Clinical and Histomorphometric Assessment of Lateral Alveolar Ridge Augmentation Using a Corticocancellous Freeze-Dried Allograft Bone Block

A common sequela of tooth loss, alveolar bone resorption interferes with rehabilitating treatments.^1–4  Implant malpositioning can cause numerous problems, such as loosening and/or fracturing of implant components, occlusal discrepancies, and compromised esthetics and phonetics.^1–3,5–7  Following tooth extraction, bone resorption will occur over a 12-month period, particularly in the first 4 months; it might range up to 5 to 7 mm buccolingually.^1–3,5,8,9  This calls for development of materials and techniques that promote predictable reconstitution of the injured tissue and restoration of function.^{1,4,6,7,10–12}  One of these is horizontal (or lateral) ridge augmentation, which has been facilitated through various approaches, including block grafting materials and guided bone regeneration.^{1–4,7,11–15} 

The particulate autograft is osteogenic (having live osteogenic cells and abundant in vital cytokines) and thus the gold standard for most craniofacial bone grafting.^1–3,10,16  However, autografts have several limitations, such as the need for extra surgeries, donor-site morbidity, potential resorption, size mismatch, and an inadequate volume of graft material.^1–3,10  Allografts are transferred between dissimilar members of the same species and are osteocunductive (acting as a scaffold for new bone formation) or osteoinductive (also having essential cytokines that can accelerate bone growth).^13,17  They are abundantly available and might eliminate the above-mentioned disadvantages of autografts. Therefore, allografts have been utilized as a substitute for or an adjunct to autografts.^1–3,10,18  The most commonly used form of allograft is the particulate form, although other forms, such as sheets, putty, gel, collagen sponge, cortical, cancellous, and recently, corticocancellous segments, are also used.¹ 

Block grafts might be adapted and rigidly fixed over the native bone surface. Therefore, they might present the advantage of minimizing the failure rate caused by graft dislocation.¹  This can be of use in horizontal ridge augmentation. Case reports have demonstrated success with both types of block allografts (freeze-dried bone allograft [FDBA] and demineralized freeze-dried bone allograft [DFDBA]) in horizontal ridge augmentation.¹  Cancellous allograft blocks have also been shown successful.^4,7,11,19  There are also a few studies on clinical bone gain of horizontal ridge augmentation using corticocancellous blocks: Toscano et al⁵  retrospectively evaluated the amount of bone gain after augmentation with a DFDBA block graft in an uncontrolled fashion and without statistical analyses.⁵  Recently, a prospective controlled clinicohistologic study evaluated 7 cases of FDBA grafts.²  There is also a computerized-tomography study on bone gain following the use of corticocancellous allograft blocks, which shows bone gains similar to those of cortical allograft and autograft blocks.¹⁵  Since the number of clinical studies on corticocancellous allograft blocks is small, and since different brands of grafts cannot be perfectly generalized to each other,^2,20  the present study was conducted. The main null hypothesis was the absence of any increases in alveolar ridge width 6 months after augmentation with an FDBA corticocancellous block. Histologic examination was carried out on biopsy specimens to measure the extent of residual graft material, newly formed bone, and level of inflammation. Finally, implant success was evaluated after a minimum of 12 months.

This prospective before-and-after clinical trial was performed on 60 measurements and 10 histologic specimens from 10 patients. The sample size was predetermined based on the findings of Buser et al¹³  to obtain sufficient powers. The study protocol was approved by the research committee of the university according to the Helsinki declaration, and signed written consent was obtained from all patients after thorough explanation of the study. Patients could leave the study at any point, in which case no changes would occur to their routine treatment protocol. Patients were subsequently selected from attendees to the Department of Periodontics and Implantology based on the inclusion criteria: the indication for lateral ridge augmentation followed by dental implantation in the same maxillary region. Excluded were ridges with insufficient height, ridges wider than 4 mm, or those narrower than 2 mm (according to cone-beam computerized tomography). Also, patients with any systemic or local conditions (eg, uncontrolled diabetes mellitus, immune deficiencies, taking immune suppressive/anticoagulant medication, bone diseases, or pregnancy) or any habits (eg, alcohol consumption, drug abuse, or cigarette smoking) affecting the health or healing capacity were excluded. Patients with poor cooperation and active/untreated periodontal diseases as well as those who were not able or willing to maintain proper oral health were excluded from the study.

According to the manufacturer (contacted personally), graft processing takes place as follows. Corticocancellous allograft blocks were prepared in accordance with the regulations of the American Association of Tissue Banks (AATB) and US Food and Drug Administration. Life and medical histories of the donor were received by experts from the relatives and medical records. Donors were excluded in the case of positive histories for numerous diseases and surgical procedures (eg, out of brain cancer, history of manipulated primary brain malignancy [surgery or shunts], human immunodeficiency infection, Creutzfeldt-Jakob disease, viral hepatitis, histories of intravenous drug users, rabies, or dementia). Some criteria for donor selection were stricter than those of AATB: for example, criminal/prison records would be considered a negative point in donor selection. Also, the time from death to the donation was reduced from the 72 hours permitted by AATB to 24 hours. After obtaining the tissue, supervised experts scrutinized it using serologic and microbiological tests. All the services and facilities (including clean rooms at pharmaceutical level) complied with current good manufacturing practices. All textures were prepared in a special environment, called class 1000, and processed in class 10–1000 sterile space. Harvested bone becomes acellular and clean under sterile conditions. After being cut to the desired size, bone was frozen. Freeze-dried bones were then lyophilized to eliminate microbial growth and minimize potential autoimmune reaction. Finally, a minimum of 25KGY was emitted to the specimen. Processed bone tissues were nontoxic as per ISO 10993-5 standard.

Half an hour before the operation, patients took 2 g amoxicillin and rinsed their mouth with 0.2% chlorhexidine.^2,12  After administering local anesthesia, a palatally inclined paracrestal (horizontal) incision was made over the edentulous ridge using a #15 scalpel. At both ends of the horizontal incision, 2 divergent periosteal vertical releasing incisions were cut, extending beyond the proximal teeth. A full-thickness flap was elevated and soft tissue remnants were removed from the cortical plate. A bone caliper (Blue & Green Co, Edmonton, Canada) was used to measure clinically the bone thickness on the mesiodistal midline of the edentulous ridge at points 2 and 5 mm apical to the ridge crest on the buccal surface. The mesiodistal midline was determined after examining the length of the ridge, dividing it by 2, finding the middle point of the ridge crest, and then drawing a vertical line perpendicular to the ridge (as the midline) from that point. Before and during the surgery, a block of corticocancellous allograft (5 mm thick, CenoBone, Tissue Regeneration Corporation, Kish Free Zone, Iran, ISO13485-BSI) was immersed in normal saline for 30 to 45 minutes to be rehydrated. After decorticating the recipient buccal plate using a round bur, the graft was placed and adjusted on the cortex. It was fixed in maximum adaptation with the bone using titanium screws (Dentium, Seoul, Korea). Sharp edges were trimmed by a bur. Any gaps between the block and cortex were filled with bone particles 500–1000 μm in size (Tissue Regeneration Corporation) mixed with saline. Ridge widths were again measured exactly as stated previously. Afterward, a 20×20 mm² resorbable collagenous membrane 0.2–0.6mm thin (derived from allogeneic pericardium, CenoMembrane, Tissue Regeneration Corporation) was placed over the graft materials. The flap was positioned over the membrane. It was sutured tension-free with 4-0 silk, according to horizontal mattress and interrupted techniques (Figure 1). Administered were amoxicillin and metronidazole (2 times a day for 10 days), analgesics (3 times or more a day) as well as 0.2% chlorhexidine mouthrinse (twice a day for 10 days). Sutures were removed 14 days later.^5,10 

About 7.0 ± 1.3 months after the first surgery (range = 6–9 months), a second surgery was carried out for implant placement (Figure 2). Similar to the first surgery, a full-thickness flap was cut and everted. The graft was inspected for any clinical signs of failure. A graft meeting the following criteria was considered successful by 2 periodontists: absence of graft exposure/radiolucency/postoperative infection, bleeding after removal of the screws, incorporation of the graft within the recipient site, and allowing implant placement.¹⁰  The absence of radiolucency was evaluated on radiographs taken only for treatment purposes (and not necessarily standardized radiographs). Ridge widths were measured at the same points as mentioned earlier, and titanium screws were removed. Depending on the surgical site's surface area, a bone core specimen was biopsied from the implantation site using a 2-mm trephine bur. Biopsy specimens were stored in 10% formalin and shipped to the laboratory for histologic evaluation.

Specimens were completely fixed by 10 days of formalin immersion. Afterward, they were decalcified by a week of 10% formic acid storage. Acid was neutralized by placement in 20% lithium bicarbonate solution for 5 minute. Blocks were split into vertical anteroposterior sections. The surface of the sectioned midline of the block was dyed with Indian ink. The block was dehydrated in graded ethanol, embedded in paraffin, and coded. The paraffin blocks were sectioned and stained with hematoxylin and eosin. An experienced pathologist examined the percentage of remaining graft material, the percentage of osteogenesis (Figure 3), and the parameters of inflammatory response (rated as no inflammatory cells [grade 0], mild inflammation represented by diffused inflammatory cells [grade 1], focal inflammation indicated by aggregation of 5 to 10 cells [grade 2], focal inflammation indicated by accumulation of 10 to 50 cells [grade 3], or a severe response indicated by a focal aggregation of more than 50 cells [Grade 4]) under light microscopy (Olympus, Tokyo, Japan).

About 15.1 ± 2.7 months after implantation (range = 12 to 18 months), implant success was determined clinically and radiographically by 2 periodontists based on clinically observed exudates or pain, a history of exudates or pain, probing ability, and radiographic bone loss.

Descriptive statistics and 95% confidence intervals (CIs) were calculated for the bone measurements. Friedman and Dunn post hoc tests were used to compare bone widths. The level of significance was predetermined as .05.

To reach the sample size, 12 patients were enrolled. Two were excluded from the study, both during the first postsurgical month because of reasons other than graft failure (car accident and a recurrent radicular abscess in a proximal tooth). Both these patients were retreated later; however, they were excluded from the study and replaced with new patients. The remaining patients were 3 women and 7 men, with an average age of 45 years.

None of the patients showed any clinical or histologic signs of graft failure in the reentry surgery. The Friedman test showed significant trends in the ridge widths, both at 2 and 5 mm distances from the ridge crest (both P = .0001, Tables 1 and 2). The Dunn test indicated significant graft surface resorptions (between the first and second surgeries) at points 2 and 5 mm away from the crest (both P < .05, Table 3).

The percentage of newly generated (and vital) bone was 33.0% ± 11.35% (95% CI = 24.88%–41.12%, median = 30%, minimum = 20.0%, maximum = 55.0%). The percentage of residual graft material was 37.50% ± 19.04% (95% CI = 23.88%–51.12%, median = 40%, minimum = 10.0%, maximum = 60.0%). About 29.5% of the biopsy span was soft tissue or empty space. The inflammation was limited to grades 1 or zero (Table 1).

Implant success was confirmed by the lack of pain or exudates and their history, and nonprobable implants in all the patients. A 2.0 ± 0.7 mm (range = 1 to 3) of radiographic bone loss was observed 12 to 18 months after implantation.

The findings of this clinical trial suggested that the use of FDBA corticocancellous blocks might be proper for lateral ridge augmentation. After 6 months, the alveolar process reached about 4-mm gain after losing about 1.5 to 2 mm of its postaugmentation width due to the graft's surface resorption. The values reported by Amooian et al² after grafting with an FDBA corticocancellous material were slightly smaller: after 6 months, they observed about 2.3 mm width gain measured at the point 2-mm apical to the crest and about 2.7-mm gain was measured at the point 5 mm away from the crest. Both of those increases were statistically significant. Toscano et al⁵  retrospectively observed about 3.5-mm increase after use of a DFDBA corticocancellous graft. However, the measuring points were not explained in that study, nor had they been defined in a reproducible and objective manner.⁵  Therefore, both interpatient and intrapatient reliability of their measurements might have been affected by the lack of standardized gauging techniques. Furthermore, they did not perform any statistical analyses and did not report any indicators of data dispersion, which are needed for statistical comparisons; and it is not known if their results were significant or not. Acocella et al¹⁰  observed about a 4-mm increase in the ridge width following the use of mineralized cortical allograft blocks (without barrier membranes). They observed about 11% surface resorption (about 0.5 mm) in the ridge surface.¹⁰  VonArx and Buser²¹  observed about 4.6-mm bone gain 6 months after placement of 5-mm autogenous block grafts, during which about 0.4-mm surface resorption occurred. Another study carried out by Buser et al¹³  showed a 2.6 -mm increase in the alveolar ridge width after using autograft blocks. These suggest that the success of the FDBA block used in the current study to improve the ridge conditions was comparable to (or better than) that of autografts and other allografts. It should be noted, however, that the extent of surface resorption was greater in this study (1 mm within 6 months) compared with what was seen by von Arx and Buser²¹  on autografts (0.36 mm within 6 months). Neither of the two studies on corticocancellous allografts evaluated surface resorption after healing of the graft. This issue might be a potential limitation of allograft blocks and should be assessed in longer follow-ups. Nissan et al^4,7,11,19  observed about 5–6 mm horizontal bone gain after grafting with cancellous allografts, with a nonsignificant 5% or 0.5 mm postaugmentation surface resorption of the grafted ridge.^4,7,11,19  Spin-Neto et al¹⁵  reported significant increases after the use of autograft cortical blocks as well as allograft cortical and corticocancellous blocks. Of these 3 graft types, only the corticocancellous groups showed a significant surface resorption of about –8.3 ± 7.1%; in the other 2 groups, the surface thickness remained unchanged (about 1% nonsignificant increase in the width, which might be an artifact of cone-beam computerized tomography method).¹⁵

Osteogenesis occurring as a creeping substitution process, together with vascularization, is the most important basis for integration of bone grafts.^1,10  Different graft materials have shown about 14% to 58% new bone formation.²  Nissan et al⁷ reported that after augmenting with cancellous block allografts, about 44% (range = 4.5%–82%) new bone was identified, while the percentages of residual graft material and connective tissue were 29% (range= 0%–69%), and 27% (range = 1%–56%), respectively. In another study on cancellous block allografts, Nissan et al¹²  found about 33% newly formed bone, 26% residual cancellous block allograft bone, and 41% marrow and connective tissue. Geurs et al²²  evaluated allogenic powder grafts and reported about 43% space and soft tissue plus 57% bone, of which 21% was newly formed bone and 36% was remnants of the graft material.²²  Strietzel et al²³  examined a synthetic hydroxyapatite bone replacement graft and observed about 52.3% newly formed bone. Iasella et al⁹  evaluated an FDBA allograft and observed about 65% bone after 6 months, including 28% vital bone and 37% nonvital graft remnants. Spin-Neto et al³  assessed a group of allograft blocks of different types versus a group of autograft blocks in lateral ridge augmentation. According to them, the fragments of necrotic bone from autografts was about 56%, which was statistically insignificantly greater than remnants of allograft blocks (43% necrotic bone).³  In terms of new bone formation, autografts induced about 27.5% vital bone while this was significantly (P = .002) less in the case of allografts (8.5% bone).³  On the other hand, soft tissue was much more prevalent (P = .000) in allografts (about 48.5% in the allograft group versus 16.5% in the autograft group).³  Cammak et al²⁴  compared the outcome of FDBA and DFDBA allografts in both sinus and ridge augmentations and reported about 42% newly formed bone in either of the graft types.²⁴  On the other hand, another study found DFDBA (with 38.5% new bone) much more successful than FDBA (with 24.5% new bone) in increasing the amount of vital bone. DFDBA is osteoinductive and is preferred over FDBA as it might have greater osteogenic potential than FDBA.^2,25  Growth and differentiation factors might be available in DFDBA preparations although some commercially available DFDBA grafts might not show a high success rate.^1,2,25–27  The graft preparation method is only one of several determinants of graft success. Some of the other factors are the precision and cleanness of the surgery, the stabilization and intimate contact of the graft block to the recipient bed achieved by fixation screws, aggressive recipient bone preparation (eg, decortication), which might improve the availability of osteoprogenitor cells and vascularization, and the use of barrier membranes.^1,13,28,29  Also, the time of histologic examination matters as well, as it is shown that replacement of fragments of necrotic bone from the graft with the newly formed bone might be a linear function of time.^6,10  Furthermore, FDBA might be provided as corticocancellous blocks, which are stronger and more appropriate for successful clinical manipulation and screw fixation compared with cancellous blocks.²  As the only other study available on corticocancellous allografts, Amooian et al²  evaluated corticocancellous FDBA blocks similar to the ones used in this study and reported about 58.5% new bone formation plus about 4% residual graft material. In the present study, about 70.5% bone was observed in specimens biopsied from the implant socket, of which 33% was newly formed vital bone and about 37.5% was nonvital fragments of the graft. Although the rate of the newly formed bone was within the range reported by other studies, it was much less than the other study on FDBA allograft blocks reporting 58% new bone and 4% allograft remnant. Besides the aforementioned items influencing the success of a graft, the biopsy technique matters as well. Amooian et al²  horizontally obtained biopsy cores from the buccal plate and filled the new socket with allograft powder. Nevertheless, in the present study, a less aggressive method was approached by acquiring the biopsy core directly from the implant socket (through the implant drill direction). Few other studies have reported very low rates of remnant materials: Wood and Mealey²⁵  showed about 9% residual graft material in their DFDBA group and about 25.5% residual graft material in their FDBA group. Toloue et al³⁰  examined calcium sulfate with FDBA and observed 2.5% residual graft material. Nevertheless, the very low rate of remaining graft materials reported by Amooian et al² still calls for future research, especially since they transplanted graft blocks, including cortical bone, and biopsied from the buccal plate. Spin-Neto et al¹⁵  evaluated 3 different block grafts (autograft cortical, allograft cortical, and allograft corticocancellous)¹⁵ ; according to them, allograft corticocancellous and autograft cortical biopsies had about 38.2% ± 12.1% and 56.7% ± 26.0% necrotic bone, respectively; whereas, necrotic bone in allograft cortical biopsies was much greater (83.7% ± 10.8%).¹⁵  The largest amount of soft tissues was seen in corticocancellous biopsies (52.5% ± 11.7%), while soft tissue was much smaller in autograft (18.1% ± 17.1%) and allograft cortical (12.3% ± 8.5%) biopsies.¹⁵ 

This study was limited by some factors. It was better to acquire a control group of autografts in order to better evaluate the outcome of allografts. Moreover, the sample size might be argued as small. Nevertheless, it was estimated based on power calculations, and the significant results verified the validity of the sample size. Another limitation was the lack of standardization of radiographs, reducing the reliability of the assessment of radiolucency when determining the implant success. Patients' radiographs might differ from case to case, depending on overall clinical needs of each patient. For ethical concerns, we could not take radiographs solely for research purposes. Also, it would be better to determine how much of each new bone was formed over the host bone surface or within the graft block.

Within the limitations of this study, it seems that lateral ridge augmentation using corticocancellous allograft blocks might be successful both clinically and histologically. Implants placed in augmented sites might have a high 12-month success rate (as the minimum of the follow-up durations in this research). Future studies with greater sample sizes and longer follow-up durations are necessary to verify our results.

Abbreviations

AATB

American Association of Tissue Banks

DFDBA

demineralized freeze-dried bone allograft

FDBA

freeze-dried bone allograft

RSA searched the literature, conceived, designed, supervised the experiments and mentored the thesis. FS mentored the thesis. VR searched the literature, analyzed statistically and interpreted/discussed the findings, drafted/revised the article, and responded to the reviewers. BI searched the literature, selected the patients, funded the study, designed and performed the experiments/measurements, and wrote the thesis. JJ performed histopathological assessments. AT searched the literature. NA determined the sample size and performed statistical analyses.

The study was self-funded by the authors. The authors declare no conflicts of interest.