Canine ex vivo tarsal arthrodesis: fixation by using a new bone tissue glue

IntroductionArthrodesis, performed as a salvage surgical procedure to treat intractable joint conditions in dogs and cats, is associated with a high incidence of complications intra and postoperative, proving the need for improved and new techniques in arthrodesis surgery. Adding a new resorbable bone glue to the arthrodesis could potentially add fixation strength and lower complications. The objectives of this experimental ex vivo biomechanical study were therefore to develop a biomechanical test model of partial tarsal arthrodesis and to determine whether the new resorbable bone glue (phosphoserine modified cement) produced measurable fixation strength in canine calcaneoquartal arthrodesis, without orthopedic implants.MethodsFour biomechanical test models with a total of 35 canine tarsal joints were used. Soft tissues were dissected to 4 different test models with variable contributions from soft tissues. The calcaneoquartal joint was prepared as in vivo arthrodesis and the glue was applied to joint surfaces as a liquid/putty (0.4 cc). After curing for 24 h, a shear force was applied to the joint (1 mm per minute) and the failure strength was recorded.ResultsCalcaneoquartal joints, where all soft tissues had been completely resected and fixated with glue (1–1.5 cm2 joint surface), withstood 2–5 mm of displacement and an average of 100 ± 58 N/cm2 of shear force (Model 1). Similar adhesive fixation strengths were obtained in Model 2 and 3 with increasing contributions from soft tissues (80 ± 44 and 63 ± 23 N/cm2, p = 0.39, ANOVA).ConclusionThe developed biomechanical model was sensitive enough to measure differences in fixation strengths between different glue formulations. The average fixation strength (60–100 N/cm2) should be strong enough to support short-term load bearing in medium sized canines (20 kg). The developed cadaver biomechanical test model is of potential use for other arthrodesis studies. The new resorbable glue can potentially contribute to stability at arthrodesis surgery, acting as a complement to today’s standard fixation, metal implants

Nuclear Magnetic Resonance and Metadynamics Simulations Reveal the Atomistic Binding of l -Serine and O-Phospho- l -Serine at Disordered Calcium Phosphate Surfaces of Biocements

Interactions between biomolecules and structurally disordered calcium phosphate (CaP) surfaces are crucial for the regulation of bone mineralization by noncollagenous proteins, the organization of complexes of casein and amorphous calcium phosphate (ACP) in milk, as well as for structure-function relationships of hybrid organic/inorganic interfaces in biomaterials. By a combination of advanced solid-state NMR experiments and metadynamics simulations, we examine the detailed binding of O-phospho-l-serine (Pser) and l-serine (Ser) with ACP in bone-adhesive CaP cements, whose capacity of gluing fractured bone together stems from the close integration of the organic molecules with ACP over a subnanometer scale. The proximity of each carboxy, aliphatic, and amino group of Pser/Ser to the Ca2+ and phosphate species of ACP observed from the metadynamics-derived models agreed well with results from heteronuclear solid-state NMR experiments that are sensitive to the 13C-31P and 15N-31P distances. The inorganic/organic contacts in Pser-doped cements are also contrasted with experimental and modeled data on the Pser binding at nanocrystalline HA particles grown from a Pser-bearing aqueous solution. The molecular adsorption is driven mainly by electrostatic interactions between the negatively charged carboxy/phosphate groups and Ca2+ cations of ACP, along with H bonds to either protonated or nonprotonated inorganic phosphate groups. The Pser and Ser molecules anchor at their phosphate/amino and carboxy/amino moieties, respectively, leading to an extended molecular conformation across the surface, as opposed to an "upright standing"molecule that would result from the binding of one sole functional group

The biological and physical performance of high strength dicalcium phosphate cement in physiologically relevant models

The chemical properties of calcium phosphate cements (CPCs) are very similar to the mineral phase of bone. CPCs are, consequently, very effective substrates (scaffolds) for tissue engineering; bone and stem cells attach readily, and can proliferate and differentiate to form new bone tissue. Unlike other CPCs that may remain largely unchanged in the body for years, such as hydroxyapatite, dicalcium phosphates are remodelled by the body and rapidly converted to new bone. Unfortunately, the dicalcium phosphates are also typically too weak to support load bearing in the human body. Our laboratory has recently developed a novel, high strength brushite CPC, (hsCPC), which can reach 10-50 fold higher failure strength than many commercially available CPCs. The aim of this thesis was to investigate the physical, chemical and biological performance of hsCPCs in physiologically relevant model of drug release, load bearing, osteoconductivity, and as a scaffold for bone tissue engineering. Multiple CPCs were compared in a model of screw augmentation to determine whether the physical properties of the cement, such as bulk strength and porosity, affected orthopedic screw holding strength. In an in vitro model of bone regeneration stem cells were grown on macroporous scaffolds that were fabricated from hsCPC. Drug releasing scaffolds were fabricated to examine whether the low porosity of hsCPC impeded drug release during a 4 week incubation period. The biological activity of an incorporated drug, Rebamipide, was examined after acute and chronic incubation periods. In the drug release study it was noted that the biological response to hsCPC was significantly better than tissue culture grade polystyrene, even in groups without drug. The mechanism underlying this biological response was further investigated by testing the effect of pyrophosphate, a common cement additive, on bone cell proliferation and differentiation. This thesis concludes that a high strength cement can produce significant improvement in screw augmentation strength, if there is sufficient cortical bone near the augmentation site. The hsCPC is also cytocompatible, and can support bone and stem cell proliferation and differentiation. hsCPC scaffolds stimulated osteogenic gene expression comparable to native bone scaffolds. hsCPC scaffolds are also capable of delivering drug for up to 4 weeks, in vitro. Finally, a cement additive, pyrophosphate, stimulated differentiation, but not proliferation of bone cells

Rebamipide Delivered by Brushite Cement Enhances Osteoblast and Macrophage Proliferation

Many of the bioactive agents capable of stimulating osseous regeneration, such as bone morphogenetic protein-2 (BMP-2) or prostaglandin E2 (PGE2), are limited by rapid degradation, a short bioactive half-life at the target site in vivo, or are prohibitively expensive to obtain in large quantities. Rebamipide, an amino acid modified hydroxylquinoline, can alter the expression of key mediators of bone anabolism, cyclo-oxygenase 2 (COX-2), BMP-2 and vascular endothelial growth factor (VEGF), in diverse cell types such as mucosal and endothelial cells or chondrocytes. The present study investigates whether Rebamipide enhances proliferation and differentiation of osteoblasts when delivered from brushite cement. The reactive oxygen species (ROS) quenching ability of Rebampide was tested in macrophages as a measure of bioactivity following drug release incubation times, up to 14 days. Rebamipide release from brushite occurrs via non-fickian diffusion, with a rapid linear release of 9.70%+/- 0.37% of drug per day for the first 5 days, and an average of 0.5%-1% per day thereafter for 30 days. Rebamipide slows the initial and final cement setting time by up to 3 and 1 minute, respectively, but does not significantly reduce the mechanical strength below 4% (weight percentage). Pre-osteoblast proliferation increases by 24% upon exposure to 0.4uM Rebamipide, and by up to 73% when Rebamipide is delivered via brushite cement. Low doses of Rebamipide do not adversely affect peak alkaline phosphatase activity in differentiating pre-osteoblasts. Rebamipide weakly stimulates proliferation in macrophages at low concentrations (118 +/- 7.4% at 1uM), and quenches ROS by 40-60%. This is the first investigation of Rebamipide in osteoblasts

Rebamipide Delivered by Brushite Cement Enhances Osteoblast and Macrophage Proliferation

Many of the bioactive agents capable of stimulating osseous regeneration, such as bone morphogenetic protein-2 (BMP-2) or prostaglandin E2 (PGE2), are limited by rapid degradation, a short bioactive half-life at the target site in vivo, or are prohibitively expensive to obtain in large quantities. Rebamipide, an amino acid modified hydroxylquinoline, can alter the expression of key mediators of bone anabolism, cyclo-oxygenase 2 (COX-2), BMP-2 and vascular endothelial growth factor (VEGF), in diverse cell types such as mucosal and endothelial cells or chondrocytes. The present study investigates whether Rebamipide enhances proliferation and differentiation of osteoblasts when delivered from brushite cement. The reactive oxygen species (ROS) quenching ability of Rebampide was tested in macrophages as a measure of bioactivity following drug release incubation times, up to 14 days. Rebamipide release from brushite occurrs via non-fickian diffusion, with a rapid linear release of 9.70%+/- 0.37% of drug per day for the first 5 days, and an average of 0.5%-1% per day thereafter for 30 days. Rebamipide slows the initial and final cement setting time by up to 3 and 1 minute, respectively, but does not significantly reduce the mechanical strength below 4% (weight percentage). Pre-osteoblast proliferation increases by 24% upon exposure to 0.4uM Rebamipide, and by up to 73% when Rebamipide is delivered via brushite cement. Low doses of Rebamipide do not adversely affect peak alkaline phosphatase activity in differentiating pre-osteoblasts. Rebamipide weakly stimulates proliferation in macrophages at low concentrations (118 +/- 7.4% at 1uM), and quenches ROS by 40-60%. This is the first investigation of Rebamipide in osteoblasts

Phosphoserine Functionalized Cements Preserve Metastable Phases, and Reprecipitate Octacalcium Phosphate, Hydroxyapatite, Dicalcium Phosphate, and Amorphous Calcium Phosphate, during Degradation, In Vitro

Phosphoserine modified cements (PMC) exhibit unique properties, including strong adhesion to tissues and biomaterials. While TTCP-PMCs remodel into bone in vivo, little is known regarding the bioactivity and physiochemical changes that occur during resorption. In the present study, changes in the mechanical strength and composition were evaluated for 28 days, for three formulations of alpha TCP based PMCs. PMCs were significantly stronger than unmodified cement (38-49 MPa vs. 10 MPa). Inclusion of wollastonite in PMCs appeared to accelerate the conversion to hydroxyapatite, coincident with slight decrease in strength. In non-wollastonite PMCs the initial compressive strength did not change after 28 days in PBS (p > 0.99). Dissolution/degradation of PMC was evaluated in acidic (pH 2.7, pH 4.0), and supersaturated fluids (simulated body fluid (SBF)). PMCs exhibited comparable mass loss (<15%) after 14 days, regardless of pH and ionic concentration. Electron microscopy, infrared spectroscopy, and X-ray analysis revealed that significant amounts of brushite, octacalcium phosphate, and hydroxyapatite reprecipitated, following dissolution in acidic conditions (pH 2.7), while amorphous calcium phosphate formed in SBF. In conclusion, PMC surfaces remodel into metastable precursors to hydroxyapatite, in both acidic and neutral environments. By tuning the composition of PMCs, durable strength in fluids, and rapid transformation can be obtained

Drug release profile of cements.

<p>The release profile of cylindrical composites in PBS, over 30 days (A) (*, § indicate p< 0.05, **, §§ indicate P<0.01, Dunnet’s t-test). The first 4 days, or 45%, of release plotted using the Higuchi model (B). After 14 days the release rate closely matched first order release (C).</p

Physical properties of cements.

<p>The setting time (A) does not change with increasing amounts of Rebamipide, however the compressive strength (B) is significantly reduced in composites containing the largest amount of Rebamipide. (*, § indicate p< 0.05, **, §§ indicate P<0.01, Dunnet’s t-test).</p

Physical and drug release characteristics of Rebamipide loaded brushite cements.

<p>Physical and drug release characteristics of Rebamipide loaded brushite cements.</p

Pyrophosphate Stimulates Differentiation, Matrix Gene Expression and Alkaline Phosphatase Activity in Osteoblasts

Pyrophosphate is a potent mitogen, capable of stimulating proliferation in multiple cell types, and a critical participant in bone mineralization. Pyrophosphate can also affect the resorption rate and bioactivity of orthopedic ceramics. The present study investigated whether calcium pyrophosphate affected proliferation, differentiation and gene expression in early (MC3T3 pre-osteoblast) and late stage (SAOS-2 osteosarcoma) osteoblasts. Pyrophosphate stimulated peak alkaline phosphatase activity by 50% and 150% at 100 mu M and 0.1 mu M in MC3T3, and by 40% in SAOS-2. The expression of differentiation markers collagen 1 (COL1), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) were increased by an average of 1.5, 2, 2 and 3 fold, by high concentrations of sodium pyrophosphate (100 mu M) after 7 days of exposure in MC3T3. COX-2 and ANK expression did not differ significantly from controls in either treatment group. Though both high and low concentrations of pyrophosphate stimulate ALP activity, only high concentrations (100 mu M) stimulated osteogenic gene expression. Pyrophosphate did not affect proliferation in either cell type. The results of this study confirm that chronic exposure to pyrophosphate exerts a physiological effect upon osteoblast differentiation and ALP activity, specifically by stimulating osteoblast differentiation markers and extracellular matrix gene expression