Scanning probe microscopy of intrafibrillar crystallites in calcified collagen

Vertebrate mineralized tissues are composite materials formed by the organized growth of carbonated apatite crystals within a matrix of collagen fibres. Calcified collagen from turkey tendon was investigated using scanning tunnelling microscopy (STM) and atomic force microscopy (AFM). Samples were treated with hydrogen peroxide to enhance the mineralized phase by removing part of the collagen matrix and the results compared with the untreated material. Plate-like crystalline entities with dimensions 35 nm × 5–8 nm by 1.5 nm were seen. These dimensions are consistent with previous reports using transmission electron microscopy (TEM) of calcified tendon and topographic imaging of tendon crystals. The resolution of the images obtained using STM is better than the previously reported pictures obtained using TEM or scanning electron microscopy (SEM). The value of 35 nm is the same as the gap region in the structure of the collagen fibrils. Stacking of plates and plate-aggregates are a dominant feature in the scanning images. These results support the concept of organized intra-fibril mineral crystals within the organic collagen matrix. Electron diffraction and X-ray diffraction were undertaken on the samples and the patterns recorded match those previously reported for carbonated apatite.     

In vitro crystallization of octacalcium phosphate on type I collagen: influence of serum albumin

The heterogeneous crystallization of octacalcium phosphate (OCP, Ca₈H₂(PO₄)₆ · 5H₂O) on demineralized Type I collagen has been studied from metastable supersaturated solutions, at 37°C and pH=6.50, using the constant composition crystal growth technique. The induction period, before OCP crystal growth, varied markedly with the degree of supersaturation of the solution. The data obtained allowed us to determine the apparent order for the precipitation and the growth mechanism of OCP on Type I collagen. Infrared spectroscopy analyses indicated the progressive mineralization of collagen and observations by scanning electron microscopy confirmed the development of OCP crystals on the collagen surface. The influence of bovine serum albumin on both the kinetics of OCP nucleation and growth has also been investigated. Because this protein was adsorbed on calcium phosphate nuclei, it exhibited two distinct effects as a function of its concentration in solution. We proposed a mechanism explaining the interaction between albumin and calcium phosphate nuclei or crystals and its incidence on the OCP crystallization kinetics. Observations by scanning electron microscopy revealed a modification of the size and the appearance of crystals grown on collagen due to the adsorption of albumin on the crystal surface. © 1999 Kluwer Academic Publishers     

Incorporation of a controlled-release glass into a calcium phosphate cement

A so-called controlled-release glass was synthesized occurring in the system CaO-Na₂O-P₂O₅. A certain sieve fraction of this glass was incorporated in a calcium phosphate cement, of which the powder contained -tricalcium phosphate (-TCP), dicalcium phosphate (DCP) and precipitated hydroxyapatite (HA). The glass appeared to retard the cement setting slightly and it reduced considerably the compressive strength after aging in aqueous solutions which were continuously refreshed. Scanning electron microscope (SEM) pictures and X-ray diffraction (XRD) patterns of the samples after 5 weeks of aging showed that the glass was not dissolved but that large brushite crystals were formed. Thereby, aging in CaCl₂ solutions resulted in more brushite formation than aging in NaCl solutions. The brushite crystals did not reinforce the cement. Neither was the aged glass-containing cement weaker than it was before the brushite formation right after complete setting. In conclusion, the incorporation of controlled-release glasses into a calcium phosphate cement and subsequent aging in aqueous solutions did not result in the formation of macropores in the cement structure, but that of brushite crystals. This incorporation reduced the compressive strength of the cement considerably. © 1999 Kluwer Academic Publishers     

Further studies of calcium phosphate growth on phosphorylated cotton fibres

Further studies using scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDX), micro-Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and solid state magic angle spinning nuclear magnetic resonance (MAS NMR) techniques of calcium phosphate growth on Ca(OH)₂-treated urea/H₃PO₃- and urea/H₃PO₄-modified cotton fibres are reported. In the case of the Ca(OH)₂-treated urea/H₃PO₃-modified fibres which have been reported in an earlier paper, further experiments subjecting the urea/H₃PO₃-modified cotton to alternative soaking treatment procedures to Ca(OH)₂ as well as different calcium phosphate growth media such as the alkaline phosphatase-catalysed hydrolysis of disodium p-nitrophenylphosphate to free phosphate have reaffirmed the importance of the Ca(OH)₂ treatment step for the stimulus and growth of calcium phosphate growth on the fibres. Studies on cotton phosphorylated by a slightly different method using urea/H₃PO₄ instead of urea/H₃PO₃ show that a phosphorylated cotton with similar properties to the urea/H₃PO₃-modified fibres can be produced. Soaking of these fibres in saturated Ca(OH)₂ solution leads to cotton coated with thin layers of calcium phosphate formed by partial hydrolysis of the PO₄ functionalities in the phosphorylated cotton which are believed to act as nucleation layers for further calcium phosphate deposition when the fibres are subsequently soaked in 1.5×SBF solution. SEM/EDX studies of the calcium phosphate coatings formed on the Ca(OH)₂-treated urea-H₃PO₄ fibres as a function of soaking time in 1.5 × SBF show that coatings deposit and become noticeably thick after approximately 9 days. XPS studies indicated the presence of carbonate species in the calcium phosphate coating deposited. In common with the calcium phosphate coated Ca(OH)₂-treated urea/H₃PO₃-modified fibres studied earlier, the average EDX-measured Ca: P ratios of the coatings formed on the Ca(OH)₂-treated urea/H₃PO₄ fibres are 1.60 and give very similar micro-FTIR spectra with evidence of carbonate which suggests that amorphous calcium deficient apatite has deposited.     

Hydroxyapatite crystal growth on calcium hydroxyapatite ceramics

Calcium hydroxyapatite (Ca-HAP) ceramics containing tricalcium phosphate (TCP) were soaked in three solutions: phosphate buffer, tris buffer, and simulated body fluid (SBF). Petal-like crystals of Ca-HAP were deposited on the Ca-HAP ceramics when (i) Ca-HAP ceramics contained -TCP, (ii) the soaking solution contained phosphate ion and (iii) the pH of soaking solution was higher than 7.3. These conditions facilitate the presence of HPO ₄ ^2– and Ca²⁺ ions, the latter from dissolution of -TCP. A well-defined X-ray diffraction pattern for the deposited Ca-HAP crystals indicates preferred growth of {002} planes. Slower crystal growth of Ca-HAP was found for SBF (pH=7.5) than in the phosphate buffer, due possibly to the lower phosphate ion content in SBF.     

Self-assembled right handed helical ribbons of the bone mineral hydroxyapatite

Osteointegration of titanium or its alloy with bone can be greatly improved by calcium phosphate coatings, and further enhanced by an extracellular matrix protein layer such as collagen. In this study, an octacalcium phosphate (OCP)/collagen composite coating layer on Ti6Al4V substrate was prepared using electrolytic deposition method. A layer of OCP mineral consisting of flake-like crystals was first formed on the Ti6Al4V substrate. Subsequently, mineralized collagen fibrils were deposited on the former OCP layer. These collagen fibrils were interconnected and well adhered on the OCP layer so that they were immobilized. The microstructure of the composite coating varied with collagen concentration in the electrolyte. This study could offer a possibility of fabricating a desired surface matrix on orthopedic implants to enhance bone formation and fixation of implants.     

Growth of calcium phosphate on surface-modified cotton

A study of the growth of amorphous calcium phosphate on surface-modified cotton fibres by a combination of scanning electron microscopy/electron diffraction X-ray analysis, micro-FTIR and X-ray photoelectron spectroscopy is reported. Cotton fibres phosphorylated by the urea/phosphorous acid method and then soaked in saturated Ca(OH)₂ for approximately one week were found to stimulate the growth of a calcium phosphate coating on their surfaces after soaking in 1.5×SBF for as little as 1 day. Ca(OH)₂ soaking of the fibres is found to produce highly crystalline clusters lodged in the fibres which were confirmed by micro-FTIR to be calcium phosphite monohydrate (CaHPO₃·H₂O). In contrast, phosphorylated fibres not subjected to the Ca(OH)₂ treatment did not exhibit calcium phosphate growth upon immersion in 1.5×SBF solution. Soaking of the Ca(OH)₂-treated fibres with time in the 1.5×SBF solution produced progressively thicker layers of calcium phosphate on the fibres as confirmed by scanning electron microscopy and X-ray photoelectron spectroscopy. In general, calcium phosphate coatings formed over 1 1–5 day period soaking in 1.5×SBF solution appeared to consist of agglomerations of a large number of small spherical particles, while coatings formed after 17 days of soaking were distinctly chunky, thick and non-uniform in appearance. Micro-FTIR indicated that CaHPO₃·H₂O clusters were still present in cotton samples even after 4 days of soaking, while after 17 days, only the infrared spectrum typical of calcium phosphate was observed. EDX-measured Ca:P ratios of the coatings, although variable, suggested amorphous calcium phosphate. The mechanism of formation of the coating is believed to involve dissolution of the CaHPO₃.H₂O clusters upon introduction of the Ca(OH)₂-treated phosphorylated cotton into the 1.5×SBF solution which elevates the Ca²⁺ ion concentration in the vicinity of the fibres so stimulating calcium phosphate formation. It is postulated that phosphite groups chemically bound to the cotton fibre surface or a calcium phosphite coating on the fibres act as nucleation sites for calcium phosphate growth in 1.5×SBF solution.     

Glucuronic acid and phosphoserine act as mineralization mediators of collagen I based biomimetic substrates

Glucuronic acid (GlcA) and phosphoserine (pS) carrying acidic functional groups were used as model molecules for glycosaminoglycans and phosphoproteins, respectively to mimic effects of native biomolecules and influence the mineralization behaviour of collagen I. Collagen substrates modified with GlcA showed a stable interaction between GlcA and collagen fibrils. Substrates were mineralized using the electrochemically assisted deposition (ECAD) in a Ca²⁺/H _x PO₄^(3−x) electrolyte at physiological pH and temperature. During mineralization of collagen–GlcA matrices, crystalline hydroxyapatite (HA) formed earlier with increasing GlcA content of the collagen matrix, while the addition of pS to the electrolyte succeeded in inhibiting the transformation of preformed amorphous calcium phosphate (ACP) to HA. The lower density of the resulting mineralization and the coalesced aggregates formed at a certain pS concentration suggest an interaction between calcium and the phosphate groups of pS involving the formation of complexes. Combining GlcA-modified collagen and pS-modified electrolyte showed dose-dependent cooperative effects.     

The growth of calcium phosphate on ceramic surfaces

Calcium phosphate ceramics are very important materials for prosthetic applications because of their excellent biocompatibility. The chemical composition of the ceramics is determining, both with respect to their capability of inducing calcium phosphate formation and regarding the crystalline phase formed. From a series of porous ceramics based on hydroxyapatite (HAP) containing metal oxides, it was found that only those containing ZrO₂ stabilized with 8% Y₂O₃ were able to induce calcium phosphate formation upon introduction into calcium phosphate supersaturated solutions. The overgrowing phase was increasingly crystalline, did not show any other characteristic X-ray peaks and infrared bands than those pertinent to a-tricalcium phosphate and had a molar CaP ratio of 32. Kinetics analysis with respect to Ca(PO₄)₂ yielded an apparent order of reaction of 5.0±0.5, suggesting polynuclear nuclei above nuclei growth. The surface energy calculated from the kinetics data for the crystalline overgrowth was found to be 88 mJ m^–2.     

Early tissue response to titanium implants inserted in rabbit cortical bone

The tissue response to screw-shaped implants of commercially pure titanium was studied 3–180 days after insertion in the rabbit tibia by means of transmission electron microscopy. Red blood cells and scattered macrophages predominated at the implant surface after 3 days. At day 7 and later intervals, multinuclear giant cells were the cell type found at the implant surface protruding into the bone marrow and in areas with no bone-titanium contact. Osteoblasts or mesenchymal cells were rarely seen at the implant surface at any time period. Two modes of mineralization could be distinguished in the interface. Firstly, the typical mineralization of osteoid seams produced by osteoblasts. Secondly, an accumulation of scattered hydroxyapatite crystals in the unmineralized collagen matrix in the interface. Mineralized tissue was observed close to the implants surface from day 14. However, the innermost 2–20 m were poorly mineralized although scattered hydroxyapatite crystals were present. The collagen fibrils did not reach the implant surface but were separated from it by an amorphous layer, being 0.3–0.5 m thick which did not decrease in width with time. An electron-dense lamina limitans-like line containing mineral was observed between the amorphous layer and the bone tissue.     

Comparative investigation of hydroxyapatite/collagen composites prepared by CaCl<Subscript>2</Subscript> addition at different time points in collagen self-assembly process

Mineralization of collagen is a common combination process mainly involving collagen self-assembly and hydroxyapatite formation. Previous measurement of turbidity showed that collagen self-assembly follows the nucleation–growth model. In the present study, 1.0 M CaCl₂ was mixed with 0.6 mg/mL collagen solution in PBS (10 mM phosphate, 80 mM NaCl, pH 7.2, and T = 35 °C) at the beginning of the lag period, at the beginning of the growth period, at t_1/2 (the time to reach half of the total turbidity change), and during the plateau period. Four different hydroxyapatite/collagen (HAp/COL) composites were prepared [COL (1), COL (2), COL (3), and COL (4)]. The optical densities increased with CaCl₂ addition, and a wave trough appeared in the mineralization kinetic curves because of amorphous/crystalline conversion. HAp formation was confirmed by Fourier transform infrared spectroscopy and X-ray diffraction measurements. Energy-dispersive spectroscopy results showed that the calcium/phosphorus ratio of COL (2) is close to that of human bone. Images obtained by scanning electron microscopy revealed that nanosized plate-like HAp of COL (2) formed and became uniformly embedded in collagen, whereas HAp formed large clusters in COL (1), COL (3), and COL (4). Quantitation analyses of collagen and HAp incorporated into composites showed that mineralization at different time points promote collagen fibril generation and have little impact on the HAp content. These results suggest that the composite fabricated by addition of CaCl₂ at the beginning of the growth period is a promising material for bone repair and implantation.     

Investigation into the formation and mechanical properties of a bioactive material based on collagen and calcium phosphate

A biomaterial composite was formed by the room-temperature precipitation of calcium phosphate (in the form of brushite) on to collagen. It was found that the addition of 1 mM O-phosphoserine (Ser P) causes the morphology of the brushite crystals to change from large plates to small needles. An increase in the surface coverage and weight fraction of brushite incorporated into the collagen was observed. The mechanical properties of this composite were tested in the wet state. The ultimate tensile strength (UTS) was 45 MPa compared with 34 MPa for the wet collagen. Osteoblastic differentiation was promoted on the surface of the material and new bone formed.     

Preparation and characterization of bioactive collagen/wollastonite composite scaffolds

A novel biodegradable collagen/wollastonite composite was prepared as three-dimensional scaffolds by freeze-drying method. Scanning electron microscope (SEM) micrographs of scaffolds showed a continuous structure of interconnected pores, and pore size was about 100 m. The tensile strength of the scaffolds was improved by incorporation of wollastonite and the in vitro bioactivity of the scaffolds was evaluated by examining the hydroxyapatite (HA) deposition on their surface in simulated body fluid (SBF). After soaking in SBF for 7 days, collagen reconstituted to fibers and HA nodules formed on collagen fibers. The result suggests that the incorporation of wollastonite could improve the mechanical strength and the in vitro bioactivity of the composite. The scaffolds could be a potential biomaterial for bone tissue engineering.     

Surface modification of calcium metaphosphate fibers

-calcium metaphosphate fibers having high aspect ratios of 10–120 with diameters of 2–10 m show high strength and good biocompatibility. When the fibers are soaked in simulated body fluid at 37 °C, however, no calcium phosphate phase is newly formed on the fibers. In the present work, by treating the fibers at 70 °C with dilute NaOH aqueous solution, the surface phase was converted successfully into the orthophosphate phase that was in fine sizes and was adhered. After soaking the treated fibers in simulated body fluid at 37 °C for 30 days, a new calcium phosphate phase was precipitated. This was attributed to the surface phase modified using dilute NaOH. The treated fibers are expected to show bone-bonding ability, i.e. bioactivity. ©2000 Kluwer Academic Publishers     

Thin hydroxyapatite layers formed on porous titanium using electrochemical and hydrothermal reaction

In our previous study, it was found that hydroxyapatite (HA) microcrystals were precipitated by hydrothermal treatment on an anodic titanium oxide film containing calcium and phosphorus (AOFCP) with an equivalent Ca/P ratio to HA, which was formed on a titanium metal anode in an aqueous electrolytic solution of dissolved calcium acetate and -glycerophosphate. In this study, the formation mechanism of the AOFCP has been clarified. Spark discharges, which occur on titanium surface with a large amount of heat generation, cause crystallization of the TiO₂ matrix of the AOFCP and incorporation of calcium and phosphorus into the matrix from these electrolytes simultaneously. The calcium and phosphorus in the matrix seem to exist as ions rather than as calcium phosphate. Also, thin HA layers consisting of the many precipitated microcrystals can be uniformly formed even on titanium with complex shapes or surface geometries such as the mesh, roughened surfaces and bead-coated porous coating by the present method.     

Calcium phosphate cement: in vitro and in vivo studies of the α-tricalcium phosphate-dicalcium phosphate dibasic-tetracalcium phosphate monoxide system

In this paper, calcium phosphate cement consisting of -tricalcium phosphate (-TCP), dicalcium phosphate dibasic (DCPD) and tetracalcium phosphate monoxide (TeCP) was investigated in vitro and in vivo. Measurements of compressive strength against soaking time in simulated body fluid (SBF) showed a rapid increase of the hardness for the first 7 days. The gained strength was retained up to 1 year and the maximal mean value was 94.7 (±14.4) MPa. X-ray diffraction (XRD) and scanning electron microscopy (SEM) presented precipitates of hydroxyapatite (HA) after mixing, also after soaking in SBF and after implantation in rat subcutaneous tissues. However, the conversion to HA happened in different ways between in vitro and in vivo exposures. Histologic examinations showed that the cement causes the same reactions at the interface with surrounding soft tissues as HA. The authors consider the cement to be a promising material as a bone substitute, bone cement or dental material, however, further studies in a paste form and in bone tissue environments are necessary.     

Piezoelectric properties of collagen-nanocrystalline hydroxyapatite composites

In this paper we did a study of the physicochemical, dielectric and piezoelectric properties of anionic collagen and collagen-hydroxyapatite (HA) composites, considering the development of new biomaterials which have potential applications in support for cellular growth and in systems for bone regeneration. The piezoelectric strain tensor element d ₁₄, the elastic constant s ₅₅, and the dielectric permittivity ₁₁ were measured for the anionic collagen and collagen-HA films. For the collagen-HA composite film (Col-HACOM) the main peaks associated to the crystalline HA is present. For the nanocrystalline composite, nanometric HA powder (103 nm particle size) (HAN), obtained by mechanical milling were used. For the composite film (Col-HAN) the HA and CaH(PO₄)₂H₂O phases were detected. One can see that the HA powder (HAN) present the main peaks associated to crystalline HA. The IR spectroscopy measurements on HA-COM and HAN powders, Col-HACOM and Col-HAN composite films and collagen film (Col) presents the main resonances associated to the modes of (PO₄)^3–, (CO₃)^2–. The IR spectra of Collagen Film (Col) shows the bands associated to amide I (C=O), amide II (N–H) and amide III (C–N) vibrational modes. The scanning electron photomicrography of the Col-HACOM and Col-HAN films, respectively, shows deposits of HA on the surface of collagen. It also shows that HACOM crystals has a dense feature, whereas the HAN crystals has soft porous surface. Energy-dispersive spectroscopy (EDS) analysis showed that the main elements of the hybrid sponge were carbon, oxygen, calcium, and phosphorus. The EDS of HACOM crystal, present in the Col-HACOM composite showed a molar ratio Ca/P = 1.71, whereas the Col-HAN composite the molar ratio of calcium and phosphorus (Ca/P = 2.14) and the amount of carbon were greater. The piezoelectric strain tensor element d ₁₄ obtained for the anionic collagen was around 0.102 pC/N. The collagen composite with nanocrystalline HA crystals (Col-HAN) present a better result (d ₁₄ = 0.040 pC/N) compared to the composite with the commercial ceramic (d ₁₄ = 0.012 pC/N). This is an indication that the nanometric particles of HA present little disturbance on the organization of the collagen fibers in the composite. In this situation the nanometric HA are the best candidates in future applications of these composites.     

Elaboration conditions influence physicochemical properties and in vivo bioactivity of macroporous biphasic calcium phosphate ceramics

Two different preparations of biphasic calcium phosphate (BCP) were characterized in vitro: BCP₁ from a mechanical mixture of hydroxyapatite (HA) and -tricalcium phosphate (-TCP) powders, and BCP₂ from calcination of a calcium-deficient apatite (CDA). The structural, physicochemical and mechanical parameters of these two preparations were investigated, and two different macroporous BCP₁ (MBCP₁) and BCP₂ MBCP₂) implants were manufactured and implanted in rabbit bone for in vivo bioactivity studies. Scanning electron microscopy observations showed that MBCP₁ implants had a significantly higher degradation rate (P<0.0001) than MBCP₂ implants. This was probably caused by the presence of calcium oxide impurities in BCP₁ and the more intimate mixture and stable ultrastructure of BCP₂. No significant difference about the newly formed bone rate in these two BCP preparations was observed. Very slight variations in sintering conditions appeared to influence the biodegradation behavior of the two MBCP implants despite their identical HA/-TCP ratios and similar porosity. Precise and complete in vitro characterization enabled us to understand and predict in vivo degradation behavior. © 1999 Kluwer Academic Publishers     

“Fabrication of arbitrarily shaped carbonate apatite foam based on the interlocking process of dicalcium hydrogen phosphate dihydrate”

Carbonate apatite (CO₃Ap) foam with an interconnected porous structure is highly attractive as a scaffold for bone replacement. In this study, arbitrarily shaped CO₃Ap foam was formed from α-tricalcium phosphate (α-TCP) foam granules via a two-step process involving treatment with acidic calcium phosphate solution followed by hydrothermal treatment with NaHCO₃. The treatment with acidic calcium phosphate solution, which is key to fabricating arbitrarily shaped CO₃Ap foam, enables dicalcium hydrogen phosphate dihydrate (DCPD) crystals to form on the α-TCP foam granules. The generated DCPD crystals cause the α-TCP granules to interlock with each other, inducing an α-TCP/DCPD foam. The interlocking structure containing DCPD crystals can survive hydrothermal treatment with NaHCO₃. The arbitrarily shaped CO₃Ap foam was fabricated from the α-TCP/DCPD foam via hydrothermal treatment at 200?°C for 24?h in the presence of a large amount of NaHCO₃.     

The processing and characterization of animal-derived bone to yield materials with biomedical applications. Part II: milled bone powders, reprecipitated hydroxyapatite and the potential uses of these materials

Further studies on the processing and use of animal-bone-derived calcium phosphate materials in biomedical applications are presented. Bone powders sourced either from the direct crushing and milling of bovine, ovine and cervine bone or after being subjected to defatting and acid digestion/NaOH reprecipitation and sodium hypochlorite hydrogen peroxide treatment of animal bones were characterized using Fourier transform infra-red (FTIR) spectroscopy, ¹³C solid state magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, atomic absorption (AA) and inductively coupled plasma (ICP) spectrometric techniques. Bone powders were trialled for their potential use as a substrate for phosphine coupling and enzyme immobilization as well as a feedstock powder for plasma spraying on titanium metal substrates. Results indicated that enzyme immobilization by phosphine coupling could be successfully achieved on milled cervine bone with the immobilized enzyme retaining some activity. It was found that the presence of impurities normally carried down with the processing of the bone materials (viz., fat and collagen) played an important role in influencing the adsorbency and reactivity of the powders. Plasma spraying studies using reprecipitated bovine-derived powders produced highly adherent coatings on titanium metal, the composition of which was mostly hydroxyapatite(Ca10(PO₄)₆(OH)₂) with low levels of -tricalcium phosphate (-Ca₃(PO₄)2) and tetracalcium phosphate (Ca₄P₂O₉) also detected. In general, animal derived calcium phosphate materials constitute a potentially cheaper source of calcium phosphate materials for biomedical applications and make use of a largely under-utilized resource from abattoir wastes. © 2000 Kluwer Academic Publishers