Maxillary sinus augmentation with a synthetic cell-binding peptide: histological and histomorphometrical results in humans

Bone substitutes should be used when sufficient amounts of autologous bone cannot be harvested from intraoral donor sites. P-15 is a highly conserved linear peptide with a 15 amino acid sequence identical to the sequence contained in the residues 766 to 780 of the alpha-chain of type I collagen. PepGen P-15 (Dentsply Friadent, Mannheim, Germany) is a combination of the mineral component of bovine bone (Osteograf/N 300) with P-15. Bio-Oss (Geistlich, Mannheim, Germany) is a deproteinized sterilized bovine bone with 75% to 80% porosity and a crystal size of approximately 10 microm in the form of cortical granules. The purpose of the present histological and histomorphometrical study was to compare maxillary sinus augmentation procedures in humans performed with PepGen P-15 with procedures associated with Bio-Oss and autologous bone. Seven patients participated in this study (3 men and 4 women; ages between 48 and 69 years, mean of 58 years) and were categorized into 3 groups. In group 1, a mixture of 50% autologous bone from an intraoral source and 50% Bio-Oss was used. In group 2, the graft materials used were 50% Bio-Oss and 50% PepGen P-15. In group 3, 50% autologous bone and 50% PepGen P-15 were used. Group 1 histomorphometry showed that the percentage of newly formed bone was 38.7% +/- 3.2%, marrow spaces represented 45.6% +/- 5%, and residual graft particles constituted the remaining 14.4% +/- 2.1%. Group 2 histomorphometry showed that newly formed bone represented 36.7% +/- 3.3%, marrow spaces represented 39.7% +/- 3.4%, and residual graft particles represented 19.6% +/- 2.1%. In group 3, newly formed bone represented 32.2% +/- 3.2%, marrow spaces represented 38% +/- 2.5%, and residual graft particles represented 28.8% +/- 1.1%. Non-statistically significant differences were found in the percentage of newly formed bone in the different groups (P = .360). Statistically significant differences were found in the percentage of residual graft materials among the different groups (group 1 vs groups 2 and 3) (P = .0001). These data demonstrate that the use of bone-replacement materials, without the addition of autologous bone, could be an alternative in sinus augmentation procedures. Such treatment would increase patient satisfaction, decrease surgical complications, and save the clinician substantial operating time.     

Histologic findings at augmented bone areas supplied with two different bone substitute materials combined with sinus floor lifting. Report of one case

This case report is focused on the histologic findings of bone tissue supplied with two different hydroxyapatites (HAs) used for maxillary sinus floor grafting in the same patient after various healing intervals. An insufficient unilateral sinus floor grafting with Bio-Oss biomaterial was followed by an additional grafting procedure with Algipore biomaterial performed 4 years later. Bone samples obtained during second-stage dental implantation contained the interesting combination of Bio-Oss, a bovine anorganic bone substitute, and Algipore, a porous algae-derived HA, in close vicinity, yet after different healing periods. Light microscopy exhibited satisfactory osseointegration of both grafting materials. However, Bio-Oss biomaterial showed no evidence of substantial remodeling after a healing period of 4.5 years. On the other hand, Algipore particles demonstrated signs of remodeling by being locally resorbed and partially replaced with newly formed bone already within 6 months.     

A method of sealing perforated sinus membrane and histologic finding of bone substitutes: a case report

To augment the atrophic posterior maxilla, a sinus bone graft has been widely used for sinus floor augmentation. Various bone substitutes have been developed and grafted in the maxillary sinus with and without membranes perforation, although autogenous bone is recommended as a gold standard of grafting materials. Membrane perforation is the most common complication associated with sinus bone graft. To repair a perforation, various methods have been developed. This case report is focused on histologic findings of 1 bovine hydroxyapatite (Bio-Oss; Geistlich Pharma AG, Wolhusen, Switzerland) and 2 kinds of human mineral allograft- Tutoplast cancellous microchips (TutoGen Medical GmbH, Neunkirchen am. Brand Germany), and irradiated allogeniccancellous bone and marrow (ICB; Rocky Mountain Tissue Bank, Aurora, CO) used for sinus graft in the same patient with membrane perforation after various healing periods. Mineral allograft showed favorable new bone regeneration with the repair of membrane perforation. This case report also describes a technique regarding how to repair completely perforated sinus membrane after the removal of a mucocele using human collagen membrane (Tutoplast pericardium; TutoGen Medical GmbH) and fibrin adhesive (Greenplast; Green Cross Co., Youngin, Korea) to stabilize collagen membrane.     

In vitro osteoclast resorption of bone substitute biomaterials used for implant site augmentation: a pilot study

PURPOSE: This observational study examined the resorptive behavior of normal neonatal rabbit osteoclasts grown on slices of bovine cortical bone as compared to samples of commercially available bone substitute biomaterials. It also examined the surface characteristics of these materials. MATERIALS AND METHODS: The 11 materials tested fell into 3 groups: (1) bone-derived, including freeze-dried human rib block, human demineralized freeze-dried bone, and deproteinated bovine bone; (2) synthetic hydroxyapatites (HA); and (3) synthetic non-HA, including coated methacrylates and coated silica glass. After 4 days in culture, 1 group of samples of each material underwent scanning electron microscopy (SEM) to evaluate resorptive pitting versus controls, while another group underwent tartrate-resistant acid phosphatase staining and light microscopy to examine osteoclast numbers and morphology. The 2 bovine-derived HA materials also underwent immunohistochemical staining and surface chemistry analysis. RESULTS: While most of these materials supported osteoclast attachment, some spreading, and survival in culture, only the bone-derived materials, with the exception of sintered deproteinated bovine bone, showed large scalloped-edged resorption pits with trails and exposed collagen when examined by SEM, although not to the same extent as unprocessed natural bone material. The HA materials and the sintered deproteinated bovine bone showed evidence of etching with smaller pits but no evidence of resorptive trail formation. The non-HA materials showed no evidence of pit formation or trails. Under immunohistochemical staining, Bio-Oss appeared to be positive for type I collagen after osteoclast activity on its surface, while Osteograf/N showed no positive staining. Surface chemistry analysis revealed nitrogen present in Bio-Oss specimens (0.17% to 0.47%), while there was no nitrogen detected in the Osteograf/N (0.00%); the percent nitrogen observed in normal bovine bone controls was 6.01% to 9.25%. DISCUSSION: The bone-derived materials supported osteoclast activity on the material surface in a way that facilitated formation of the more complex resorption pits in vitro. Assuming the rate of pit formation observed in vitro mimics that observed in vivo, the quantity and type of osteoclastic remodeling seen on non-bone-derived materials--and perhaps sintered bone-derived materials--would be extremely slow to negligible. Physiologic removal of non-bone-derived bone substitutes in vivo may occur by methods other than osteoclast resorption. CONCLUSIONS: Allogenous and xenogenous bone-derived materials that undergo delayed physiologic resorption may be more appropriately used with a staged surgical approach when used in sites intended to support osseointegrated dental implants. The combination of collagen staining and the presence of nitrogen suggest that there may be residual protein in Bio-Oss.     

Human histologic and histomorphometric analysis comparing OsteoGraf/N with PepGen P-15 in the maxillary sinus elevation procedure: a case report

The use of the anorganic bovine bone mineral OsteoGraf/N combined with demineralized freeze-dried bone allograft has received widespread use in sinus elevations. This composite graft material has proven to be suitable, predictable, and successful for the placement and integration of endosseous implants in the edentulous, atrophic maxilla. In this case study, the current materials and accepted methodology were compared with the latest tissue-engineered bone replacement graft material, PepGen P-15. PepGen P-15 is a combination of OsteoGraf/N and a synthetic peptide (P-15) that mimics the cell-binding domain of Type-I collagen responsible for cell migration, differentiation, and proliferation. The radiographic, histologic, and histomorphometric evaluations of the sinus grafted with PepGen P-15 showed enhanced bone formation within a shorter time interval compared with the composite graft material of OsteoGraf/N and demineralized freeze-dried bone allograft.     

Surface- and nonsurface-dependent in vitro effects of bone substitutes on cell viability

The aim of the present in vitro study was to evaluate the influence of different bone substitute materials (BSM) on the viability of human primary osteoblasts (PO), bone marrow mesenchymal cells (BMMC), and nonadherent myelomonocytic cells (U937). Six different bone substitute materials were tested: Bio-Oss Spongiosa (BOS), Tutodent Chips (TC), PepGen P-15 (P-15), Ostim (OM), BioBase (BB), and Cerasorb (CER). Cells were cultivated on comparable volumes of BSM in 96-well plates. Cell culture-treated polystyrol (Nunclon Delta surface; C) served as positive control. After 2 h and 3, 6, 10, and 14 days, viability of cells was evaluated using a standardized ATP viability assay (CellTiter Glo). Nonsurface-dependent effects of the materials were separately tested using nonadherent U937 suspension cells. For statistical analysis, the Mann-Whitney test was used. Results were considered statistically significant at P < 0.05. Cell viability of PO increased significantly on TC, C, and CER followed by BB. No changes were found for P-15 and decreasing viability for BOS and OM. BMMC showed similar results on C, TC, CER, and P-15. Lower viability for BB and no viability could be detected for BOS and OM (Mann-Whitney test, respectively). Nonadherent cells displayed increasing viability in presence of CER, BB, and BOS. No changes were observed for TC and P-15, whereas for OM, no viability was detected after a maximum cultivation period of 3 days. It was concluded that granular hydroxyapatite (HA; TC, BOS, P-15) and alpha- and beta-tricalciumphosphate (CER, BB) support, whereas nanosized HA (OM) limit or even inhibit surface- and nonsurface-related cell viability in the in vitro model used.     

Maxillary sinus augmentation using a synthetic cell-binding peptide: a histologic and transmission electron microscopy case study in man

PepGen P-15 is a combination natural anorganic bovine-derived hydroxyapatite matrix coupled with a synthetic cell-binding peptide (P-15). This material has improved bone formation in periodontal osseous defects and bone regenerative procedures. There were 3 specimens retrieved 18 months after a sinus lifting procedure using PepGen P-15. These specimens were treated to be observed under light microscopy and transmission electron microscopy. Light microscopy showed that most of the particles were surrounded by newly formed bone. In some areas, osteoid matrix was present. No acute inflammatory infiltrate was present. In transmission electron microscopy, all phases of bone formation (i.e., osteoid matrix, woven bone, and lamellar bone) were observed in the newly formed bone around the biomaterial particles. In some regions, this newly formed bone seemed to present interdigitations connecting to or entering into the particle surface. To our knowledge, this is the first report presenting data on transmission electron microscopy of PepGen P-15 used in a sinus augmentation procedure in man. Our results confirm previous reports on the clinical effectiveness of this material.     

Evaluation of the use of iliac cancellous bone and anorganic bovine bone in the reconstruction of the atrophic maxilla with titanium mesh: a clinical and histologic investigation.

The present article describes a titanium mesh procedure used for bone augmentation in the treatment of severe maxillary atrophy. A mix of iliac cancellous bone and anorganic bovine bone in a 1:1 ratio is proposed for achieving the best bone quality at the time of implant placement, which is performed 5 to 6 months after the augmentation surgery. This procedure provides for 3-stage surgery using a titanium mesh (which is removed 4 to 5 months later) to retain the cancellous bone/Bio-Oss mixture. Bone specimens taken 5 to 6 months after the augmentation procedure showed bone regeneration and the presence of vessels, indicating bone vitality.     

Healing of onlay mandibular bone grafts covered with collagen membrane or bovine bone substitutes: a microscopical and immunohistochemical study in the sheep

The objective of this study was to evaluate the role of collagen membrane and Bio-Oss coverage in healing of an onlay graft to the mandible. Twelve adult sheep each received an onlay bone graft (experiment 1), bone graft+Bio-Gide (experiment 2), and bone graft+Bio-Oss/Bio-Gide (experiment 3) on the lateral surface of the mandible. The animals were euthanized at 4, 8, 12 or 16 weeks after surgery, and findings were analysed by routine microscopy and immunohistochemistry for proliferation (Ki67) and apoptotic (Caspase-3) markers. Grafts were fully incorporated in all specimens. Pronounced resorption was observed in experiment 1. Minimal loss of graft volume was seen in experiment 2 specimens without membrane displacement. A remarkable increase in the augmented region of the mandible was observed in experiment 3. A high number of osteoclasts were expressed within the grafts during the early healing period, and thereafter declined markedly. Osteoblasts within the grafts expressed a moderate level of Ki67 at 8 weeks, which thereafter declined markedly. The strongest expression of Caspase-3 on the bone surface was observed after 16 weeks. In conclusion, the effect of collagen membrane coverage on bone graft volume maintenance was dependent on membrane stability during healing. An autogenous bone graft covered with Bio-Oss particles resulted in a remarkable increase in augmented lateral surface of the mandible. The late stage of bone graft healing was associated with a high apoptotic induction pathway of osteoblasts lining the surfaces of the new bone, demonstrated by strong positive Caspase-3 immunoreactivity.     

Vertical ridge augmentation of the atrophic posterior mandible with interpositional bloc grafts: bone from the iliac crest vs. bovine anorganic bone. Clinical and histological results up to one year after loading from a randomized-controlled clinical trial

Objectives: To compare two different techniques for vertical bone augmentation of the posterior mandible: bone blocs from the iliac crest vs. anorganic bovine bone blocs used as inlays.
Materials and methods: Ten partially edentulous patients having 5–7 mm of residual crestal height above the mandibular canal had their posterior mandibles randomly allocated to both interventions. After 4 months implants were inserted, and after 4 months, provisional prostheses were placed. Definitive prostheses were delivered after 4 months. Histomorphometry of samples trephined at implant placement, prosthesis and implant failures, any complication after loading and peri-implant marginal bone-level changes were assessed by masked assessors. All patients were followed up to 1 year after loading.
Results: Four months after bone augmentation, there was statistically significant more residual graft (between 10% and 13%) in the Bio-Oss group. There were no statistically significant differences in failures and complications. Two implants could not be placed in one patient augmented with autogenous bone because the graft failed whereas one implant and its prosthesis of the Bio-Oss group failed after loading. After implant loading only one complication (peri-implantitis) occurred at one implant of the autogenous bone group. In 16 months (from implant placement to 1 year after loading), both groups lost statistically significant amounts of peri-implant marginal bone: 0.82 mm in the autogenous bone group and 0.59 mm in the Bio-Oss group; however, there were no statistically significant differences between the groups.
Conclusions: Both procedures achieved good results, but the use of bovine blocs was less invasive and may be preferable than harvesting bone from the iliac crest.     

Histologic and histomorphometric evaluation of an implant retrieved 8 years after insertion in a sinus augmented with anorganic bovine bone and anorganic bovine matrix associated with a cell-binding peptide: a case report

Few histologic and histomorphometric reports are present in the literature regarding the peri-implant bone response around implants inserted in sinuses grafted with different biomaterials. Anorganic bovine bone (ABB) and anorganic bovine matrix with the addition of an active cell-binding peptide (PepGen P-15) are xenogenic materials that have been reported to present biocompatibility and osteoconductivity. A monolateral sinus augmentation procedure with ABB (50%) and PepGen P-15 (50%) was performed in a 54-year-old man. Two titanium implants with a sandblasted and acid-etched surface were inserted after 6 months. After an additional 6 months, a fixed prosthetic restoration was fabricated. One implant fractured in the coronal portion after an 8-year loading period and was removed using a 5-mm trephine bur. Few particles of both grafting materials were present in the peri-implant bone. No graft material particles were found in contact with the implant surface, and bone was always interposed between the graft materials and surface. No inflammatory cell infiltrate, multinucleated giant cells, or foreign body reaction cells were found. The tissues around the implant were composed of 51.4% ± 4.8% bone, 6.2% ± 0.7% ABB particles, 2.4% ± 0.5% PepGen P-15, and 40.0% ± 7.1% marrow spaces. The bone-implant contact percentage was 78.4% ± 4.1%. A sinus augmentation procedure using ABB and PepGen P-15 produced bone formation with subsequent implant osseointegration, which was still present after 8 years of implant loading.     

Cortical bone regeneration with a synthetic cell-binding peptide: a histologic and histomorphometric pilot study

PepGen P-15 is a combination natural anorganic bovine-derived hydroxyapatite matrix (ABM) coupled with a synthetic cell-binding peptide (P-15). This material has been reported to enhance bone formation in periodontal osseous defects. The aim of this study was to assess the effect of ABM/P-15 on the healing of cortical bone defects in rabbits. Five New Zealand rabbits were used. Two 8-mm bone defects were created in each tibia. Eight defects were filled with PepGen P-15, 8 defects with PepGen P-15 Flow, and 4 defects were used as a control group. A total of 20 defects were created. All rabbits were killed at 4 weeks. Block sections containing the defects were retrieved and the specimens processed for light microscopy examination. Newly formed bone was present in both test groups, whereas, in the control-group, only a scarce quantity of newly formed bone was present and the cortical defects had not been filled by the regenerated bone. Statistical evaluation showed that there were statistically significant differences between control sites and sites treated with P-15 and P-15 Flow (P = 0.0001), and also between sites treated with P-15 and P-15 Flow (P = 0.0001), respectively. No acute inflammatory infiltrate cells were visible in both of these groups. Both PepGen P-15 and PepGen P-15 Flow enhanced new bone formation in the cortical drilled defects, whereas control defects showed very little newly formed bone.     

Morphological and chemical analysis of bone substitutes by scanning electron microscopy and microanalysis by spectroscopy of dispersion energy

This study evaluated the morphological and chemical composition of the following bone substitutes: cancellous and cortical organic bovine bone with macro and microparticle size ranging from 1.0 to 2.0 mm and 0.25 to 1.0 mm, respectively; inorganic bovine bone with particle size ranging from 0.25 to 1.0 mm; hydroxyapatite with particle size ranging from 0.75 to 1.0 mm; and demineralized freeze-dried bone allograft with particle size ranging from 0.25 to 0.5 mm. The samples were sputter-coated with gold in an ion coater, the morphology was observed and particle size was measured under vacuum by scanning electron microscopy (SEM). The chemical composition was evaluated by spectroscopy of dispersion energy (EDS) microanalysis using samples without coating. SEM analysis provided visual evidence that all examined materials have irregular shape and particle sizes larger than those informed by the manufacturer. EDS microanalysis detected the presence of sodium, calcium and phosphorus that are usual elements of the bone tissue. However, mineral elements were detected in all analyzed particles of organic bovine bone except for macro cancellous organic bovine bone. These results suggest that the examined organic bovine bone cannot be considered as a pure organic material.     

3种骨代用品修复骨缺损的组织学定量分析

目的探讨无机牛骨(Bio-Oss),携带羧氨基的无机牛骨(PepGen P-15TM)及人工合成磷酸三钙(Cera-sorb)的骨缺损修复能力。方法健康成年雄性杂种犬18只,在两侧下颌骨制备4个直径9 mm、深3 mm的骨缺损,把3种骨代用品Bio-Oss,Cerasorb和PepGen P-15TM随机植入3个骨缺损中,分别作为Bio-Oss组、Cera-sorb组和PepGen P-15TM组;剩余1个骨缺损不植入骨代用品,作为空白对照组。术后1、3、6个月取样制片,以组织形态计量学法分析骨缺损修复率。结果 1、3、6个月时3个骨代用品组骨缺损区新骨形成率均高于空白对照(P<0.05);1个月时PepGen P-15TM组骨缺损新骨形成率(24.77%)高于Cerasorb组(15.33%)和Bio-Oss组(16.22%)(P<0.05);6个月时PepGen P-15TM组存留率高于Cerasorb组和Bio-Oss组(P<0.05)。结论3种骨代用品均具有骨缺损修复能力,且PepGen P-15TM的早期骨修复能力强于Bio-Oss及Cerasorb,但3者的长期骨修复能力无明显差异。3种材料中PepGen P-15TM存留率最高,但不影响新骨形成。     

Comparison of osteogenic potential between apatite-coated poly(lactide-co-glycolide)/hydroxyapatite particulates and Bio-Oss

Previously, we developed a poly(lactide-co-glycolide)/nano-hydroxyapatite (PLGA/HA) composite that overcame the limitations of conventional ceramic bone substitutes. This was achieved by introducing a bone-like apatite layer on the composite to further enhance its osteogenic potential. In this study, we compared the osteogenic potential of the apatite-coated PLGA/HA particulates to that of Bio-Oss, a deproteinized bovine bone material. A mixture of fibrin gel and either apatite-coated PLGA/HA particulates or Bio-Oss was implanted into critical-size rat calvarial defects. As a control, fibrin gel was implanted alone into the defects. At eight weeks after treatment, histological examination showed new bone formation around the grafting materials, and bone formation was similar between the two groups. In the control group, bone was not regenerated and the defects were filled with fibrous tissues. This study showed that a synthetic bone graft material, apatite-coated PLGA/HA particulates, had a comparable bone regeneration potential to the bovine-derived bone graft material, Bio-Oss.     

Comparison of inorganic bovine bone mineral particles with porous hydroxyapatite granules and cranial bone dust in the reconstruction of full-thickness skull defect.

Twenty adult rabbits were used to evaluate the biocompatibility and osteoconductivity of Bio-Oss, an inorganic bovine bone mineral, in the reconstruction of full-thickness skull defects. Skull defects were treated with either autogenous bone dust, porous hydroxyapatite granules, Bio-Oss particles, or were left untreated as controls. Histological examination of decalcified sections showed incorporation of Bio-Oss into the host tissue without a significant inflammatory reaction. Measurement of the profile area occupied by the bone revealed that Bio-Oss, hydroxyapatite, and the control had the same amount of bone ingrowth, whereas autogenous bone dust produced a greater amount of bone (p < 0.01). We conclude that Bio-Oss, like porous hydroxyapatite, has sufficient osteoconductive properties and can also be used as a bone substitute material.     

Human histologic and electromicroscopic analysis with synthetic peptide enhanced hydroxyapatite in the maxillary sinus elevation procedure: a case report

Elevation of the sinus floor to augment the alveolar process to place implants is an effective treatment. The traditional use of autologous bone implies a secondary donor site and the related morbidity, and, hence, is not readily accepted by many patients. The new graft material, synthetic peptide enhanced hydroxyapatite (PepGen P-15 Flow, Ceramed Dental, Lakewood, CO, DENTSPLY Int.,York, PA), shows the ease in use and ability to generate new bone growth in the sinus elevation procedure. Histologic and electromicroscopic analysis was performed on the new bone formed after the application.     

Shh修饰骨髓间充质干细胞与不同骨移植材料复合的体外观察

目的：研究两种不同骨移植材料对Shh基因修饰的骨髓间充质干细胞（MSCs）生长和增殖活性的影响。方法：差异贴壁法培养大鼠MSCs,携带音狸因子（Shh）腺相关病毒2型（rAAV2-Shh）转导MSCs,然后与两种不同的骨移植材料—聚D,L-乳酸/羟基磷灰石（PDLLA/HA）和β-磷酸三钙（β-TCP）体外复合。MTT法检测MSCs的增殖活性和黏附能力。扫描电镜观察MSCs在骨移植材料上的生长情况。结果：β-TCP组黏附和增殖活性均强于PDLLA/HA组（P〈0.05）。此结果在扫描电镜观察中得到同样的证实。结论：与PDLLA/HA比较,β-TCP具有更良好的生物相容性。     

Sinus grafting using recombinant human tissue factor, platelet-rich plasma gel, autologous bone, and anorganic bovine bone mineral xenograft: histologic analysis and case reports

PURPOSE: The purpose of this study was to analyze healthy bone formation by means of histology and immunohistochemistry after grafting with a mixture of autologous ground calvarial bone, inorganic xenograft, platelet-rich plasma (PRP), and recombinant human tissue factor (rhTF). MATERIALS AND METHODS: Maxillary sinus floor augmentation was performed on 3 patients by grafting with 5 to 10 mL of a paste consisting of autologous powder from calvarial bone (diameter < 1 mm), 50% v/v anorganic bovine bone mineral xenograft (PepGen P-15, a new tissue-engineered bone replacement graft material), PRP (1.8 x 10(6) platelets/mm3 plasma), and about 1 microg rhTF. Six and 10 months after grafting, bone cores were extracted for implant fixation and analyzed. RESULTS: Histology demonstrated a high degree of inorganic xenograft integration and natural bone regeneration. Both the xenograft and newly synthesized bone were colonized with osteocytes and surrounded by osteoblasts. Six-month-old bone cores demonstrated a ratio of synthesized bone to xenograft particles ratio of 0.5, whereas 10-month-old cores demonstrated a ratio of 2. A low degree of inflammation could also be observed using S100A8 immunohistochemistry. DISCUSSION: Autologous grafting in edentulous patients is a complex procedure; the successful substitution of synthetic analogs for ground bone is a major challenge. CONCLUSION: In this investigation, it was shown that inorganic xenograft in the harvested bone paste could be safe for patients and had high bone regeneration capacity over time. The sinus graft showed intense bone formation 6 months after grafting and a further increase in bone growth 10 months after grafting.