Pre-clinical in vivo models for the screening of bone biomaterials for oral/craniofacial indications: focus on small-animal models.

Preclinical in vivo experimental studies are performed for evaluating proof-of-principle concepts, safety and possible unwanted reactions of candidate bone biomaterials before proceeding to clinical testing. Specifically, models involving small animals have been developed for screening bone biomaterials for their potential to enhance bone formation. No single model can completely recreate the anatomic, physiologic, biomechanic and functional environment of the human mouth and jaws. Relevant aspects regarding physiology, anatomy, dimensions and handling are discussed in this paper to elucidate the advantages and disadvantages of small-animal models. Model selection should be based not on the 'expertise' or capacities of the team, but rather on a scientifically solid rationale, and the animal model selected should reflect the question for which an answer is sought. The rationale for using heterotopic or orthotopic testing sites, and intraosseous, periosseous or extraskeletal defect models, is discussed. The paper also discusses the relevance of critical size defect modeling, with focus on calvarial defects in rodents. In addition, the rabbit sinus model and the capsule model in the rat mandible are presented and discussed in detail. All animal experiments should be designed with care and include sample-size and study-power calculations, thus allowing generation of meaningful data. Moreover, animal experiments are subject to ethical approval by the relevant authority. All procedures and the postoperative handling and care, including postoperative analgesics, should follow best practice.

OsteoMacs: Key players around bone biomaterials.

Osteal macrophages (OsteoMacs) are a special subtype of macrophage residing in bony tissues. Interesting findings from basic research have pointed to their vast and substantial roles in bone biology by demonstrating their key function in bone formation and remodeling. Despite these essential findings, much less information is available concerning their response to a variety of biomaterials used for bone regeneration with the majority of investigation primarily focused on their role during the foreign body reaction. With respect to biomaterials, it is well known that cells derived from the monocyte/macrophage lineage are one of the first cell types in contact with implanted biomaterials. Here they demonstrate extremely plastic phenotypes with the ability to differentiate towards classical M1 or M2 macrophages, or subsequently fuse into osteoclasts or multinucleated giant cells (MNGCs). These MNGCs have previously been characterized as foreign body giant cells and associated with biomaterial rejection, however more recently their phenotypes have been implicated with wound healing and tissue regeneration by studies demonstrating their expression of key M2 markers around biomaterials. With such contrasting hypotheses, it becomes essential to better understand their roles to improve the development of osteo-compatible and osteo-promotive biomaterials. This review article expresses the necessity to further study OsteoMacs and MNGCs to understand their function in bone biomaterial tissue integration including dental/orthopedic implants and bone grafting materials.

VEGF incorporated into calcium phosphate ceramics promotes vascularisation and bone formation in vivo

Bone formation and osseointegration of biomaterials are dependent on angiogenesis and vascularization. Angiogenic growth factors such as vascular endothelial growth factor (VEGF) were shown to promote biomaterial vascularization and enhance bone formation. However, high local concentrations of VEGF induce the formation of malformed, nonfunctional vessels. We hypothesized that a continuous delivery of low concentrations of VEGF from calcium phosphate ceramics may increase the efficacy of VEGF administration.VEGF was co-precipitated onto biphasic calcium phosphate (BCP) ceramics to achieve a sustained release of the growth factor. The co-precipitation efficacy and the release kinetics of the protein were investigated in vitro. For in vivo investigations BCP ceramics were implanted into critical size cranial defects in Balb/c mice. Angiogenesis and microvascularization were investigated over 28 days by means of intravital microscopy. The formation of new bone was determined histomorphometrically. Co-precipitation reduced the burst release of VEGF. Furthermore, a sustained, cell-mediated release of low concentrations of VEGF from BCP ceramics was mediated by resorbing osteoclasts. In vivo, sustained delivery of VEGF achieved by protein co-precipitation promoted biomaterial vascularization, osseointegration, and bone formation. Short-term release of VEGF following superficial adsorption resulted in a temporally restricted promotion of angiogenesis and did not enhance bone formation. The release kinetics of VEGF appears to be an important factor in the promotion of biomaterial vascularization and bone formation. Sustained release of VEGF increased the efficacy of VEGF delivery demonstrating that a prolonged bioavailability of low concentrations of VEGF is beneficial for bone regeneration.

The effect of a slow mode of BMP-2 delivery on the inflammatory response provoked by bone-defect-filling polymeric scaffolds

We investigated the inflammatory response to, and the osteoinductive efficacies of, four polymers (collagen, Ethisorb, PLGA and Polyactive) that bore either an adsorbed (fast-release kinetics) or a calcium-phosphate-coating-incorporated (slow-release kinetics) depot of BMP-2. Titanium-plate-supported discs of each polymer (n = 6 per group) were implanted at an ectopic (subcutaneous) ossification site in rats (n = 48). Five weeks later, they were retrieved for a histomorphometric analysis of the volumes of ectopic bone and foreign-body giant cells (a gauge of inflammatory reactivity), and the degree of polymer degradation. For each polymer, the osteoinductive efficacy of BMP-2 was higher when it was incorporated into a coating than when it was directly adsorbed onto the material. This mode of BMP-2 carriage was consistently associated with an attenuation of the inflammatory response. For coated materials, the volume density of foreign-body giant cells was inversely correlated with the volume density of bone (r(2) = 0.96), and the volume density of bone was directly proportional to the surface-area density of the polymer (r(2) = 0.97). Following coating degradation, other competitive factors, such as the biocompatibility and the biodegradability of the polymer itself, came into play.

Functionalisation of PLLA nanofiber scaffolds using a possible cooperative effect between collagen type I and BMP-2: impact on colonization and bone formation in vivo

The reconstruction of large bone defects after injury or tumor resection often requires the use of bone substitution. Artificial scaffolds based on synthetic biomaterials can overcome disadvantages of autologous bone grafts, like limited availability and donor side morbidity. Among them, scaffolds based on nanofibers offer great advantages. They mimic the extracellular matrix, can be used as a carrier for growth factors and allow the differentiation of human mesenchymal stem cells. Differentiation is triggered by a series of signaling processes, including integrin and bone morphogenetic protein (BMP), which act in a cooperative manner. The aim of this study was to analyze whether these processes can be remodeled in artificial poly-(l)-lactide acid (PLLA) based nanofiber scaffolds in vivo. Electrospun matrices composed of PLLA-collagen type I or BMP-2 incorporated PLLA-collagen type I were implanted in calvarial critical size defects in rats. Cranial CT-scans were taken 4, 8 and 12 weeks after implantation. Specimens obtained after euthanasia were processed for histology and immunostainings on osteocalcin, BMP-2 and Smad5. After implantation the scaffolds were inhomogeneously colonized and cells were only present in wrinkle- or channel-like structures. Ossification was detected only in focal areas of the scaffold. This was independent of whether BMP-2 was incorporated in the scaffold. However, cells that migrated into the scaffold showed an increased ratio of osteocalcin and Smad5 positive cells compared to empty defects. Furthermore, in case of BMP-2 incorporated PLLA-collagen type I scaffolds, 4 weeks after implantation approximately 40 % of the cells stained positive for BMP-2 indicating an autocrine process of the ingrown cells. These findings indicate that a cooperative effect between BMP-2 and collagen type I can be transferred to PLLA nanofibers and furthermore, that this effect is active in vivo. However, this had no effect on bone formation. The reason for this seems to be an unbalanced colonization of the scaffolds with cells, due to insufficient pore size.

Impact of pore size on the vascularization and osseointegration of ceramic bone substitutes in vivo

The repair of bone defects with biomaterials depends on a sufficient vascularization of the implantation site. We analyzed the effect of pore size on the vascularization and osseointegration of biphasic calcium phosphate particles, which were implanted into critical-sized cranial defects in Balb/c mice. Dense particles and particles with pore sizes in the ranges 40-70, 70-140, 140-210, and 210-280 mum were tested (n = 6 animals per group). Angiogenesis, vascularization, and leukocyte-endothelium interactions were monitored for 28 days by intravital microscopy. The formation of new bone and the bone-interface contact (BIC) were determined histomorphometrically. Twenty-eight days after implantation, the functional capillary density was significantly higher with ceramic particles whose pore sizes exceeded 140 mum [140-210 mum: 6.6 (+/-0.8) mm/mm(2); 210-280 mum: 7.3 (+/-0.6) mm/mm(2)] than with those whose pore sizes were lesser than 140 mum [40-70 mum: 5.3 (+/-0.4) mm/mm(2); 70-140 mum: 5.6 (+/-0.3) mm/mm(2)] or with dense particles [5.7 (+/-0.8) mm/mm(2)]. The volume of newly-formed bone deposited within the implants increased as the pore size increased [40-70 mum: 0.07 (+/-0.02) mm(3); 70-140 mum: 0.10 (+/-0.06) mm(3); 140-210 mum: 0.13 (+/-0.05) mm(3); 210-280 mum: 0.15 (+/-0.06) mm(3)]. Similar results were observed for the BIC. The data demonstrates pore size to be a critical parameter governing the dynamic processes of vascularization and osseointegration of bone substitutes. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007.

Oral implants placed in bone defects treated with Bio-Oss, Ostim-Paste or PerioGlas: an experimental study in the rabbit tibiae

OBJECTIVES: To compare the histological features of bone filled with Bio-Oss, Ostim-Paste or PerioGlas placed in defects in the rabbit tibiae by evaluating bone tissue composition and the integration of titanium implants placed in the grafted bone. MATERIAL AND METHODS: Two cylindrical bone defects, about 4 mm in diameter and 6 mm in depth, were created in the tibiae of 10 rabbits. The defects were filled with either Bio-Oss, PerioGlas, Ostim-Paste or left untreated, and covered with a collagen membrane. Six weeks later, one titanium sandblasted and acid-etched (SLA) implant was inserted at the centre of each previously created defect. The animals were sacrificed after 6 weeks of healing. RESULTS: Implants placed in bone previously grafted with Bio-Oss, PerioGlas or Ostim-Paste obtained a larger extent of osseointegration, although not statistically significant, than implants placed in non-grafted bone. The three grafting materials seemed to perform in a similar way concerning their contribution towards implant osseointegration. All grafting materials appeared to be osteoconductive, thus leading to the formation of bridges of mineralized bone extending from the cortical plate towards the implants surface through the graft scaffold. CONCLUSIONS: Grafting with the above-mentioned biomaterials did not add any advantage to the osseointegration of titanium SLA implants in a self-contained defect.

Comparative study of biphasic calcium phosphates with different HA/TCP ratios in mandibular bone defects. A long-term histomorphometric study in minipigs

Three biphasic calcium phosphate (BCP) bone substitute materials with hydroxyapatite (HA)/tricalcium phosphate (TCP) ratios of 20/80, 60/40, and 80/20 were compared to coagulum, particulated autogenous bone, and deproteinized bovine bone mineral (DBBM) in membrane-protected bone defects. The defects were prepared in the mandibles of 24 minipigs that were divided into four groups of six with healing times of 4, 13, 26, and 52 weeks, respectively. The histologic and histomorphometric evaluation focused on differences in amount and pattern of bone formation, filler degradation, and the interface between bone and filler. Collapse of the expanded polytetrafluoroethylene barrier membrane into the coagulum defects underlined the necessity of a filler material to maintain the augmented volume. Quantitatively, BCP 20/80 showed bone formation and degradation of the filler material similar to autografts, whereas BCP 60/40 and BCP 80/20 rather equaled DBBM. Among the three BCP's, the amount of bone formation and degradation of filler material seemed to be inversely proportional to the HA/TCP ratio. The fraction of filler surface covered with bone was highest for autografts at all time points and was higher for DBBM than BCP 80/20 and 60/40 at the early healing phase. TRAP-positive multinucleated cells were identified on BCP and DBBM surfaces without showing typical signs of resorption lacunae.

Variation of the mechanical properties of PMMA to suit osteoporotic cancellous bone

Poly(methyl methacrylate) (PMMA) is by far the most frequently used bone substitute material for vertebroplasty. However, there are serious complications, such as cement leakage and an increased fracture rate of the adjacent vertebral bodies. The latter may be related to the mechanical properties of the augmented segment within the osteoporotic spine. A possible counter-measure is prophylactic augmentation at additional levels, but this aggravates the risk for the patient. Introduction of pores is a possible method to reduce the inherent high stiffness of PMMA. This study investigates the effect of porosity on the mechanical properties of PMMA bone cement. Different fractions of a highly viscous liquid were mixed into the PMMA during preparation. An open-porous material with adjustable mechanical properties resulted after removal of the aqueous phase. Different radiopacifiers were admixed to investigate their suitability for vertebroplasty. The final material was characterized mechanically by compressive testing, microscopically and radiologically. In addition, the monomer release subsequent to hardening was measured by means of gas chromatography. The Young's modulus in compression could be varied between 2800 +/- 70 MPa and 120 +/- 150 MPa, and the compression ultimate strength between 170 +/- 5 MPa and 8 +/- 9 MPa for aqueous fractions ranging between 0 and 50% of volume. Only a slight decrease of the Young's modulus and small changes of ultimate strength were found when the mixing time was increased. An organic hydrophilic and lipophilic radiopacifier led to a higher Young's modulus of the porous material; however, the ultimate strength was not significantly affected by adding different radiopacifiers to the porous cement. The radiopacity was lost after washing the aqueous phase out of the pores. No separation occurred between the aqueous and the PMMA phase during injection into an open porous ceramic material. The monomer released was found to increase for increasing aqueous fractions, but remained comparable in magnitude to standard PMMA. This study demonstrates that a conventional PMMA can be modified to obtain a range of mechanical properties, including those of osteoporotic bone.

Properties of an injectable low modulus PMMA bone cement for osteoporotic bone

The use of polymethylmethacrylate (PMMA) cement to reinforce fragile or broken vertebral bodies (vertebroplasty) leads to extensive bone stiffening. Fractures in the adjacent vertebrae may be the consequence of this procedure. PMMA with a reduced Young's modulus may be more suitable. The goal of this study was to produce and characterize stiffness adapted PMMA bone cements. Porous PMMA bone cements were produced by combining PMMA with various volume fractions of an aqueous sodium hyaluronate solution. Porosity, Young's modulus, yield strength, polymerization temperature, setting time, viscosity, injectability, and monomer release of those porous cements were investigated. Samples presented pores with diameters in the range of 25-260 microm and porosity up to 56%. Young's modulus and yield strength decreased from 930 to 50 MPa and from 39 to 1.3 MPa between 0 and 56% porosity, respectively. The polymerization temperature decreased from 68 degrees C (0%, regular cement) to 41 degrees C for cement having 30% aqueous fraction. Setting time decreased from 1020 s (0%, regular cement) to 720 s for the 30% composition. Viscosity of the 30% composition (145 Pa s) was higher than the ones received from regular cement and the 45% composition (100-125 Pa s). The monomer release was in the range of 4-10 mg/mL for all porosities; showing no higher release for the porous materials. The generation of pores using an aqueous gel seems to be a promising method to make the PMMA cement more compliant and lower its mechanical properties to values close to those of cancellous bone.

Which biomaterials may promote periodontal regeneration in intrabony periodontal defects? A systematic review of preclinical studies.

OBJECTIVE To systematically analyze the regenerative effect of the available biomaterials either alone or in various combinations for the treatment of periodontal intrabony defects as evaluated in preclinical histologic studies. DATA SOURCES A protocol covered all aspects of the systematic review methodology. A literature search was performed in Medline, including hand searching. Combinations of searching terms and several criteria were applied for study identification, selection, and inclusion. The preliminary outcome variable was periodontal regeneration after reconstructive surgery obtained with the various regenerative materials, as demonstrated through histologic/ histomorphometric analysis. New periodontal ligament, new cementum, and new bone formation as a linear measurement in mm or as a percentage of the instrumented root length were recorded. Data were extracted based on the general characteristics, study characteristics, methodologic characteristics, and conclusions. Study selection was limited to preclinical studies involving histologic analysis, evaluating the use of potential regenerative materials (ie, barrier membranes, grafting materials, or growth factors/proteins) for the treatment of periodontal intrabony defects. Any type of biomaterial alone or in various combinations was considered. All studies reporting histologic outcome measures with a healing period of at least 6 weeks were included. A meta-analysis was not possible due to the heterogeneity of the data. CONCLUSION Flap surgery in conjunction with most of the evaluated biomaterials used either alone or in various combinations has been shown to promote periodontal regeneration to a greater extent than control therapy (flap surgery without biomaterials). Among the used biomaterials, autografts revealed the most favorable outcomes, whereas the use of most biologic factors showed inferior results compared to flap surgery.