Hala Zreiqat

The influence hydroxyapatite nanoparticle shape and size on the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatite-PCL composites.

We developed a composite biphasic calcium phosphate (BCP) scaffold by coating a nanocomposite layer, consisting of hydroxyapatite (HA) nanoparticles and polycaprolactone (PCL), over the surface of BCP. The effects of HA particle size and shape in the coating layer on the mechanical and biological properties of the BCP scaffold were examined. Micro-computerized tomography studies showed that the prepared scaffolds were highly porous (approximately 91%) with large pore size (400-700 microm) and an interconnected porous network of approximately 100%. The HA nanoparticle (needle shape)-composite coated scaffolds displayed the highest compressive strength (2.1 +/- 0.17 MPa), compared to pure HA/beta-TCP (0.1 +/- 0.05 MPa) and to the micron HA - composite coated scaffolds (0.29 +/- 0.07 MPa). These needle shaped scaffolds also showed enhanced elasticity and similar stress-strain profile to natural bone. Needle shaped coated HA/PCL particles induced the differentiation of primary human bone derived cells, with significant upregulation of osteogenic gene expression (Runx2, collagen type I, osteocalcin and bone sialoprotein) and alkaline phosphatase activity compared to other groups. These properties are essential for enhancing bone ingrowth in load-bearing applications. The developed composite scaffolds possessed superior physical, mechanical, elastic and biological properties rendering them potentially useful for bone tissue regeneration.

The incorporation of strontium and zinc into a calcium-silicon ceramic for bone tissue engineering.

In this study we developed novel scaffolds through the controlled substitution and incorporation of strontium and zinc into a calcium-silicon system to form Sr-Hardystonite (Sr-Ca(2)ZnSi(2)O(7), Sr-HT). The physical and biological properties of Sr-HT were compared to Hardystonite (Ca(2)ZnSi(2)O(7)) [HT]. We showed that Sr-HT scaffolds are porous with interconnected porous network (interconnectivity: 99%) and large pore size (300-500 microm) and an overall porosity of 78%, combined with a relatively high compressive strength (2.16+/-0.52 MPa). These properties are essential for enhancing bone ingrowth in load-bearing applications. Sr-HT ceramic scaffolds induced the attachment and differentiation of human bone derived cells (HOB), compared to that for the HT scaffolds. Sr-HT scaffolds enhanced expression of alkaline phosphatase, Runx-2, osteopontin, osteocalcin and bone sialoprotein. The in vivo osteoconductivity of the scaffolds was assessed at 3 and 6 weeks following implantation in tibial bone defects in rats. Histological staining revealed rapid new growth of bone into the pores of the 3D scaffolds with the Sr-HT and HT, relative to the beta-tricalcium phosphate (beta-TCP). In vivo, HT and Sr-HT produced distinct differences in the patterns of degradation of the materials, and their association with TRAP positive osteoclast-like cells with HT appearing more resistant compared to both Sr-HT and beta-TCP.

The synergistic effect of hierarchical micro/nano-topography and bioactive ions for enhanced osseointegration

Both surface chemistry and topography have significant influence on good and fast osseointegration of biomedical implants; the main goals in orthopeadic, dental and maxillofacial surgeries. A surface modification strategy encompassing the use of bioactive trace elements together with surface micron/nano-topographical modifications was employed in this study in an attempt to enhance the osseointegration of Ti alloy (Ti-6Al-4V), a commonly used implant. Briefly, we developed strontium-substituted hardystonite (Sr-HT) ceramic coating with a hierarchical topography where the nanosized grains were superimposed in the micron-rough coating structure. Its ability to induce new bone formation was evaluated by an in vivo animal model (beagle dogs). Hardystonite (HT), classic hydroxyapatite (HAp) coated and uncoated Ti-alloy implants were parallelly investigated for comparison. In addition, we investigated the effects of surface topography and the dissolution products from the coatings on the in vitro bioactivity using canine bone marrow mesenchymal stem cells (BMMSCs) cultured on the implant surface as well as using extracts of the coated implants. Micro-CT evaluation, histological observations, biomechanical test (push-out test) and sequential fluorescent labeling and histomorphometrical analysis consistently demonstrated that our developed Sr-HT-coated Ti-alloy implants have the highest osseointegration, while the uncoated implants had the lowest. The osseointegration ability of HAp-coated Ti alloy was inferior to that seen for HT- and Sr-HT-coated Ti alloy. We demonstrated that the dissolution products, particularly strontium (Sr) from the Sr-HT-coated implants, enhanced the ALP activity and in vitro mineralization ability, while the micro/nano-topography was more related to the promotion of cell adhesion. Those results suggest that our developed Sr-HT coatings have the potential for future use as coatings for orthopedic/dental and maxillofacial devices.

S100A8 and S100A9 in Human Arterial Wall IMPLICATIONS FOR ATHEROGENESIS

Atherogenesis is a complex process involving inflammation. S100A8 and S100A9, the Ca2+-binding neutrophil cytosolic proteins, are associated with innate immunity and regulate processes leading to leukocyte adhesion and transmigration. In neutrophils and monocytes the S100A8-S100A9 complex regulates phosphorylation, NADPH-oxidase activity, and fatty acid transport. The proteins have anti-microbial properties, and S100A8 may play a role in oxidant defense in inflammation. Murine S100A8 is regulated by inflammatory mediators and recruits macrophages with a proatherogenic phenotype. S100A9 but not S100A8 was found in macrophages in ApoE-/- murine atherosclerotic lesions, whereas both proteins are expressed in human giant cell arteritis. Here we demonstrate S100A8 and S100A9 protein and mRNA in macrophages, foam cells, and neovessels in human atheroma. Monomeric and complexed forms were detected in plaque extracts. S100A9 was strongly expressed in calcifying areas and the surrounding extracellular matrix. Vascular matrix vesicles contain high levels of Ca2+-binding proteins and phospholipids that regulate calcification. Matrix vesicles characterized by electron microscopy, x-ray microanalysis, nucleoside triphosphate pyrophosphohydrolase assay and cholesterol/phospholipid analysis contained predominantly S100A9. We propose that S100A9 associated with lipid structures in matrix vesicles may influence phospholipid-Ca2+ binding properties to promote dystrophic calcification. S100A8 and S100A9 were more sensitive to hypochlorite oxidation than albumin or low density lipoprotein and immunoaffinity confirmed S100A8-S100A9 complexes; some were resistant to reduction, suggesting that hypochlorite may contribute to protein cross-linking. S100A8 and S100A9 in atherosclerotic plaque and calcifying matrix vesicles may significantly influence redox- and Ca2+-dependent processes during atherogenesis and its chronic complications, particularly dystrophic calcification.

Porous diopside (CaMgSi2O6) scaffold: A promising bioactive material for bone tissue engineering

Diopside (CaMgSi(2)O(6)) powders and dense ceramics have been shown to be bioactive biomaterials for bone repair. The aim of this study is to prepare bioactive diopside scaffolds and examine their physicochemical and biological properties. X-ray diffraction, scanning electron microscopy (SEM), micro-computerized tomography and energy-dispersive spectrometry were used to analyse the composition, microstructure, pore size and interconnectivity of the diopside scaffolds. The mechanical strength and stability as well as the degradation of the scaffolds were investigated by testing the compressive strength, modulus and silicon ions released, respectively. Results showed that highly porous diopside scaffolds with varying porosity and high interconnectivity of 97% were successfully prepared with improved compressive strength and mechanical stability, compared to the bioglass and CaSiO(3) scaffolds. The bioactivity of the diopside scaffolds was assessed using apatite-forming ability in simulated body fluids (SBF) and by their support for human osteoblastic-like cell (HOB) attachment, proliferation and differentiation using SEM, and MTS and alkaline phosphatase activity assays, respectively. Results showed that diopside scaffolds possessed apatite-forming ability in SBF and supported HOB attachment proliferation and differentiation. Bioactive diopside scaffolds were prepared with excellent pore/structure art, and improved mechanical strength and mechanical stability, suggesting their possible applications for bone tissue engineering regeneration.

The effect of mesoporous bioactive glass on the physiochemical, biological and drug-release properties of poly(DL-lactide-co-glycolide) films

Poly(lactide-co-glycolide) (PLGA) has been widely used for bone tissue regeneration. However, it lacks hydrophilicity, bioactivity and sufficient mechanical strength and its acidic degradation by-products can lead to pH decrease in the vicinity of the implants. Mesoporous bioactive glass (MBG) with highly ordered structure (pore size 2-50nm) possesses higher bioactivity than non-mesoporous bioactive glass (BG). The aim of this study is to investigate the effect of MBG on the mechanical strength, in vitro degradation, bioactivity, cellular response and drug release of PLGA films and optimize their physicochemical, biological and drug-delivery properties for bone tissue engineering application. The surface and inner microstructure, mechanical strength and surface hydrophilicity of MBG/PLGA and BG/PLGA films were tested. Results indicated that MBG or BG was uniformly dispersed in the PLGA films. The incorporation of MBG into PLGA films significantly improved their tensile strength, modulus and surface hydrophilicity. MBG/PLGA resulted in an enhanced mechanical strength, in vitro degradation (water absorbance, weight loss and ions release), apatite-formation ability and pH stability in simulated body fluids (SBF), compared to BG/PLGA. MBG/PLGA and BG/PLGA films enhanced human osteoblastic-like cells (HOBs) attachment, spreading and proliferation compared to PLGA. HOBs differentiation was significantly upregulated when cells were cultured on 30 MBG/PLGA for 14 days, compared to 30 BG/PLGA. MBG/PLGA enhanced the accumulative release of dexamethazone (DEX) at early stages (0-200h) compared to BG/PLGA, however, after 200h, DEX-release rates for MBG/PLGA was slower than that of BG/PLGA. The contents of MBG in PLGA films can control the amount of DEX released. Taken together, MBG/PLGA films possessed excellent physicochemical, biological and drug-release properties, indicating their potential application for bone tissue engineering by designing 3D scaffolds according to their corresponding compositions.

Biological response of human bone cells to zinc-modified Ca-Si-based ceramics.

Calcium silicate (CaSiO(3)) ceramics have received considerable attention in recent years due to their excellent bioactivity and degradability. However, their poor chemical stability limits their biological applications. Hardystonite (Ca(2)ZnSi(2)O(7)) ceramics are Ca-Si-based materials developed by incorporating zinc into the Ca-Si system to improve their chemical stability. However, the biological responses of Ca(2)ZnSi(2)O(7) to bone cells are unknown. The objective of this study is to investigate and compare the in vitro responses of human osteoblast-like cells (HOBs) and osteoclasts when cultured on Ca(2)ZnSi(2)O(7) and CaSiO(3) ceramic disks. The ability of Ca(2)ZnSi(2)O(7) ceramics to support HOB attachment, cytoskeleton organization, proliferation and differentiation was assessed by scanning electron microscopy, confocal microscopy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, alkaline phosphatase activity and quantitative real-time polymerase chain reaction. Our results show that Ca(2)ZnSi(2)O(7) supported HOB attachment with a well-organized cytoskeleton structure, and significantly increased cellular proliferation and differentiation compared to CaSiO(3). In addition, Ca(2)ZnSi(2)O(7) showed increased expression levels of osteoblast-related mRNAs (alkaline phosphatase, collagen type I, osteocalcin, receptor activator of NF(kappa)B ligand and osteoprotegerin) compared to CaSiO(3). Ca(2)ZnSi(2)O(7) ceramic supported the formation of mature and functional osteoclasts and formed resorption imprints. On CaSiO(3) ceramics, the cells failed to differentiate from the monocytes into osteoclasts. Taken together, these results indicate that Hardystonite ceramics are conducive to both types of bone cells, osteoblast-like cells and osteoclasts, suggesting their potential use for skeletal tissue regeneration and as coatings onto currently available orthopedic and dental implants.

Factors regulating osteoclast formation in human tissues adjacent to peri-implant bone loss: expression of receptor activator NFκB, RANK ligand and osteoprotegerin

Aseptic bone loss adjacent to orthopedic joint implants is a common cause of joint implant failure in humans. This study investigates the expression of key regulators of osteoclast formation, receptor activator NFkappaB (RANK), Receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin (OPG), in the peri-implant tissues of patients with osteolysis compared with levels in synovial tissues from osteoarthritic and healthy subjects. Immunohistochemical studies demonstrated that significantly higher levels of RANKL protein (p<0.05) were found in the peri-implant tissues of patients with implant failure than in similar tissues from osteoarthritic and healthy subjects. In contrast, OPG protein levels were similar in all tissues. RANKL, expressed as mRNA and protein, was predominantly associated with cells containing wear particles. Dual labeling studies showed that the cells expressing RANKL protein were macrophages. In situ hybridization studies confirmed that mRNA encoding for these proteins is also expressed by cells in the peri-implant tissues. In addition, RANK mRNA was expressed in cells that contained wear particles. These findings show that abnormally high levels of RANKL are expressed in peri-implant tissues of patients with prosthetic loosening and that these abnormal levels of RANKL may significantly contribute to aseptic implant loosening.

The responses of osteoblasts, osteoclasts and endothelial cells to zirconium modified calcium-silicate-based ceramic

In this study we have developed Ca(3)ZrSi(2)O(9) (Baghdadite) ceramics by incorporating Zirconium in Ca-Si system and determined their biological properties. Ca(3)ZrSi(2)O(9) ceramics possess apatite-formation ability in simulated body fluid, indicating their potential bioactivity. The response of human osteoblast like cells (HOB), osteoclast and endothelial cells when cultured on Ca(3)ZrSi(2)O(9) ceramics was investigated. Scanning electron microscopy and immunofluorescence studies demonstrated that this material supports HOB cell attachment with organized cytoskeleton structure. Compared to CaSiO(3), Ca(3)ZrSi(2)O(9) ceramics induced increased HOB proliferation and differentiation as shown by increased methyltetrazidium salt (MTS), alkaline phosphatase activity, and mRNA expression levels of bone-related genes (Collagen type I, alkaline phosphatase, Bone Sialoprotein, receptor activator of NF-kappaB ligand and osteoprotegerin). Ca(3)ZrSi(2)O(9) ceramics supported the fusion of monocytes to form functional osteoclasts with their characteristic features of f-actin ring structures and the expression of alpha(v)beta(3) integrin consistent with functional activity. Osteoclasts cultured on Ca(3)ZrSi(2)O(9) expressed increased levels of osteoclast-related genes; Cathepsin K, Carbonic Anhydrase II, Matrix metalloproteinase-9, receptor activator of NF-kappaB and Calcitonin Receptor, consistent with the formation of functional osteoclasts. In addition to HOB and osteoclasts, Ca(3)ZrSi(2)O(9) supported the attachment of endothelial cells, which expressed the endothelial cell markers; ZO-1 and VE-Cadherin. Results presented here indicate that Ca(3)ZrSi(2)O(9) ceramics have the potential for applications in bone tissue regeneration.

Preparation and analysis of macroporous TiO2 films on Ti surfaces for bone-tissue implants.

This article describes the preparation and analysis of macroporous TiO2 films on Ti surfaces, for application in bone tissue-Ti implant interfaces. These TiO2 bioceramic films have a macroporous structure consisting of monodisperse, three-dimensional, spherical, interconnected pores adjustable in the micron size range. Micron-sized polystyrene (PS) bead templates are used to precisely define the pore size, creating macroporous TiO2 films with 0.50, 16, and 50 microm diameter pores, as shown by scanning electron microscopy. X-ray photoelectron spectroscopy shows the films to be predominantly composed of TiO2, with approximately 10% carbon. X-ray diffraction reveal rutile as the main phase when fired to the optimal temperature of 950 degrees C. Preliminary experiments find that the in vitro proliferation of human bone-derived cells (HBDC) is similar on all three pore sizes. However, higher [3H]thymidine incorporation by the HBDC is observed when they are grown on 0.50- and 16-microm pores compared to the 50-microm pores, suggesting an enhanced cell proliferation for the smaller pores.