Pericardium Membrane from Zimmer Dental was inserted. The mucoperiosteal flap was repositioned and sutured with Vicryl 4/0 (Ethilon; Johnson& Johnson Medical GmbH, Norderstedt, Germany). Follow-up appointments were carried out on the first and tenth postoperative day. After 10 days, the sutures were removed.

Graft Material

Puros cancellous particulate allograft (Zimmer Dental) was used as graft material. This product is homologous bone treated with the Tutoplast process:21

  1. Lipids are removed from all grafts in an ultrasonic acetone bath, as they may interfere with the healing process; this step also inactivates enveloped viruses such as HIV and Hepatitis C virus.
  2. Bacteria are destroyed using an osmotic treatment.
  3. Soluble proteins are eliminated, and nonenveloped viruses and bacterial spores are destroyed using an oxidative treatment with hydrogen peroxide (H2O2), but collagen is preserved.
  4. Final acetone wash for dehydrating the tissue and eliminating residual prions.
  5. Irradiation using low dose of gamma irradiation (17.8 to 20.1 kGy) to sterilize the tissue. This process guarantees maintenance of collagen tissue, and the preservation of bone tissue architecture thanks to low-dose gamma irradiation.

Volumetric Evaluation—Posthealing

After a healing period of 4 months (T3), patients underwent a second CBCT investigation (GXCB-500; Gendex Dental System, Biberach, Germany) to evaluate the quantity of regenerated bone using the same radiological guide as described before. The cross-section image passing through the center of the metal cylinder was selected to compare bone height in the radiograph taken at T1 and T3. The same para-axial image was used to compare bone thickness. The measurement was taken at the level of the distal bone crest of the mesial tooth. A single measure of bone height and thickness for each implant site was taken, and the differences of distances (T3 − T1) both in height and in thickness were calculated for each patient. Two examiners performed bone-level measurements after a calibration exercise demonstrating 96.5% concordance within ±0.5 mm for measurements (Asahi Roentgen, Japan).

The following parameters were assessed:

  1. Bone height: The vertical height from the implant platform to the most coronal point of the buccal bone.
  2. Bone width 0 and bone width 2: The horizontal thickness of the buccal bone measured at 0 and 2 mm apical to the platform, respectively.
  3. Gengival height: The vertical height from the implant platform to the marginal soft-tissue level.
  4. Gengival width 0 and gengival width 2: The horizontal thickness of the buccal mucosa measured at 0 and 2 mm apical to the platform, respectively.
Fig. 1
Fig. 1:
Trephine bur drilling bone in the regenerated site (A), the collected bone sample (8 mm high and 2 mm wide) (B), and sample of a regenerated site resin embedded and stained by Goldner stain (×40) (C).

Positive and negative values of BH/GH indicate bone/soft-tissue levels coronal and apical to the implant platform, respectively.

To assess the reliability and reproducibility of the measurement method, 2 calibrated examiners independently measured the parameters 10 times on 10 randomized CBCT scans. The intraexaminer and interexaminer reliability of the measurements was expressed as the intraclass correlation coefficient (0.95–0.99). Subsequently, a single examiner performed all the measurements and data collection.

Table 1
Table 1:
Reports the Outcomes Regarding the Amount of Bone Devoid of Stroma

Third-Stage Surgery—Implant Insertion and Biopsy

After 5 months (T4), a mucoperiosteal flap was elevated, and bone drills (Trephine Bur 2 mm, Biomet 3i) were used to obtain samples of the regenerated bone (Fig. 1, A and B). Preoperative CBCT was used to identify pristine bone.

Preoperative antibiotic prophylaxis was administered as previously described for T2 surgery.

Each bone sample was at least 8 mm in height. An implant (Full Osseotite NT; Biomet 3i, Palm Beach Gardens, FL) was contextually inserted in each regenerated site. Postoperative follow-up appointments were carried out on the first and tenth postoperative day. After 10 days, the sutures were removed.

Processing of Bone Biopsy Samples

The histological bone specimens were prepared adopting an acrylic resin embedding method ideal for bone biopsies not previously decalcified. This method preserves tissue morphology overcoming the limits of traditional processes of decalcification and paraffin embedding, avoiding manipulations that may cause contraction and distortion of the material. The mineralized component of bone maintains the correct steric relationship, especially in those areas where grafted bone takes the form of spicules and fragments. The resin used for embedding was a cold-polymerizing resin (4°C). It is a combination of a water-soluble resin (HEMA) and a plasticizer (Hydroxyethane). Each sample was collected, fixed in ether for at least 24 hours, and dehydrated in absolute ethanol at room temperature. The material was placed in the infiltration solution (resin + hardener I-accelerator benzoyl peroxide hydrate) under vacuum for at least 72 hours. Samples were then embedded in resin. The embedding solution was prepared adding the hardener II (accelerator—tetramethylaniline) in the infiltration solution. A Teflon mould was used for embedding in which the polymerization reaction occurs in the absence of air at 4°C. After polymerization, the specimens were prepared fixing the block of resin onto a support with a special fast-acting resin glue. Semithin sections (2–3 μm) were obtained from each block using a semiautomatic rotary microtome with a tungsten blade. Then, the sections were stained with Gill's hematoxylin, Toluidine blue, and Goldner's trichrome. The Goldner method is a modified Masson's trichrome stain, specifically modified for bone tissue, and it stains mineralized bone in green, osteoid in red, and nuclei in blue. The samples were then ready to be microscopically observed (20X–40X) and evaluated through histomorphometric analysis to have an exact percentage of regenerated bone.

Histomorphometric Analysis

Histomorphometric analysis allows for characterization of many biological structures through the measurement of morphological elements of various types that are classified in known parameters. Image J software (https://imagej.nih.gov/ij/), with an algorithm of deconvolution of colors, was used to assess the percentage of bone in the samples distinguishing stroma/osteoid and regenerated mineralized bone. Slides were scanned with Aperio XT (20x) (Leica Biosystems), and the images of all biopsies were acquired and saved as Tiff image files.

The Masson's trichrome module was used to separate the images in 3 channels, RGB. The green channel was used for bone quantification, and the red channel for osteoid tissue. The threshold value of background deviation was chosen, and then, the images were transformed in a binary system, and the region of interest (ROI) were delimited to be counted. A previous measurement was recorded using Aperio ScanScope on each image. Total bone was automatically identified and counted, and each area of osteoid tissue was manually selected by the pathologist to avoid including fibrous tissue or areas of necrosis.

Follow-up Analysis

Intraoral periapical radiographs were accomplished to assess interproximal bone levels at implant placement (T4) and at the 6-year follow-up appointment (T5). Radiographs were obtained with the parallel long-cone technique. The implant-abutment interface was used as the reference point for bone-level measurements. Interproximal bone levels were assessed from these reference points to the most coronal bone levels at the mesial and distal surfaces of each implant. Two examiners performed bone-level measurements after a calibration exercise demonstrating 95.7% concordance within ± 0.5 mm for measurements.

Results

Ten consecutive patients (7 women and 3 men, mean age 59.9 years, range 48–84) requiring the extraction of compromised elements, postextraction site implementation, and subsequent implant placement were enrolled in this study. All subjects were healthy and nonsmoker; 3 women were under treatment with calcium and vitamin D for mild osteoporosis with bone mineral density T-score values lower than −2.5.

Four patients had a tooth to be extracted because of periodontal disease (degree 3 mobility) and 6 for a fractured root. In total, 12 sites have been evaluated (3 monoradicular and 9 multirooted teeth, 5 upper and 7 lower teeth); in fact, 2 patients required 2 implants each in different sites.

Radiographic Analysis

The 3-dimensional X-ray analysis of the 12 postextractive sites showed a mean vertical bone augmentation of 4.1 mm (range: 1.9–5 mm) in the lower jaw and of 3.35 mm (range: 2.3–4 mm) in the maxilla. The mean horizontal bone augmentation in the lower jaw was 2.02 mm (range: 1.5–2.8 mm) and 2.15 mm (range: 1.6–2.8 mm) in the maxilla.

Histological Analysis Results

The histological analysis of the 12 samples (Fig. 1C) showed an intense bone metabolic activity with active osteoblasts next to the graft surface, at the level of the native bone-graft interface, and in the grafted area (Fig. 2). The grafted material was partially replaced by new regenerated bone, and a partially mineralized osteoid matrix was visible with new vessels. In 3 cases, connective tissue (Fig. 2D) prevailed over mineralized bone tissue. An active osteoid matrix edge (Fig. 2) was observed in 10 samples. In 2 samples, the grafted materials were completely replaced by new regenerated bone. No samples presented histological signs of inflammation (Fig. 2). A great rearrangement of the grafted materials was present in all samples except one.

Fig. 2
Fig. 2:
In the first line, histological characteristics of different bone samples were shown. The grafted material (brown) was partially replaced by new regenerated bone (green) and a partially mineralized osteoid matrix (red) (×40); in the second line, the program for automatic deconvolution of the colors highlights bone sample in white; and in the third line, areas of osteoid tissue made by the pathologist to avoid including areas of necrosis and stroma in the count. (AC) Patient 2: 82.65% of total new bone and 0.74% of osteoid tissue; (DF) patient 4 (an 84-year-old man): 48.60% of total new bone and 0.02% of osteoid tissue and much adipose tissue; (GI) patient 6: 62.23% of mature bone and 2.72% of osteoid rib; and (JL) patient 8 (a 62-year-old woman with osteopenia): 5.40% of total new bone and 0.03% of osteoid rib tissue. Arrows indicate the grafted material.

Histomorphometric Analysis Results

For histomorphometric analysis, only 9 samples were taken into account (only those related to the multirooted sites) to standardize the samples: 4 maxillary sites and 5 sites of the lower jaw. The histomorphometric study included 4 men and 5 women (3 of them had osteoporosis), aged between 48 and 84 years (mean: 59 years) (Table 1). The medium total bone was 60.01% (range 25.69%–88.49%), and the mature bone was 98.41% (range 94.48%–99.98%). No differences were found in bone formation between maxillary and mandibular sites.

Follow-up Results

At the 6-year follow-up, one patient with 2 implants was lost from follow-up because she died. All the other implants were stable and in function. No technical or biological complications were detected.

The mean bone resorption was 0.14 mm (range 0–0.5) on the mesial side and 0.21 mm on the distal side 0.07 mm.

Patients anecdotally reported good satisfaction with their implant rehabilitations.

Discussion

According to the results of this study, mineralized human bone allograft seems a viable bone substitute to regenerate bone defects. The 6-year follow-up analysis also showed stable periimplant bone levels, and no implants were lost. No signs of periimplant disease were found.

The present research applied an innovative histological procedure of resin embedding. This allowed to highlight in red the areas of strong metabolic bone activity, which would be impossible to detect using the hematoxylin-eosin staining usually used in similar studies. In this case, it would have been impossible to understand whether the bone was metabolically active or not.

The histomorphometric analysis showed bone growth exceeding the 50% in all patients except 2. These 2 patients were a 71-year-old woman with osteoporosis and an 84-year-old man. The rest of the patients had a percentage of total bone between 50% and 60%; this was considered a greatly successful result, considering that the samples were taken at 5 months only of maturation.

In addition, the presence of osteoid tissue was a sign of strong metabolic activity, and its presence was important to assess bone growth. Men had a higher percentage of osteoid than women, except the 84-year-old man. The 3 women with osteoporosis and older than 60 years had a very low percentage of osteoid: 0.04%, 0.03%, and 0.05%. This suggests that in these patients, bone formation is reduced and a complete bone formation might not be achieved.

The mean bone vertical regeneration was of 4.1 mm in the lower jaw and 3.35 mm in the maxilla. These results are similar to those obtained by Elnayef et al22 in a systematic review on vertical ridge augmentation. They found a mean vertical augmentation of 3.83 mm, but they did not evaluate the horizontal augmentation that in our research was of 2.02 mm in the lower jaw.

Solvent dehydrated human allograft bone has been especially used as bone substitute in subjects requiring maxillary lateral sinus augmentation. In particular, Monje et al compared dehydrated versus freeze-dried human allograft finding good histomorphometric results after 6 months: the mineralized tissue in the Puros group was of 31.9%.11 Similar percentages were also obtained by other authors.7,23

Compared with the above-mentioned studies, the present research showed greater bone growth exceeding the 50% in all patients except 2. This may be due to the very favorable morphology of postextractive sites compared with sinus augmentation. Monje observed that the turnover process using Puros is accelerated, and this may be due to the Tutoplast technique that consents the preservation of collagen promoting the osteoconductive ability of the bone graft.

In addition, tooth extraction was gently performed using a piezoelectric tool. This technique may have favor bone regeneration. Piezosurgery presents a selective micrometric cutting to obtain an extremely conservative avulsion with the preservation of the alveolus walls.24

One of the limits of this study is the lack of a control group. The regeneration technique applied was not compared with nonregenerated postextractive sites or with postextractive sites where different regeneration techniques were used.

Another limit is represented by the small sample size (12 sites). A study with a larger population would provide more conclusive results. In addition, split-mouth comparative studies including other materials would be necessary to better compare results between bone substitute materials.

Conclusions

The results obtained by the radiographic, histological, and histomorphometric analysis demonstrated excellent bone regeneration both in terms of quantity and quality when Puros allograft material was used in postextractive sites. Cancellous particulate allograft bone demonstrated stable results over a 6-year period.

Disclosure

The authors claim to have no financial interest, either directly or indirectly, in the products or information listed in the article.

Approval

The present research was conducted in accordance with the Declaration of Helsinki, and the local Ethical Committee of Genoa University (Genoa, Italy) approved the study (prot. 62549).

Roles/Contributions by Authors

D. Baldi: concept/design, data acquisition, analysis, and final approval. P. Pesce: paper drafting, data analysis, data acquisition, and final approval. B. Musante: concept/design, data acquisition, and final approval. F. Pera: data analysis and final approval. E. Fulcheri: concept/design, data acquisition, analysis, and final approval. F. Romano: data analysis and final approval. M. Menini: concept/design, paper drafting, analysis, and final approval.

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Keywords:

bone regeneration; dental implants; Puros

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