The bone tissue responses to prehydrated and collagenated cortico-cancellous porcine bone grafts: a study in rabbit maxillary defects

Background: Bone substitutes should have osteoconductive properties and be completely replaced with new bone with time. Adding collagen gel to prehydrated and collagenated porcine bone (PCPB) particles results in a sticky and moldable material which facilitates clinical handling. However, the possible influence of the gel on the bone tissue response is not known. Purpose: The objective of the study was to evaluate the bone tissue responses to PCPB graft with or without collagen gel and to evaluate the resorption/degradation properties of the biomaterials. Materials and Methods: Fourteen rabbits were used in the study. Bilateral bone defects, 5 × 8 × 3 mm, were created in the maxilla and filled with PCPB + collagen gel (test) or with PCPB only (control) and covered with a collagen membrane. Animals were killed after 2 (n = 3), 4 (n = 3), and 8 weeks (n = 8) for histological and morphometrical evaluations. Results: There were no differences between test and control defects. Both materials showed bone formation directly on the particles by typical osteoblastic seams. The bone area increased with time (2–8 weeks) for both sides, from 16.2% (control) and 19.2% (test) to 42.7 and 43.8%, respectively. The PCPB, whether mixed with collagen gel or not, was resorbed by osteoclasts as well as part of remodeling with the formation of osteons within the particles. Morphometry showed a decrease of PCPB area from 19.4% (control) and 23.8% (test) after 2 weeks to 3.7 and 9.3% after 8 weeks, respectively. Conclusions: Mixing collagen gel and PCPB to facilitate the clinical handling does not influence the bone tissue responses to the material, which exhibited osteoconductive properties and was resorbed with time.     

ACP and Surgicel in bone hemostasis. A comparative experimental and histologic study

The search for a biodegradable material which may prevent or stop bleeding in bone tissue and ensure healing is of continuous interest. ACP, an absorbable collagen paste, is a new hemostatic agent. The present study was devised to compare ACP with another commonly used hemostatic agent, Surgicel, in order to determine the tissue response during bone healing. The materials were implanted in the mandibular bone of New Zealand white rabbits. The observation times were 1, 2, 4 and 8 weeks. The result showed that the healing was uneventful for both the hemostatic agents even though healing was slightly retarded compared to natural healing. Both the hemostatic agents were completely resorbed within 4-8 weeks and also the bone healing was completed after this observation period.     

Paste-like inorganic bone matrix: preclinical testing of a prototype preparation in the porcine calvaria

Objective: To evaluate the osteoconductive properties and the volume stability of an injectable paste‐like inorganic bone matrix (PBM) in porcine calvaria defects. Material and methods: We created six circumferential defects in the calvaria of 12 adult iberico pigs. The defects were filled with either PBM, Bio‐Oss® of different particle size, carrier alone, or left empty. PBM was composed of Bio‐Oss® with a particle size ranging from 250 to 500 μm and a hydrogel‐carrier of carboxymethylcellulose and collagen. After 6 and 12 weeks of healing, the animals were sacrificed and undecalcified ground sections were prepared and subjected to histologic and histomorphometric analysis. To quantify the osteoconductive properties of PBM, bone volume per tissue volume (BV/TV) in the defect area was determined. To determine the volume stability, bone substitute volume per tissue volume (BSV/TV) was measured. Results: After 6 weeks, PBM particles in the center of the defect were surrounded by fibrous connective tissue, which was later replaced by bone. BV/TV in the PBM group increased from 29.7±12.7% (minimum 12.2%, maximum 43.7%) after 6 weeks to 43.9±14.9% (minimum 27.8%, maximum 63.9%) after 12 weeks (Mann–Whitney test; P=0.6). According to the Friedman test, BV/TV in groups containing Bio‐Oss® of different particle sizes, the carrier and the empty defects was similar to the results obtained with PBM (6 weeks P=0.8; 12 weeks P=0.22). BSV/TV in the PBM group was stable over time, with 10.1±9% (minimum 3.3%, maximum 27.6%) and 16.5±12.9% (minimum 1%, maximum 32.7%), after 6 and 12 weeks, respectively (P=0.72). BSV/TV in the PBM group was comparable to the results obtained with the Bio‐Oss® particles of different sizes (Friedman test; 6 weeks P=0.0503; 12 weeks P=0.56). Conclusions: The results of this preclinical study showed that the PBM is osteoconductive and maintains the augmented volume, similar to commercial Bio‐Oss®. These data suggest that the osteoconductive properties of Bio‐Oss® are maintained at the smaller particle size and in the presence of the carrier.     

The evaluation of processed cancellous bovine bone as a bone graft substitute

OBJECTIVE: To determine the ability of a novel bovine cancellous bone xenoimplant to act as an osteoconductive graft in an ovine femoral defect model. An autograft harvested from the xenoimplant site was placed in a contralateral limb defect for comparison. MATERIAL AND METHODS: The xenoimplant used had been rendered immunologically inert by a novel defatting and deproteinating process. Following surgical implantation of cores into condylar cancellous bone defects, fluorochrome labels were administered to 12 sheep at 2 1/2, 4 1/2 and 8 weeks. Incorporation of the xenoimplants and autografts into the host bone was compared radiographically and histomorphometrically at 10 weeks. RESULTS: Radiographically, the degree of osteointegration was comparable. Histomorphometric data, consisting of labelled surface (LS) estimates, confirmed osteoconductive properties of both the xenoimplants and autografts. Remodelling activity was greatest in the xenoimplants at 2 1/2, weeks. At 4 1/2 weeks, there was more activity in the autograft, but by 8 weeks they were performing similarly. Xenoimplant-LS estimates were comparable or greater than those of the autograft at all times. CONCLUSIONS: Processed bovine cancellous bone xenoimplants were osteoconductive in this model and show promise for development as a biomaterial in human and veterinary orthopaedic surgery.     

Utilization of autogenous bone, bioactive glasses, and calcium phosphate cement in surgical mandibular bone defects in Cebus apella monkeys

PURPOSE: The purpose of the present study was to evaluate the histologic results of bone cavities that were surgically created in the mandibles of Cebus apella monkeys and filled with autogenous bone, PerioGlas, FillerBone, or Bone Source. MATERIALS AND METHODS: Surgical cavities 5 mm in diameter were prepared through both mandibular cortices in the mandibular angle region. The cavities were randomly filled, and the animals were divided into groups according to the material employed: Group 1 cavities were filled with autogenous corticocancellous bone; group 2 cavities were filled with calcium phosphate cement (BoneSource); and group 3 and group 4 cavities were filled with bioactive glass (FillerBone and PerioGlas, respectively). After 180 days the animals were sacrificed, and specimens were prepared following routine laboratory procedures for hematoxylin/eosin staining and histologic evaluation. RESULTS: The histologic analysis showed that autogenous bone allowed total repair of the bone defects; bioactive glasses (FillerBone and PerioGlas) allowed total repair of the defects with intimate contact of the remaining granules and newly formed bone; and the cavities filled with calcium phosphate cement (BoneSource) were generally filled by connective fibrous tissue, and the material was almost totally resorbed. DISCUSSION: The autogenous bone, FillerBone, and PerioGlas provided results similar to those in the current literature, showing that autogenous bone is the best choice for filling critical-size defects. Synthetic implanted materials demonstrated biocompatibility, but the bio-glasses demonstrated osteoconductive activity that did not occur with calcium phosphate (Bone-Source). CONCLUSION: According to the methodology used in this study, it can be concluded that the utilization of autogenous bone and bioactive glasses permitted the repair of surgically created critical-size defects by newly formed bone; the synthetic implanted materials demonstrated biocompatibility, and the bioactive glasses demonstrated osteoconductive activity. The PerioGlas was mostly resorbed and replaced by bone and the remaining granules were in close contact with bone; the FillerBone showed many granules in contact with the newly formed bone; BoneSource did not permit repair of the critical-size defects, and the defects were generally filled by connective fibrous tissue.     

Tissue engineering of bone: search for a better scaffold

BACKGROUND: Large bone defects still represent a major problem in orthopedics. Traditional bone-repair treatments can be divided into two groups: the bone transport (Ilizarov technology) and the graft transplant (autologous or allogeneic bone grafts). Thus far, none of these strategies have proven to be always resolving. As an alternative, a tissue engineering approach has been proposed where osteogenic cells, bioceramic scaffolds, growth factors and physical forces concur to the bone defect repair. Different sources of osteoprogenitor cells have been suggested, bone marrow stromal cells (BMSC) being in most cases the first choice. METHODS AND RESULTS: In association with mineral tridimensional scaffolds, BMSC form a primary bone tissue which is highly vascularized and colonized by host hemopoietic marrow. The chemical composition of the scaffold is crucial for the osteoconductive properties and the resorbability of the material. In addition, scaffolds should have an internal structure permissive for vascular invasion. Porous bioceramics [hydroxyapatite (HA) and tricalcium phosphate] are osteoconductive and are particularly advantageous for bone tissue engineering application as they induce neither an immune nor an inflammatory response in the implanted host. Earlier, we first reported a cell-based tissue engineering procedure to treat three patients with long bone segmental defects. Cells were loaded on a 100% HA porous ceramic. These scaffolds proved to have good osteoconductive properties resulting in a good functional recovery, but they have not been resorbed after more than 5 years from the implant. In addition, due to the high density of the mineral and the relatively low porosity (50-60%), it was very difficult to monitor the patient recovery during the post-surgery time using X-rays. CONCLUSIONS: We report here some pre-clinical testing of new scaffolds. To compare these second generation ceramic scaffolds more suitable for a tissue engineering approach we had to first establish animal models and analysis procedures including the use of X-ray-computed microtomography associated with X-rays synchroton radiation.     

Evaluation of a novel biphasic calcium phosphate in standardized bone defects: a histologic and histomorphometric study in the mandibles of minipigs

OBJECTIVE: A novel biphasic calcium phosphate (CaP) granulate consisting of hydroxyapatite (HA) and beta-tricalciumphosphate (TCP) was compared with pure HA and pure TCP and with autograft as positive control. MATERIALS AND METHODS: Four standardized bone defects were prepared in both mandibular angles of 16 minipigs and grafted with autogenous bone chips, HA, HA/TCP (60% : 40%), or TCP. Histologic and histomorphometric analysis of bone formation and graft degradation followed healing periods of 2, 4, 8, and 24 weeks. RESULTS: 2 weeks: more bone formation in defects filled with autograft than with the three CaP materials (P<0.05). 4 weeks: bone formation differed significantly (P<0.05) between all four materials (autograft>TCP>HA/TCP>HA). 8 weeks: more bone formation in defects with autograft and TCP than with HA/TCP (P<0.05), and HA/TCP had more bone formation than HA (P<0.05). 24 weeks: no difference in bone formation between the groups. Autograft and TCP resorbed quickly and almost completely over 8 weeks, whereas HA/TCP and HA showed limited degradation over 24 weeks. CONCLUSION: All defects healed with mature lamellar bone and intimate contact between bone and the remaining graft material. The rate of bone formation corresponded to the content of TCP in the CaP materials.     

A histologic and histomorphometric evaluation of anorganic bovine bone retrieved 9 years after a sinus augmentation procedure

BACKGROUND: Anorganic bovine bone (ABB) has been shown to have osteoconductive properties and no inflammatory or adverse responses as grafting materials used in sinus augmentation procedures. Despite these successful results, histologic data in humans over the long-term period are scarce. The purpose of this study was to analyze the histomorphometric data 9 years after surgery in a case of maxillary sinus augmentation using ABB. METHODS: The histologic evaluation was performed in five different thin sections of the specimen, comparing histomorphometric measures for newly formed bone, marrow spaces, biomaterial particles remnants, and number of osteocytes embedded in both trabecular bone and bone tissue near the ABB. The investigation was carried out by means of scanning electron microscopy and brightfield and circularly polarized light microscopy. RESULTS: We observed a mean amount of newly formed bone of 46.0% +/- 4.67%, ABB remnants of 16.0% +/- 5.89%, and marrow spaces of 38.0% +/- 8.93%. The osteocyte index was 4.43 for bone around ABB and 3.27 in the trabecular bone at a distance from the particles. CONCLUSIONS: After 9 years, the tissue pattern appeared composed by residual ABB particles in close contact to the newly formed bone. The bone mineralized matrix around the ABB had collagen fibers randomly oriented and more osteocytes embedded. The results demonstrate both a high level of osteoconductivity and a "biomimetic" behavior over the long term.     

Ten-year follow-up in a maxillary sinus augmentation using anorganic bovine bone (Bio-Oss). A case report with histomorphometric evaluation

Several bone grafting materials have been used in sinus augmentation procedures. Bio-Oss (deproteinized and sterilized bovine bone) has shown to have osteoconductive properties and no inflammatory or adverse responses have been published. In spite of these successful results, histologic data regarding bone augmentation using Bio-Oss in humans is scarce. The purpose of this study was to analyse the amount of Bio-Oss ossification in a case of maxillary sinus augmentation, recording and comparing histomorphometric data 8 months, 2 and 10 years after surgery. This long-term histologic evaluation of retrieved specimens has been performed, comparing histomorfometric measures at different times. Eight months after surgery we observed in 20 different thin sections of the specimen a mean amount of bone tissue (including medullar spaces) of 29.8% (and 70.2% of Bio-Oss) +/- 2.6. At 2 years the bone tissue increased to 69.7% + 2.7 and 10 years after surgery it was 86.7% +/- 2.8. The comparison of the means for each time has shown a highly significant increasing trend in bone formation associated with Bio-oss resorption: at 8 months, 2 and 10 years.     

Tissue response to composite ceramic hydroxyapatite/demineralized bone implants

This study evaluated the tissue reactions to two materials: ceramic hydroxyapatite (CHA), and a composite material of demineralized bone powder (DBP) and CHA (ratio of 4:1) in a collagen vehicle. The materials were tested in a subcutaneous pocket, a mandibular onlay, and in a calvarial onlay model. Specimens were evaluated histologically at 7, 10, 14, and 21 days postimplantation. Ceramic hydroxyapatite, implanted subcutaneously, elicited a fibrous response with minimal inflammation, but did not induce bone formation. In specimens of subcutaneously implanted composite material, induced bone was evident in association with the DBP. In CHA onlay specimens, there was a small amount of reactive bone extending from the host bone into the implant. In composite onlays, bone filled the entire body of the implant. The results of this study indicate that CHA particles were not osteoinductive in heterotopic sites and that osteoconductive ingrowth was minimal in onlays. Bone was induced by DBP even when mixed with CHA particles and implanted in subcutaneous and intraosseous sites. It was concluded that composite implants may provide a means of combining the osteoinductive properties of DBP with the bulk and structural support of osteoconductive CHA particles.     

Bone-implant contact at calcium phosphate-coated and porous titanium oxide (TiUnite)-modified oral implants

BACKGROUND: Calcium phosphate (CP)-coated implants are usually referred to as having osteoconductive properties, whereas titanium implants with a native oxide layer are considered less osteoconductive. Often smooth titanium oxides (TOs) are compared to relatively rough CP structures. The objective of this study was to evaluate osteoconduction by comparing bone-implant contact at a relatively smooth, highly crystalline CP coating with a structured, porous TO (TiUnite)-modified surface. MATERIAL AND METHODS: Ten adult Hound Labrador mongrel dogs were used. Four titanium implants (Nobel Biocare) with CP-coated (2) or TO-modified (2) surfaces were installed 12 weeks following mandibular premolar and molar teeth extraction. The implants were alternated within and between jaw quadrants in consecutive animals. Mucosal flaps were advanced and sutured leaving the implants in a submerged position. The animals were injected with fluorescent bone labels at 3 and 4 weeks postsurgery, and pre-euthanasia to monitor progress of bone formation. The animals were euthanized at 8 weeks postsurgery and block biopsies were prepared for histologic and histometric analysis. RESULTS: There were no remarkable differences in bone formation and apparent bone-implant contact comparing the TO-modified and CP-coated surfaces. However, the measured average bone-implant contact was 71% and 57% (P=0.027) for TO-modified and CP-coated implants, respectively. CONCLUSIONS: We conclude that the TO surface exhibits osteoconductive properties exceeding that of the CP surface. One or several of the chemical and physical properties of the TO surface may result in the remarkable bone formation along its surface. This study indicated that crystallinity and/or chemistry may be important.     

Superior effect of MD05, beta-tricalcium phosphate coated with recombinant human growth/differentiation factor-5, compared to conventional bone substitutes in the rat calvarial defect model

BACKGROUND: MD05 consists of beta-tricalcium phosphate (beta-TCP) coated with recombinant human growth/differentiation factor-5 (rhGDF-5) and is under evaluation as an osteoinductive and osteoconductive bone graft material for use in dental and maxillofacial applications. The objective of this study was to compare the bone regenerative properties of MD05 with those of conventional commercially available bone substitutes. METHODS: Full-thickness, 6-mm diameter, calvarial critical-size defects (two per animal) were created in adult Sprague-Dawley rats. Groups of rats were implanted with the following: 1) MD05; 2) bovine bone mineral; 3) bovine bone mineral with collagen; 4) bovine bone mineral with synthetic peptide, 5) beta-TCP (from two different manufacturers); or 6) no filling material (sham controls). Blinded macroscopic analysis, histopathologic analysis, and histomorphometric analysis were carried out 6 weeks after implantation. RESULTS: New bone formation assessed histomorphometrically was about five times greater with MD05 than with the other bone substitutes tested, and bone repair was well advanced in MD05-filled defects after 6 weeks. The extent of fibrous tissue and residual implant were significantly lower in the MD05 group. In contrast to the other materials, the use of MD05 was associated with the complete osseous bridging of the defect and with the presence of normal bone marrow. The osteoinductive effect of rhGDF-5 was apparent from the more pronounced bone ingrowth observed with MD05 compared to the beta-TCP carrier alone. All implants showed good biocompatibility. CONCLUSION: MD05 achieved superior bone regeneration compared to conventional materials and is a promising new bone substitute for dental and maxillofacial applications.     

Bone healing and graft resorption of autograft, anorganic bovine bone and beta-tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs

OBJECTIVE: The purpose was to qualitatively and quantitatively compare the bone formation and graft resorption of two different bone substitutes used in both orthopedic and oral surgery, with autogenous bone as a positive control. MATERIALS AND METHODS: Three standardized bone defects were prepared in both mandibular angles of 12 adult minipigs. The defects were grafted with either autograft, anorganic bovine bone (ABB), or synthetic beta-tricalcium phosphate (beta-TCP). Sacrifice was performed after 1, 2, 4, and 8 weeks for histologic and histomorphometric analysis. RESULTS: At 2 weeks, more new bone formation was seen in defects filled with autograft than with ABB (P approximately 0.0005) and beta-TCP (P approximately 0.002). After 4 weeks, there was no significant difference between beta-TCP and the two other materials. Defects grafted with ABB still exhibited less bone formation as compared with autograft (P approximately 0.004). At 8 weeks, more bone formation was observed in defects grafted with autograft (P approximately 0.003) and beta-TCP (P approximately 0.00004) than with ABB. No difference could be demonstrated between beta-TCP and autograft. beta-TCP resorbed almost completely over 8 weeks, whereas ABB remained stable. CONCLUSION: Both bone substitutes seemed to decelerate bone regeneration in the early healing phase as compared with autograft. All defects ultimately regenerated with newly formed bone and a developing bone marrow. The grafting materials showed complete osseous integration. Both bone substitutes may have a place in reconstructive surgery where different clinical indications require differences in biodegradability.     

Osteogenesis of hydroxyapatite and tricalcium phosphate used as a bone substitute.

Hydroxyapatite (HA) and tricalcium phosphate (TCP) are useful for grafting and augmentation of bone tissue. In this study, conventional and histochemical transmission electron microscopy were used to study osteogenic events at the interface between the implanted materials and adjacent tissue from 1 to 4 weeks postoperatively. The microscopic results indicated that TCP was resorbed more rapidly than HA after implantation, with a notable breakdown of material and replacement by mesenchymal cells with ultrastructural features resembling osteoprogenitor cells and collagen up to 4 weeks postoperatively. Alkaline phosphatase and acid phosphatase reactivity in the tissues helped to identify and differentiate the histologic differences observed between HA and TCP.     

珊瑚转化多孔羟基磷灰石复合rhBMP-2人工骨的动物实验研究

目的：寻找新的骨代用材料。方法：采用珊瑚转化多孔羟基磷石（ＣＨＡ）作为移植物支架,用Ｉ型胶原作为ｒｈＢＭＰ－２的缓释载体及赋形剂,将三者复合制成新型人工骨,进行了大鼠标准颅骨缺损骨移植实验。结果：ＣＨＡ／胶原具有良好的生物相容性和骨引导作用。ｒｈＢＭＰ－２／ＣＨＡ／胶原具有骨引导和骨诱导双重特性,其成骨效应明显优于单纯ＣＨＡ／胶原植入。结论：该复合人工骨有可能成为临床修复骨缺损的有效材料。     

Collagen gel and membrane in guided tissue regeneration in periodontal fenestration defects in dogs

Abstract The effect of a collagen gel matrix as a submembranous space-maintaining material was evaluated in guided tissue regeneration procedures. In 4 dogs, contralateral surgical circular fenestration defects, 5 mm in diameter, were produced at the midbuccal aspect of the alveolar bone in 8 maxillary canines. Removal of bone, PDL and cementum was complete. Experimental sites were filled with collagen gel and covered with collagen membranes; control sites were covered with collagen membranes and the underlying space was spontaneously filled with blood. Mucogingival flaps were repositioned. Histological and histomorphometric observations, 6 weeks post-surgery, indicated that defects covered by collagen membranes presented the most impressive regeneration with almost complete coverage of the denuded root by new cementum (98.4%) and new bone (63.2%). In the experimental defects. 83.5% coverage of new cementum with only 21.9% new bone regeneration was observed. These results suggest that collagen gel. interfered with healing by PDL and bone-derived cells in the submembranous space.     

Histomorphometric analysis of rat alveolar wound healing with hydroxyapatite alone or associated to BMPs

Several materials and techniques have been proposed to improve alveolar wound healing and decrease loss of bone height and thickness that normally follow dental extraction. The objective of this research was the histologic analysis of bone morphogenetic proteins implanted into dental alveoli of rats after extraction. A total of 45 adult male Wistar rats were divided into three groups of 15 animals each: control (no treatment), implanted with pure hydroxyapatite (HA, 3 mg) and implanted with hydroxyapatite plus bone morphogenetic proteins (HA/BMPs, 3 mg). Five animals from each group were sacrificed at 7, 21 and 42 days after extraction for the histometric analyses of the osteoconductive potential of hydroxyapatite associated or not with BMPs. After dissection, fixation, decalcification and serial microtomy of 6-micron thick sections, the samples were stained with hematoxylin-eosin for histologic and histometric analyses. Both HA and HA/BMPs caused a delay in wound healing compared to control animals, evaluated by the percentage of bone tissue in the alveoli. The treatment with HA/BMPs had the greatest delay at 21 days, even though it produced values similar to the control group at 42 days. The materials did not improve alveolar repair in the normal period of wound healing and the association of HA/BMPs did not have osteoconductive properties with granulated hydroxyapatite as the vehicle.     

Tomodensitometric and histologic evaluation of the combined use of a collagen membrane and a hydroxyapatite spacer for guided bone regeneration: a clinical report

In this report, the problems of insufficient bone and soft tissue after extraction of maxillary incisors were addressed concurrently prior to endosseous implant placement, by combining the use of a diphenylphosphorylazide-cross-linked Type I collagen membrane and a resorbable space-making biomaterial composed of 200-micron porous hydroxyapatite granules blended in Type I collagen and chondroitin-4-sulfate. Upon flap reflection 8 months postsurgery, the horizontal deficiencies were almost completely resolved, membranes completely resorbed and the defects filled with hard, bonelike tissue, with a few superficial hydroxyapatite granules. Histologic evaluation of the bone biopsies obtained at the implantation sites revealed dense, well-reconstructed alveolar bone with a few traces of hydroxyapatite granules that had been completely resorbed. Tomodensitometric evaluation indicated that bone regeneration ranged from 14% to 58%, with an average bone gain of 29.77%. Four nonsubmerged ITI titanium implants placed in the augmented bone have been in function for more than 5 years, with no clinical or radiographic signs of hard or soft tissue breakdown. Bacterial sampling at dental sites with periodontitis 1 month prior to periodontal therapy and at implant sites for up to 30 months demonstrated rapid colonization of implant surfaces by periodontopathogens without causing any detrimental effect to implant integration.