In situ observation of fracture behavior of canine cortical bone under bending.

Cortical bone provides many important body functions and maintains the rigidness and elasticity of bone. A common failure mode for bone structure is fracture under a bending force. In the current study, the fracture behavior of canine cortical bone under three-point bending was observed in situ using an atomic force microscope (AFM), a scanning electron microscope (SEM), and an optical microscope to examine the fracture process in detail. Nanoindentation was carried out to determine the elastic modulus and hardness of different building blocks of the canine cortical bone. The results have shown that the special structure of Haversian systems has significant effects on directing crack propagation. Although Haversian systems contain previously believed weak points, and micro-cracks initiate within Haversian systems, our findings have demonstrated that macro-cracks typically form around the boundaries of Haversian systems, i.e. the cement lines. Micro-cracks that developed inside Haversian systems have the functions of absorbing and dissipating energy and slow down on expanding when interstitial tissue cannot hold any more pressure, then plastic deformation and fracture occur.

Bovine osteoblasts cultured on polyanionic collagen scaffolds: an ultrastructural and immunocytochemical study.

Collagen is the most abundant protein in the body and is also the most important component of the extracellular matrix. Collagen has several advantages as a biomaterial such as lack of toxicity, biocompatibility, biodegradability, and easy reabsorption. In this study, we examined bovine osteoblasts cultured on native or anionic collagen scaffolds prepared from bovine pericardium after selective hydrolysis of glutamine and asparagine side chain amides for periods from 24 (BP24) and 48 h (BP48). The cells were cultured in control and mineralization medium at 37 °C in the presence of 5% CO(2). Transmission and scanning electron microscopy, energy dispersive spectroscopy, and an immunocytochemical marker were used for analysis. Cells with an irregular morphology forming a confluent multilayer were observed on matrices kept in control medium. Most of these cells presented a polygonal or elongated flattened morphology. Several spherical deposits of calcium crystal associated with phosphorus were observed on the native and BP48 matrices. Similar results were observed in samples kept in control medium except with lower calcium/phosphorus ratio. Vesicles actively expelled from the cell membrane were also seen (do this vesicles corresponds to calcium/phosphorus deposits). Osteocalcin was clearly visible on matrices kept in mineralization medium and was more expression on the surface of BP48 matrices. The results showed that anionic collagen is able to support osteoblastic differentiation, regardless of the medium used. Finally, the BP48 matrix promoted better osteoblast differentiation than the native matrix.

The effects of a soluble polymer and bone wax on sternal healing in an animal model.

PURPOSE: This study compares the effects of a soluble polymer hemostatic material and bone wax on sternal bone healing. DESCRIPTION: Median sternotomies were performed on 20 New Zealand White rabbits, and sufficient polymer (Ostene; Ceremed Inc, Los Angeles CA) or bone wax (Bone Wax; Ethicon Inc, Somerville, NJ) was applied to achieve bone hemostasis. After 6 weeks, sternal healing was assessed using roentgenograms, histology, and mechanical strength testing. EVALUATION: Roentgenograms revealed normal bone healing in the polymer-treated group and nonunion in the bone wax group. Histology showed normal bone healing in the polymer group, with fibrotic scar tissue and the absence of new bone formation in the bone wax group. Mechanical strength testing showed that polymer-treated sternal segments were twice as strong as those treated with bone wax. They had a significantly higher flexural strength (2.53 +/- 0.43 vs. 1.29 +/- 0.37 megapascal [MPa]; p < 0.001) and Young's modulus (0.315 +/- 0.056 vs 0.146 +/- 0.031 MPa; p < 0.001). CONCLUSIONS: The application of the polymer hemostatic material to the sternum resulted in significantly stronger union compared with the use of bone wax.

Infection rates and healing using bone wax and a soluble polymer material.

The effects of using a newly available water-soluble polymer bone hemostatic material in a contaminated environment were assessed in a rabbit tibial defect model. Infection rates and healing of polymer-treated bone were compared with the infection and healing of bone wax-treated bone and untreated controls after a bacterial challenge. Defects created in 24 rabbit tibias were treated with the polymer or bone wax, or left without a hemostatic agent. The defects were inoculated with Staphylococcus aureus ATCC-29213 (2.5 x 10(4) colony-forming units). After 4 weeks, all defects treated with bone wax were infected and osteomyelitis had developed, and none had evidence of bone healing. In the polymer and control groups, two defects in each group (25%) had osteomyelitis develop. The remaining six defects in each group (75%) showed no osteomyelitis and exhibited normal bone healing. The polymer-treated defects had a considerably lower rate of osteomyelitis and positive bone cultures compared with the bone wax-treated group. There were no differences between the polymer-treated and control groups in the rates of osteomyelitis, positive cultures, or bone healing. The use of a soluble polymer as an alternative to bone wax may decrease the rates of postoperative bone infections.

Platelet-rich and platelet-poor plasma: development of an animal model to evaluate hemostatic efficacy.

Hemostasis is important for any surgical procedure. One method uses autologous platelet-rich and/or platelet-poor plasma sprayed on the wound site. Although effective, there are little quantitative data available to fully document the extent to which these autologous products function as hemostats. Also, limitations in current animal models make quantitative study of topical hemostats difficult. A porcine partial-thickness skin wound model was developed to compare the hemostatic ability of these treatments with untreated control wounds. Rectangular partial-thickness dermal wounds were created in the back of a pig, which was then sprayed with activated platelet-rich plasma, activated platelet-poor plasma, or left untreated. Bleeding was quantified by two methods: 1) gravimetric measurement of exudate transfer to a sponge over a 15-minute interval, and 2) iron assay of the exudate over this same interval. Values for treated wounds were normalized to those of control wounds to minimize interanimal variability. Both gravimetric and iron assay measurements demonstrated that platelet-rich plasma was effective within 5 minutes after application with normalized bleeding values of approximately 35% and 20%, respectively, of the untreated controls. Corresponding values for platelet-poor plasma were approximately 90% and 65%, respectively, with differences only significant for the iron assay method measured on 10- and 15-minute wound exudate. Although both platelet-rich and platelet-poor plasma demonstrated hemostatic potential, the effect was more robust with the former. Iron assay was a more accurate method of measuring bleeding than gravimetric analysis.

Microindentation test for assessing the mechanical properties of cartilaginous tissues.

Mechanical properties of the fresh control, frozen, and vitrified cartilaginous (cartilage and meniscus) samples were measured by microindentation. Indentation depth, elastic modulus, and indentation yield strength were obtained from the microindentation loading curves. Indentation deformation behavior was studied using Hertz contact model. The stress distribution of cartilaginous tissues under indentation loading was analyzed by finite element technique. It was found that fresh tissue shows the lowest indentation depth and the highest elastic modulus and indentation yield strength, followed by vitrified and frozen tissues. The vitrified tissue shows slightly lower but comparable mechanical properties with control tissue. The vitrification technique used in this study can preserve live cells with superior mechanical properties that make it an ideal technique for use in orthopedic and other biomedical applications. The microindentation tests and corresponding analysis methods used in this study offer a simple way to evaluate the mechanical properties of cartilaginous tissues. It suits small sample sizes and it may be used for other biological tissues.

A comparison of ProOsteon, DBX, and collagraft in a rabbit model.

Many bone graft substitutes (BGSs) have been developed and are commercially available. These products differ in the tailoring of their properties, including size, form, osteoconductivity, osteoinductivity, and resorption kinetics. Differential enhancement of these properties may optimize the performance of these materials for varying applications. BGSs offer an opportunity to lessen morbidity of harvesting and use of autogenous and/or allograft bone. The purpose of this study is to quantitatively compare the magnitude of bony ingrowth and biodegradation of different commercially available BGS materials in a rabbit femoral defect model. BGSs from each of three classes (ceramic (ProOsteon), demineralized bone matrix (DBX), and composite (Collagraft)) were implanted in cylindrical defects in bilateral femoral condyles of 12 adult New Zealand White rabbits. Each of the three BGS materials and the empty controls were compared. The specimens were harvested at 3 months postimplantation for radiographic and histologic evaluation. Histomorphometry yielded resorption of graft material remaining in the index defect. Magnitude of bony ingrowth was assessed based on an 8-bit 256 densitometry model. Histomorphometric analysis of the data demonstrated statistical differences in the resorption and magnitude of bony ingrowth of the three BGS materials. The three BGS were significantly different for ingrowth (p = 0.046) when using the Wilcoxon Test. The ceramic graft material averaged 47% bony ingrowth. Rabbit-based DBX material showed extensive osseous ingrowth (35%) and the composite graft material demonstrated significant bony ingrowth (56%). The control, as anticipated, showed the least amount of bony ingrowth (29%). Fisher's Exact Test yielded statistical differences (p = 0.0003) when comparisons for resorption were conducted. An ideal BGS material should be biocompatible, be able to withstand the local load environment for a given application, degrade in concert with bony replacement, and be both osteoinductive and osteoconductive. This in-vivo, head-to-head comparison of three commercially available BGS materials in an animal model compares these characteristics and demonstrates differences between them, which may act as a guide in the use of these products in human applications.

Sterilizing Bacillus pumilus spores using supercritical carbon dioxide.

Supercritical carbon dioxide (SC CO(2)) has been evaluated as a new sterilization technology. Results are presented on killing of B. pumilus spores using SC CO(2) containing trace levels of additives. Complete killing was achieved with 200 part per million (ppm) hydrogen peroxide in SC CO(2) at 60 degrees C, 27.5 MPa. Addition of water to SC CO(2) resulted in greater than three-log killing, but this is insufficient to claim sterilization. Neither ethanol nor isopropanol when added to SC CO(2) affected killing.

Arterial embolization using poly-N-acetyl glucosamine gel in a rat kidney model.

Aqueous solutions of poly-N-acetyl glucosamine (p-GlcNAc) exhibit a liquid-gel transition at physiological pH and temperature. This feature inspired the authors to conduct a study to evaluate the macro- and histological changes of rat kidneys after embolization using either p-GlcNAc gel injection into the renal artery or ligation of the renal artery. The procedures were performed in 46 rats through open abdominal surgeries. Animals were sacrificed at 3 days and at 1, 3, 5, and 8 weeks postoperatively. The results of both macro-observation and histological study showed that p-GlcNAc gels were effective in causing necrosis and subsequent fibrosis in all embolized kidneys. The data indicate that p-GlcNAc gel may have promise as an effective agent for therapeutic embolization.

Micro/nanoscale mechanical and tribological characterization of SiC for orthopedic applications.

Micro/nanomechanical and tribological characterization of SiC has been carried out. For comparison, measurements on SiC, CoCrMo, Ti-6Al-4V, and stainless steel have also been made. Hardness and elastic modulus of these materials were measured by nanoindentation using a nanoindenter. The nanoindentation impressions were imaged using an atomic force microscope (AFM). Scratch, friction, and wear properties were measured using an accelerated microtribometer. Scratch and wear damages were studied using a scanning electron microscope (SEM). It is found that SiC exhibits higher hardness, elastic modulus, scratch resistance as well as lower friction with fewer and smaller debris particles compared to other materials. These results show that SiC possesses superior mechanical and tribological properties that make it an ideal material for use in orthopedic and other biomedical applications.

In vitro analysis of anionic collagen scaffolds for bone repair.

Collagen has been extensively described as a beneficial material in bone tissue engineering due to its biocompatibility, biodegradability, low antigenicity, and high tensile strength. However, collagen scaffolds in their pure form have some drawbacks and improvements in the physical, chemical, and biologic properties of collagen are necessary to overcome those inadequacies. Recently, the selective hydrolysis of carboxyamides of asparagine and glutamine residues of collagen has been employed to increase the number of negative sites and enhance the piezoelectric properties of collagen. Anionic collagen scaffolds were prepared by use of a hydrolysis treatment for either 24 h [bovine pericardium (BP 24)] or 48 h (BP 48). Bovine osteoblasts were cultured on them and on native matrices to understand the cellular interactions responsible for the good osteoconductivity and biocompatibility reported with in vivo tests. Based on the data obtained on cell adhesion, alkaline phosphatase (ALP) and extracellular matrix macromolecule production, and cellular proliferation through histological analysis, we may conclude that the materials tested reveal sufficient biocompatibility level for bone repair. Further, the evidence of some connection between ALP activity and the mineralization process should be emphasized. BP 48 presented the most promising results stimulating in vitro mineralization, ALP production, and possible osteoblast differentiation.

Is there an inhibitory effect of COX-2 inhibitors on bone healing?

The use of the new selective cyclooxygenase-2 (COX-2) inhibitors (such as celecoxib and rofecoxib) for the treatment of pain and inflammation caused by fractures, cementless total joint replacements, soft tissue healing to bone, and spinal fusion surgeries has been controversial due to the convincing data collected from nonspecific NSAIDs such as indomethacin and naproxen regarding their inhibitory effects on bone healing and the similar effects of COX-2 specific NSAIDs in animal models. Is there a significant inhibitory effect of COX-2 inhibitors on bone healing in humans? To answer this question, we reviewed existing scientific evidence (based mainly on a MedLine search) of the potential effects of COX-2 inhibitors on bone healing. The literature shows that COX-2 inhibitors do have inhibitory effects on bone healing in animal models, but the effects of COX-2 inhibitors on similar processes in humans remain largely unknown.

Vitreous preservation of articular cartilage grafts.

Articular cartilage has proved refractory to satisfactory cryopreservation using conventional freezing methods. Therefore, an ice-free cryopreservation method by vitrification was tested. Osteochondral plugs from New Zealand White rabbits were preserved using either a freezing method or an ice-free vitrification method of cryopreservation. Preserved and fresh control plugs were implanted in the tibial plateau of allogeneic recipients. A modified O'Driscoll grading scale, based on gross pathology, histopathology, and histochemistry, was used to evaluate the explants.The histology of fresh and vitrified explants was essentially the same, while the frozen cryopreserved explants were devoid of chondrocytes and only fibroblastlike cells were observed. The O'Driscoll grading indicated that both fresh and vitrified plugs performed significantly better than frozen plugs (p < or =.05). The results demonstrate the feasibility of vitrification as a storage method for cartilaginous tissues.

The mechanisms of the inhibitory effects of nonsteroidal anti-inflammatory drugs on bone healing: a concise review.

Nonsteroidal anti-inflammatory drug (NSAID) use continues to expand at a remarkable rate due both to the broad spectrum of clinical applications for these medications and to the relatively recent introduction of the popular COX-2-selective inhibitors. The use of NSAIDs is particularly prevalent in patients with a variety of musculoskeletal conditions and injuries. Reports of impaired bone healing associated with NSAID use, therefore, are a particular cause for concern. Animal and in vitro studies have demonstrated impaired bone healing in the presence of traditional NSAIDs, as measured by a variety of different parameters. More recently, initial studies investigating the effects of COX-2-selective inhibitors on bone healing have yielded similar results. With mounting evidence that NSAIDs do in fact interfere with proper bone healing in various animal models, questions have arisen regarding the potential mechanism through which NSAIDs produce this outcome and whether these results can be translated to clinical settings. A likely pathway for these observed effects results from an understanding of the steps involved in bone healing itself. These steps include an inflammatory response, bone resorption, and new bone formation. Investigations over the past several decades have elucidated a role for prostaglandins (PGs) in each of these areas. Specifically, PGs have been shown to elicit and participate in inflammatory responses, increase osteoclast activity and subsequent bone resorption, and increase osteoblast activity and new bone formation. This apparent integral role for PGs in the process of bone healing, coupled with the knowledge that NSAIDs act by inhibiting the production of PGs, results in an understanding of the likely mechanism through which NSAIDs impart their deleterious effects on bone healing. By inhibiting the COX enzymes and the subsequent production of PGs, NSAIDs not only achieve their desired anti-inflammatory effects but also inhibit the increased production of PGs that is necessary for bone healing to occur. Despite this understanding of the potential mechanism through which NSAIDs inhibit bone healing in a laboratory setting, few studies exist that show whether these inhibitory effects are also evident clinically. Thus, further studies will need to decipher whether similar inhibitory effects occur in a clinical setting.

Animal models for therapeutic embolization.

Embolization techniques have been performed in different animals to accumulate basic data before a clinical trial. Choosing the right embolization model for a specific project is critical. However, there are several variables when defining the best model for embolization research such as the size of the animal to be used, the target organs, the route of introducing the embolization agent, and the feasible methods of evaluation. Commonly used research animals for endovascular embolization include rabbits, dogs, and rats. Frequently used target organs are the kidney and the liver. Most models use a transcatheter for introducing the embolus and occasionally open surgery and direct arterial injection are used. Basic methods of evaluation are straightforward, and commonly include macro observation of the embolized organs, angiogram, and histology. This article concisely reviews the available animal models and their evaluation for embolization research to help researchers to choose the appropriate model.

Thermogelling biodegradable copolymer aqueous solutions for injectable protein delivery and tissue engineering.

This paper reports on the thermogelling, biodegradable polymer formulations based on poly(DL-lactic acid-co-glycolic acid)/(poly(ethylene glycol) graft copolymers for in vivo biomedical applications using animal models. The description includes diabetic control by sustained insulin delivery and cartilage repair by chondrocyte cell delivery. With one injection of the poly(DL-lactic acid-co-glycolic acid)/(poly(ethylene glycol) graft copolymers insulin formulation, the blood glucose level could be controlled from 5 to 16 days in diabetic rats by varying the polymer composition. The cartilage defect was notably repaired using chondrocyte suspension in the thermogelling PLGA-g-PEG compared with a control. This report shows that thermogelling biodegradable PLGA/PEG graft copolymer system can be a promising platform for protein and cell-based therapy.