Calcium Phosphate-rich Layer Research Articles

The Effect of Iron Oxide Insertion on the In Vitro Bioactivity, and Antibacterial Properties of the 45S5 Bioactive Glass.

The aging population and increasing incidence of trauma among younger age groups have heightened the increasing demand for reliable implant materials. Effective implant materials must demonstrate rapid osseointegration and strong antibacterial properties to ensure optimal patient outcomes and decrease the chance of implant rejection. This study aims to enhance the bone-implant interface by utilizing 45S5 bioglass modified with various concentrations of Fe3O4 as a coating material. The effect of the insertion of Fe3O4 into the bioglass structure was studied using Raman spectroscopy which shows that with the increase in Fe3O4 concentration, new vibration bands associated with Fe-related structural units appeared within the sample. The bioactivity of the prepared glasses was evaluated using immersion tests in simulated body fluid, revealing the formation of a calcium phosphate-rich layer within 24 h on the samples, indicating their potential for enhanced tissue integration. However, the sample modified with 8 mol% of Fe3O4 showed low reactivity, developing a calcium phosphate-rich layer within 96 h. All the bioglasses showed antibacterial activity against the Gram-positive and Gram-negative bacteria. The modified bioglass did not present significant antibacterial properties compared to the bioglass base.

Synthesis and characterization of bioscaffolds using freeze drying technique for bone regeneration

Abstract Bioscaffolds composed of polymeric biomaterials must be bio-compatible, absorbable and biodegradable upon implantation into humans. The aim of the current study is to synthesize highly porous bioscaffold that provides the appropriate environment for the regeneration of bone tissue. To accomplish this, bioscaffold was prepared by Freeze drying method using Beta-tricalcium phosphate, chitin and gelatin. The Scaffold was characterized by X-ray diffractometry (XRD), Fourier transform infrared spectroscopy (FTIR), and Field emission scanning electron microscopy (FESEM). The in-vitro bioactivity was tested by calcium phosphate rich layer on surface of soaked scaffold in different pH. FESEM demonstrated that the prepared scaffold had interconnected pores with a pore size of 50–60 nm. The XRD showed the composite structure and FTIR revealed the incorporation of Beta-tricalcium phosphate into chitin and gelatin. Porosity calculated by Archimedes principles was 76%. The degradation of scaffold biomaterial was pH dependent. The acidic pH had high weight loss initially ranging from 35% to 40% compared to neutral pH. However, the degradation rate was slowed down after 10 h. Thus, our study preliminarily concludes that Beta-tricalcium phosphate, chitin and gelatin incorporated bio-scaffolds could be an ideal scaffold for bone regeneration. However, further research is needed to confirm the use of synthesized scaffold for bone tissue engineering or bone defects.

Manufacturing and Characterization of Highly Porous Bioactive Glass Composite Scaffolds Using Unidirectional Freeze Casting

The aim of this work is the fabrication of bioactive and degradable scaffolds for bone tissue engineering. Freeze casting is used to obtain macropores. Alongside, highly bioactive 45S5 Bioglass, gelatin and chitosan are used as biocompatible binder and stabilizing agent, respectively. By varying the cooling rate between 2 and 4 K min−1 and whether the slurry is allowed to form a gelled network at 7 °C before freeze casting or not, samples with a porosity of 75% are achieved. X‐ray tomography analysis shows smallest pore sizes between 73 and 77 µm and a rather lamellar structure parallel to the freezing direction for the non‐gelled samples, whereas the gelled samples have smallest pores between 96 and 120 µm and show a rather cellular structure. Compression tests reveal compressive strengths from 2 (non‐gelled) to 3 MPa (gelled), while the quasielastic moduli of the gelled samples (44–46 MPa) clearly exceed values of the non‐gelled (20–23 MPa). Thus, it is concluded that the modified pore structure caused by the gelling process markedly improves the mechanical properties of the samples. After seven days in SBF under physiological conditions, a calcium phosphate rich layer is detected on the samples surface, revealing the bioactivity of the scaffolds.

Reinforcement of freeze-dried chitosan scaffolds with multiphasic calcium phosphate short fibers

The composite scaffolds of the chitosan and multiphasic calcium phosphate (HW) short fibers were prepared by freeze drying and characterized by X-ray diffractometry (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM and FE-SEM). The mechanical properties of the scaffolds were assessed by compression test. The incorporation of HW fibers consisting three phases of hydroxyapatite (HA), beta-tricalcium phosphate (β-TCP) and calcium pyrophosphate (CPP) into the chitosan matrices was associated with an increase in pore size, density and compressive strength and modulus, and a decrease in porosity and swelling ratio of the scaffolds. The strongest composite scaffolds in this study with a chitosan: HW fibers weight ratio of 1:1 showed a mean porosity of 69% and a mean strength and modulus of 420kPa and 3.87MPa, respectively. The in vitro bioactivity of the composites was confirmed by the formation of a calcium phosphate rich layer on the surface of soaked scaffolds in simulated body fluid. The findings of this initial work indicate that the chitosan–multiphasic calcium phosphate short fibers may be a suitable material for bone scaffolding.

Imaging Physicochemical Reactions Occurring at the Pore Surface in Binary Bioactive Glass Foams by Micro Ion Beam Analysis

In this work, the physicochemical reactions occurring at the surface of bioactive sol-gel derived 3D glass scaffolds via a complete PIXE characterization were studied. 3D glass foams in the SiO(2)-CaO system were prepared by sol-gel route. Samples of glass scaffolds were soaked in biological fluids for periods up to 2 days. The surface changes were characterized using particle induced X-ray emission (PIXE) associated to Rutherford backscattering spectroscopy (RBS), which are efficient methods to perform quantitative chemical maps. Elemental maps of major and trace elements at the glass/biological fluids interface were obtained at the micrometer scale for every interaction time. Results revealed interconnected macropores and physicochemical reactions occurring at the surface of pores. The micro-PIXE-RBS characterization of the pores/biological fluids interface shows the glass dissolution and the rapid formation of a Ca rich layer with the presence of phosphorus that came from biological fluids. After 2 days, a calcium phosphate-rich layer containing magnesium is formed at the surface of the glass scaffolds. We demonstrate that quantities of phosphorus provided only by the biological medium have a significant impact on the development and the formation of the phosphocalcic layer.

Orthophosphate nanostructures in SiO 2–P 2O 5–CaO–Na 2O–MgO bioactive glasses

Vibrational spectroscopy, 29Si and 31P magic-angle spinning nuclear magnetic resonance spectroscopy and high resolution transmission electron microscopy were used to investigate structural aspects of SiO 2–P 2O 5–CaO–Na 2O–MgO glasses. The experimental results show that for the two compositions, 25.3SiO 2–10.9P 2O 5–32.6CaO–31.2MgO and 33.6SiO 2–6.40P 2O 5–19.0CaO–41.0MgO, phosphorous is present in a nano-crystalline form with interplanar distances in the 0.21–0.26 nm range. The two glasses develop a surface CaP-rich layer and the presence of any intermediate silica-rich layer was not detected. It was suggested that the phosphate nano-regions may play a key role in the initial stages of the bioactive process, acting as nucleation sites for the calcium phosphate-rich layer.

Micro-PIXE–RBS methods highlighting the influence of phosphorus on the in vitro bioactivity of sol–gel derived glass particles in the SiO2–CaO–P2O5 system

Ion beam analysis methods were used to characterize the interface of bioactive glasses with surrounding biological fluids. Glass particles in the SiO2–CaO and SiO2–CaO–P2O5 compositions were made by sol–gel processing and soaked in biological fluids for periods up to 4 days. The surface changes were characterized using PIXE–RBS, which are efficient methods for multielemental analysis and accurate trace elements quantification. Elemental maps of major and trace elements were obtained at a micrometer scale and revealed the bone bonding ability of the materials. Glass particles are quickly coated with a thin calcium phosphate-rich layer containing traces of magnesium. After a few days, SiO2–CaO–P2O5 glass particles are entirely changed into calcium phosphates, whereas SiO2–CaO particles exhibit a different behavior: the previously Ca–P enriched periphery has been dissolved and glass particles consist of a silicate network. Calculation of the Ca–P atomic ratios at the glass/biological fluids interface provides us with an explanation for this: an enduring apatitic phase seems to be formed at the periphery of SiO2–CaO–P2O5 glass particles. Presence of phosphorus in the glass matrix thus has an influence on the amplitude and the kinetics of reaction of the bioactivity process. It might result in an improved chemical bond with living tissues.

Biological activity of a SiO2–CaO–P2O5 sol–gel glass highlighted by PIXE–RBS methods

It is proposed in this study to observe the influence of P2O5 on the formation of the apatite-like layer in a bioactive glass via a complete PIXE characterization. A glass in the SiO2–CaO–P2O5 ternary system was elaborated by sol–gel processing. Glass samples were soaked in biological fluids for periods up to 10 days. The surface changes were characterized by a micro-analytical technique of particle induced X-ray emission (PIXE) associated to Rutherford backscattering spectroscopy (RBS), in which multi-elemental distributions are efficiently analyzed. Elemental maps of major and trace elements were obtained at a micrometer scale and revealed the bone bonding ability of the material. A calcium phosphate-rich layer containing magnesium appears a few days after the interaction. We demonstrate that the presence of phosphorus in the material has an impact on the development and the formation rate of the bone-like apatite layer. Indeed, the Ca/P atomic ratio at the glass/biological fluids interface is closer to the nominal value of pure apatite compared to P2O5-free glasses. It would permit, in vivo, an improved chemical bond between the biomaterials and bone.

In Vitro studies of bioactive glass/polyhydroxybutyrate composites

Bioactive materials can help bone reparation and regeneration by offering support to bone growth. In vitro studies of bioactive glass/polyhydroxybutyrate composites were carried out to evaluate the influence of the composition on the bioactivity through the presence of calcium phosphate (Ca-P) on the layer formed when the substrates were immerse in simulated body fluid (SBF). The in vitro tests were carried out by soaking the composites bioactive glass/polyhydroxybutyrate 30/70 and 40/60 in SBF. The surface of the composites was analyzed by Scanning Electron Microscopy (SEM) with Energy Dispersive Spectroscopy (EDS) and also via x ray Diffraction (XRD). The solutions were analyzed by Inductively Couple Plasma (ICP). SEM images show a formation of a Ca-P rich layer on surface of composites. XRD results characterized the layer as calcium hydrogen phosphate. Ca/P ratios found via EDS results show a value close to 1.67. According to ICP results, the Ca e P ions are from SBF.

Microstructure and macroscopic properties of bioactive CaO–SiO2–PDMS hybrids

CaO-SiO2-PDMS (polydimethylsiloxane) hybrid materials were synthesized as crack-free monoliths presenting in vitro bioactivity, i.e. able to be coated with a calcium phosphate-rich layer after having been soaked in simulated body fluid (SBF). A wide physical-chemical characterization of these materials was carried out to relate their microscopic structure and macroscopic properties. The effect of PDMS and the amounts of water used for the tetraethoxysilane (TEOS) hydrolysis on the mechanical properties of hybrid materials was investigated by three-point bending tests. For a given amount of water, as PDMS content in hybrids increased, the elastic modulus decreased. Furthermore, keeping the PDMS content constant, when the amount of H2O decreased, the elastic modulus increased. Regarding in vitro bioactivity and mechanical properties, the hybrid material obtained with molar ratios H2O/TEOS = 2 and TEOS/PDMS = 3.5 proved to be the best candidate for either soft tissue substitution or metallic implant coating since the hybrid material would promote bond to bone formation, simultaneously dampening the mechanical charges.

The biocompatibility and osteoconductivity of a cement containing β–TCP for use in vertebroplasty

A new composite bone cement designated "G2B1" was developed for percutaneous transpedicular vertebroplasty. G2B1 contains beta tricalcium phosphate particles and methylmethacrylate-methylacrylate copolymer as the powder components, and methylmethacrylate, urethane dimethacrylate, and tetrahydrofurfuryl methacrylate as the liquid components. Biocompatibility and osteoconductivity were evaluated using scanning electron microscopy, contact microradiography, and Giemsa surface staining 4, 8, 12, 26, and 52 weeks after implantation into rat tibiae. To evaluate osteoconductivity, affinity indices (%) were calculated. Scanning electron microscopy and contact microradiography revealed that bone contact with G2B1 was attained within 4 weeks (affinity index: 50.2 +/- 11.8 at 4 weeks) and at most of the margin within 26 weeks (affinity index: 87.4 +/- 7.2 at 26 weeks). Specifically, G2B1 contacted bone via a wide calcium-phosphate-rich layer, and its degradation started within 8 weeks, mainly in the marginal area. Giemsa surface staining showed that there was almost no inflammatory reaction around the G2B1. These results indicate that G2B1 is a biocompatible and osteoconductive bone cement.

Micro-PIXE characterization of interactions between a sol–gel derived bioactive glass and biological fluids

Bioactive glasses possess the ability to bond to living tissues through the formation of a calcium phosphate-rich layer at their interface with living tissues. This paper reports the different steps of this bioactivity process via a complete micro-PIXE characterization of a sol–gel derived SiO 2–CaO bioactive glass in contact with biological fluids for different delays. Multi-elemental cartography at the glass/biological fluids interface together with major and trace elements quantification permit a better understanding of the five reaction stages involved in the bioactivity mechanisms. The presence of phosphorus was detected at the periphery of the material within 6 h of interaction with biological fluids. A calcium phosphate-rich layer containing magnesium is formed after a few days of interaction and presence of bone-like apatite is deduced from the calculation of the Ca/P ratio at the material interface. That is of deep interest for clinical applications, because this biologically active behavior results in the formation of a strong interfacial bond between the glass and host tissues, and will stimulate bone–cell proliferation.

Solution Mediated Effect of Bioactive Glass in Poly (Lactic-Co-Glycolic Acid)- Bioactive Glass Composites on Osteogenesis of Marrow Stromal Cells

A previous study demonstrated that the incorporation of bioactive glass (BG) into poly (lactic-co-glycolic acid) (PLGA) can promote the osteoblastic differentiation of marrow stromal cells (MSC) on PLGA by forming a calcium phosphate rich layer on its surface. To further understand the mechanisms underlying the osteogenic effect of PLGA-BG composite scaffolds, we tested whether solution-mediated factors derived from composite scaffolds/hybrids can promote osteogenesis of marrow stromal cells. The dissolution product from PLGA-30%BG scaffold stimulated osteogenesis of MSC, as was confirmed by increased mRNA expression of osteoblastic markers such as osteocalcin (OCN), alkaline phosphatase (ALP), and bone sialoprotein (BSP). The three-dimensional structure of the scaffolds may contribute to the production of cell derived factors which promoted distant MSC differentiation. Thus PLGA-BG composites demonstrates significant potential as a bone replacement material.

Effect of pH and ionic strength on the reactivity of Bioglass® 45S5

Bioglass® 45S5 is a silica-based melt-derived glass, used in medical field as a bone regenerative material because of the deposition of a layer of hydroxy carbonate apatite (HCA) on the surface of the glass when immersed in body fluid.The present paper studies the early steps of reaction of 2-μm sized particles of Bioglass®, in solutions buffered with TRIS at different pH, by means of ICP-ES and FTIR spectroscopy. Only at pH 8 could a total reconstruction of the glass be observed, with the formation of both a silica and a calcium phosphate rich layers. At higher pH, selective dissolution of the glass was hindered by the immediate precipitation of a layer of calcium phosphate, whereas at lower pH a total breakdown of the glass occurred and no calcium phosphate precipitation was noted. The use of the ATR-liquid cell allowed the observation of the reaction in real time, and this showed that the process of silica formation is not separable from cation leaching from the glass, as well as the formation of the calcium phosphate rich layer.

Bioactive Ceramic And Model Surfaces Adsorb Ligands and Factors That Stimulate Cellular Function

Abstract Microscopy and microanalysis techniques have played an important role in our understanding of how biomaterials interact with their environment. In first part of this study, we will focus on the behavior of bioactive glass, whereas in the second a model surface will be investigated. Upon implantation bioactive glass undergoes a series of reactions that leads to the formation of a calcium phosphate-rich layer. Most in vitro studies of the changes that occur on the surface of bioactive glass have employed the use of buffer solutions with compositions reflecting the ionic composition of interstitial fluid. Although these studies have documented the physical and chemical changes associated with bioactive glass immersed in aqueous media, they do not reveal the effect of serum proteins and cells which are present at the implantation site. In the present study, we document, using atomic force microscopy (AFM) and Rutherford backscattering spectrometry (RBS), significant differences in reaction layer composition, thickness, morphology and kinetics of formation arising from the presence of serum proteins.

Bioactive glass serves as a substrate for maintenance of phenotype of nucleus pulposus cells of the intervertebral disc

The objective of the study was to investigate the capability of bioactive glass 45S5 to serve as a substrate for nucleus pulposus cells in vitro. Nucleus pulposus cells were isolated from adult rabbit discs and seeded onto bioactive glass. At selected time intervals, the cells and glass were evaluated. We found that the cells rapidly attached to the substrate, colonizing it within 12 h. By 21 days, they had formed a lawn of cells over the glass substrate. DNA measurements showed a progressive increase in cell number with time. The phenotype was maintained; the cells expressed aggrecan, and collagen type II and I, but were negative for collagen type X. CD44, a cell-surface glycoprotein that binds hyaluronate, was also expressed by these cells. Electron dispersive X-ray analysis and Fourier transform infrared spectroscopy revealed calcium phosphate-rich layer formation on the substrate surface. The results of this study suggest that nucleus pulposus cell proliferation may be an anchorage dependent event, and that the cells use the calcium phosphate-rich layer to facilitate cell adhesion, and subsequent proliferation. These findings point to the importance of the conditioned bioactive glass as a substrate for nucleus pulposus cells.

In vitro bioactivity of glass and glass-ceramics of the 3CaO·P 2O 5–CaO·SiO 2–CaO·MgO·2SiO 2 system

A glass of nominal composition (wt%) 40.0 CaO–34.5 SiO 2–16.5 P 2O 5–8.5 MgO–0.5 CaF 2 has been obtained (G13). The glass showed in vitro bioactivity evidenced by the formation on its surface of a calcium phosphate-rich layer when soaked in a solution with ionic composition analogous to human plasma. By thermal treatments of G13, a glass-ceramic (GC13) containing apatite, diopside, althausite and akermanite as crystalline phases was developed. GC13 as-made did not show in vitro bioactivity. However, after chemical treatment of GC13 with 1 m HCl (GC13-HCl), the in vitro studies showed the formation of an apatite-like layer covering certain areas of the material surface. The influence of both chemical and morphological factors on the in vitro bioactivity has been studied.

Effect of magnesium content on the in vitro bioactivity of CaO-MgO-SiO2-P2O5 sol-gel glasses

Three glasses in the system CaO-MgO-SiO 2 -P 2 O 5 , with high silica content (80 mol% SiO 2 ) and 0, 3 and 7 mol% of MgO, respectively, have been prepared by the sol-gel method. The in vitro bioactivity of pellets, prepared by compacting the glass powders, was assessed by determining the changes in surface morphology and composition after soaking in a simulated body fluid (SBF) for up to 14 days at 37 °C. Formation of a calcium phosphate rich layer on the glasses surface was followed by scanning electron microscopy (SEM), X-ray energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD). Three effects of MgO in the glass upon the newly formed layer were detected: (i) it slows down the rate of formation of the layer, (ii) it increases the thickness of the layer and (iii) a whitlockite-like phase appeared together with the apatite-like phase formed on the surface of bioactive materials when exposed to physiological solutions.

Formation of surface reaction products on bioactive glass and their effects on the expression of the osteoblastic phenotype and the deposition of mineralized extracellular matrix

The objective of the study was to examine the effect of alkali ion release, pH control and buffer capacity on the expression of the osteoblastic phenotype. In addition, we determined the importance of modifications of the surface of porous bioactive glass (BG) on the activity of rat calvaria osteoblasts in vitro. We found that at a low tissue culture medium (TCM) volume to BG surface area (Vol/SA) ratio, the products of glass corrosion elevated the pH of the TCM to a value that adversely affected cellular activity; thus, the matrix synthesized by the cells was non-mineralized. On the other hand, when the Vol/SA was high and the buffer capacity of the medium was not exceeded, the cells generated a mineralized extracellular matrix. Addressing the second issue, we observed that modification of the composition of the BG surface markedly influenced osteoblast activity. BG that was coated with either a calcium phosphate-rich layer only or a serum protein layer changed the phenotypic characteristics of the osteoblasts. The presence of either of these surfaces lowered the alkaline phosphatase activity of the attached cells; this finding indicated that the osteoblast phenotype was not conserved. However, when the BG was coated with a bilayer of calcium phosphate and serum proteins, the alkaline phosphatase (AP) activity was elevated and the extracellular matrix contained characteristic bone markers. Our findings indicate that the calcium phosphate-rich layer promotes adsorption and concentration of proteins from the TCM, and it is utilized by the osteoblasts to form the mineralized extracellular matrix.

Changes in the elemental composition of Bioglass during its surface development in the presence or absence of proteins

The present study examined the elemental distribution with depth from the surface of Bioglass 45S5 immersed in simulated body fluid (SBF) in the presence or absence of proteins derived from foetal calf serum for 960 h using energy dispersive spectroscopy (EDS). The changes in elemental composition of the surface of Bioglass were also analysed with EDS for different immersion times (10 min to 960 h) before and after immersion in SBF with or without proteins. The presence of proteins consistently altered the elemental composition of Bioglass with depth from the surface, although the development of a silica rich layer was similar in both the presence or absence of proteins. In the presence of proteins the accumulation of calcium phosphate at the surface of Bioglass was slower, formed a narrower layer and had lower concentrations of calcium and phosphate than the calcium phosphate rich layer that developed in SBF alone.