Growth Of Calcium Phosphate Research Articles

Study on the Effects of Type I Collagen Combined with Noncollagenous Proteins on Hydroxyapatite Formation in vitro using SPM and GIXD

AbstractThe effects of the components of extracellular matrix on the bone formation and the kinetics of crystal growth of calcium phosphate have remained unknown. In this paper, we reported a method to investigate the role of Type I collagen and the interactions with other ECM proteins such as fibronectin and elastin during biomimic mineralization process in vitro. The early stage of mineralization was characterized by scanning probe microscopy (SPM) and shear modulation force microscopy (SMFM). The late stage of mineralization was investigated by synchrotron grazing incident x-ray diffraction (GIXD). The results demonstrate the cooperative interaction between type I collagen and noncollagenous proteins such as fibronectin or elastin could be essential for the biomineralization.

Bioinspired mineralization and cell adhesion on surface functionalized poly(vinyl alcohol) films

Poly(vinyl alcohol) (PVA) films, when surface functionalized by phosphorylation, induced biomimetic nucleation and growth of calcium phosphate in a simulated physiological environment. The surface phosphorylation on PVA was ensured by attenuated total reflectance infrared spectroscopy. The morphology of the calcium phosphate phase grown on surface-phosphorylated PVA (PPVA) was analysed using scanning electron microscopy coupled with an energy-dispersive X-ray detector. The primary nucleation of calcium phosphate occurs in 3 days and secondary nucleation occurs after 10 days. The energy-dispersive X-ray analysis shows that the Ca/P ratio of the coating increases with time of exposure to the simulated physiological fluid and reaches 1.67 at 10 days. The PPVA supports in vitro cell adhesion and promotes in vitro biomineralization in the presence of cells, evaluated using human osteosarcoma cells.

Bioinspired synthesis of self-assembled calcium phosphate nanocomposites using block copolymer-peptide conjugates

Thermoreversibly gelling block copolymers conjugated to hydroxyapatite-nucleating peptides were used to template the growth of inorganic calcium phosphate in aqueous solutions. Nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), transmission electron microscopy, x-ray diffraction, and small-angle scattering were used to characterize these samples and confirm that the peptides promoted the growth of hydroxyapatite as the inorganic phase. Three different polymer templates were used with varying charges on the polymer chains (nonionic, anionic, and zwitterionic), to investigate the role of charge on mineralization. All of the polymer-inorganic solutions exhibited thermoreversible gelation above room temperature. Nanocomposite formation was confirmed by solid-state NMR, and several methods identified the inorganic component as hydroxyapatite. Small angle x-ray scattering and electron microscopy showed thin, elongated crystallites. Thermogravimetric analysis showed an inorganic content of 30–45 wt% (based on the mass of the dried gel at ∼200 °C) in the various samples. Our work offers routes for bioinspired bottom-up approaches for the development of novel, self-assembling, injectable nanocomposite biomaterials for potential orthopedic applications.

Coating Electrospun Poly(ε-caprolactone) Fibers with Gelatin and Calcium Phosphate and Their Use as Biomimetic Scaffolds for Bone Tissue Engineering

Electrospinning was employed to fabricate fibrous scaffolds of poly(epsilon-caprolactone) in the form of nonwoven mats. The surfaces of the fibers were then coated with gelatin through layer-by-layer self-assembly, followed by functionalization with a uniform coating of bonelike calcium phosphate by mineralization in the 10 times concentrated simulated body fluid for 2 h. Transmission electron microscopy, water contact angle, and scanning electron microscopy measurements confirmed the presence of gelatin and calcium phosphate coating layers, and X-ray diffraction results suggested that the deposited mineral phase was a mixture of dicalcium phosphate dehydrate (a precursor to apatite) and apatite. It was also demonstrated that the incorporation of gelatin promoted nucleation and growth of calcium phosphate. The porous scaffolds could mimic the structure, composition, and biological function of bone extracellular matrix. It was found that the preosteoblastic MC3T3-E1 cells attached, spread, and proliferated well with a flat morphology on the mineralized scaffolds. The proliferation rate of the cells on the mineralized scaffolds was significantly higher (by 1.9-fold) than that on the pristine fibrous scaffolds after culture for 7 days. These results indicated that the hybrid system containing poly(epsilon-caprolactone), gelatin, and calcium phosphate could serve as a new class of biomimetic scaffolds for bone tissue engineering.

Accelerating calcium phosphate growth on NaOH-treated poly-(lactic- co-glycolic acid) by evaporation-induced surface crystallization

Poly(lactic- co-glycolic acid) (PLGA) is a promising material for the regeneration of bone tissue, but its surface properties are not optimal for the application. Coating the surface of PLGA with a continuous layer of calcium phosphate is an effective approach to address the limitation. Current coating techniques for PLGA require immersion in supersaturated calcium phosphate solutions for days to weeks. In this study, we report a simple technique to accelerate the coating process to only 2 h immersion in supersaturated solutions. PLGA pellets were first treated with NaOH to increase their hydrophilicity. The NaOH-treated PLGA pellets were repeatedly dipped in a supersaturated calcium phosphate solution and dried in air. After 10 times of the dip-and-dry treatment, a layer of calcium phosphate crystallites uniformly covered the surfaces of the pellets. After the crystallite-covered pellets were immersed in the supersaturated solution for 2 h, about 5-μm thick continuous calcium phosphate coatings formed on the surfaces. The dip-and-dry technique was also applied on a variety of metals and porous structures. An evaporation-induced surface crystallization process was suggested as the mechanism for the dip-and-dry treatment.

Comparison of chitosan apatite composites synthesized by different methods

AbstractThis article presents some results concerning chitosan apatite composites obtained by two different methods. First method is based on phosphorylation, calcification and soaking in different calcium phosphate growth media (1.5 × SBF (simulated body fluid), Ca‐PTris (calcium phosphorous tris) and the second method is based on mixing of calcium deficient apatite powder with a chitosan solution. This research is focused on studying and understanding the effect of using different methods on Ca:P ratio of composites. The Ca:P ratio of composites formed in 1.5 × SBF are closer to that of bone when compared with the Ca:P ratio of composites formed in Ca‐PTris solution and Ca:P ratio of composites formed by mixing of calcium deficient apatite powder with a chitosan solution. The Ca:P ratio of the composite (1.75) immersed in 1.5 × SBF for 35 day is similar to the theoretical value of hydroxyapatite (1.67) and equal to the theoretical value of human bone (1.75). POLYM. COMPOS., 2009. © 2008 Society of Plastics Engineers

Electrochemical processes of nucleation and growth of calcium phosphate on titanium supported by real‐time quartz crystal microbalance measurements and X‐ray photoelectron spectroscopy analysis

Real-time, in situ electrochemical quartz crystal microbalance (EQCM) measurements are conducted to better understand the electrocrystallization of calcium phosphates (CaP) on CP-Ti. X-ray photoelectron spectroscopy is used to identify the exact phase deposited, so that reliable estimation of the electrochemical processes involved is made. Analysis of the integrated intensity of the oxygen shake-up peaks, in combination with the determination of Ca/P and O/Ca atomic ratios, enables to determine unambiguously that the octacalcium phosphate (OCP) is formed. Its role as a precursor to hydroxyapatite (HAp) is discussed. After an incubation period, the process by which OCP is formed follows a Faradaic behavior. The incubation time may be related to the need for local increase of pH before precipitation from solution can occur. The standard enthalpy of activation is approximately 40 kJ/mol, which excludes diffusion-controlled processes from being rate determining. The OCP deposit has thickness approximately 0.61 microm, apparent density approximately 0.95 g/cm3, 63.6% porosity, and deposition rate of 23.5 ng/(cm2 s) or 15 nm/min. The low-equivalent weight value of 20.5 g/equiv, and the associated remarkably high number of electrons transferred in the reaction n approximately 24, indicates that most of the current is consumed either by electrolysis of water or by a complex set of parasitic reactions. The low-solubility product allows precipitation of CaP even at relatively low concentrations of calcium and phosphate/hydrogen phosphate ions. It is shown that HAp most likely forms via transformation of precursor phases, such as OCP, rather than directly.

Bioactive Porous Titanium: An Alternative to Surgical Implants

Porous titanium implants have been used to improve implant-bone attachment by the ingrowth of bone tissue within the porous structure. Despite the efficient bone adhesion of porous titanium implants, chemical bonds are required at bone-implant interface. These implants can become bioactive by a biomimetic precipitation process. The aim of this work was to enhance the bioactivity of pure porous titanium implants by biomimetic process. The samples immersed in a simulated body fluid promoted the nucleation and growth of calcium phosphate (Ca-P) crystals, such as hydroxyapatite (Hap), on the material surface. Scanning electron microscopy, energy dispersive spectroscopy, and Fourier transform infrared spectroscopy analyses revealed that a Ca-P deposition occurred without the need of pretreatments to improve the surface bioactivity. This present study indicates the potential for growing a bone-like Hap layer on porous titanium implants by biomimetic processes.

Bone tissue engineering with premineralized silk scaffolds

Silk fibroin biomaterials are being explored as novel protein-based systems for cell and tissue culture. In the present study, biomimetic growth of calcium phosphate on porous silk fibroin polymeric scaffolds was explored to generate organic/inorganic composites as scaffolds for bone tissue engineering. Aqueous-derived silk fibroin scaffolds were prepared with the addition of polyaspartic acid during processing, followed by the controlled deposition of calcium phosphate by exposure to CaCl 2 and Na 2HPO 4. These mineralized protein-composite scaffolds were subsequently seeded with human bone marrow stem cells (hMSC) and cultured in vitro for 6 weeks under osteogenic conditions with or without BMP-2. The extent of osteoconductivity was assessed by cell numbers, alkaline phosphatase and calcium deposition, along with immunohistochemistry for bone-related outcomes. The results suggest increased osteoconductive outcomes with an increase in initial content of apatite and BMP-2 in the silk fibroin porous scaffolds. The premineralization of these highly porous silk fibroin protein scaffolds provided enhanced outcomes for the bone tissue engineering.

Mechanism of calcium disilicide‐induced calcification of crystalline silicon surfaces in simulated body fluid under zero bias

A dry-etch spark ablation method was used to produce calcium disilicide (CaSi2/Si) layers on silicon surfaces, and their biomineralization under zero bias was followed by means of scanning electron microscopy, X-ray energy dispersive analysis, and Raman spectroscopy. CaSi2/Si wafers are bioinert at 25 degrees C and bioactive at 37 degrees C. Mechanistic insights regarding biomineralization were derived from an analysis of film growth morphology and chemical composition after various soaking periods in standard simulated body fluid (SBF). Changes in CaSi2/Si calcification behavior as a function of reaction temperature and pH, SBF concentration, and various surface modification processes were also employed for this purpose. During CaSi2/Si calcification under zero bias, calcium phosphate (CaP) growth is strongly dependent on the structural degradation of CaSi2/Si grains. Surface silanol groups, initially present on the as-prepared material, cannot induce CaP nucleation, which begins only upon delamination of CaSi2/Si layers. The calcium phosphate phases, which are present during various growth stages, possibly include a combination of Mg-substituted whitlockite, monetite, and tricalcium phosphate.

Synthesis of chitin calcium phosphate composite in different growth media

AbstractThis paper presents some results concerning chitin calcium phosphate composites obtained in different growth media. The synthesis of chitin calcium phosphate composite was carried out by phosphorylation, calcification, and soaking in different calcium phosphate growth media. This research is focused on studying and understanding the effect of using different growth media on composite samples. Hydroxyapatite was determined in the composite samples synthesized by using both simulated body fluid (SBF) and calcium phosphorus tris (Ca‐PTris) solution. Calcium/phosphorus (Ca:P) ratio of the composite synthesized by using SBF was found higher than that of composite synthesized by using Ca‐PTris solution as calcium phosphate growth media. The Ca:P ratio of the composite (1.72) immersed in 1.5× SBF for 35 day is similar to the theoretical value of hydroxyapatite (1.67) and closer to the theoretical value of human bone (1.75). POLYM. COMPOS., 29:84–91, 2008. © 2007 Society of Plastics Engineers

A Novel Approach of Homogenous Inorganic/Organic Composite through In Situ Precipitation in Gelatine/Poly(Acrylic Acid) Gel

A new in situ precipitation technique was developed to promote high-affinity nucleation and growth of calcium phosphate in the polymer hydrogel. Gelatine/poly(acrylic acid)/hydroxyapatite (HA) composite has been prepared using template-driven reaction. Nano-sized hydroxyapatite particles were distributed within organic template homogenously, furthermore, inorganic particles were fine and uniform. During the composite process, 3D network of organic matrices and homogenous distributed nucleation sites played an important role in the superfine interaction of HA and hydrogel. This method provides an efficient approach toward inorganic/organic nanocomposites with high-uniformity decentralization for biomimetic replant applications. This paper discussed the mechanism of the reaction, and the concept of in situ precipitation in gel was brought forward.

Calcium phosphate formation on plasma immersion ion implanted low density polyethylene and polytetrafluorethylene surfaces

The flexible structure of polymers has enabled them to be useful in a wide variety of medical applications due to the possibility to tailor their properties to suit desired applications. For a long time, there has been an increasing interest in utilizing polymers as matrices for calcium phosphate-based composites with applications in hard tissue implants. On the other side, polymers with application as heart valves, urea catheters and artificial vessels present a case where the formation of minerals (namely calcification) should be avoided. The modification of polymer surfaces by various ion beam treatments for reducing the calcification, as for example plasma immersion ion implantation (PIII), is well known and has a long time effect. This work is part of a wider investigation of the ability of plasma immersion ion implanted polymers to induce calcium phosphate formation from an aqueous solution resembling the human blood plasma. In the experiment described in this paper, topographical and chemical changes were inserted on the surfaces of two conventional polymers (low density polyethylene and polytetrafluorethylene) by PIII with nitrogen ions, and under conditions mimicking the natural mineral formation processes. The effect of the plasma modification on the calcium phosphate nucleation and growth from the aqueous solution was ambiguous. We suppose that the complex combination of surface characteristics influenced the ability of the plasma treated polymer films to induce the formation of a calcium phosphate layer.

Effects of pH and initial Ca2+−H2PO4 − concentration on fibroin mineralization

In the present study, the effects of pH and initial Ca2+−H2PO4 − (Ca-P) concentration on fibroin mineralization were studied. The crystal growth of calcium phosphates was regulated by regenerated silk fibroin for 8 h (at pH 4.0, 7.0 and 10.0, respectively). Meanwhile, different concentrations of Ca2+ were employed at a certain pH value, keeping the initial Ca-P molar ratio constant at 1.67, i.e., the stoichiometry of hydroxyapatite [Ca10(PO4)6(OH)2, HAP]. The products were characterized by X-ray diffractometry (XRD), scanning electron microscopy (SEM) and thermogravimetric analysis (TGA). The results demonstrated that, compared to pH 4.0 and 10.0, pH 7.0 promoted the transformation of brushite (CaHPO4·2H2O, DCPD) to HAP. In the composites of mineralized fibroin, DCPD is the main inorganic phase at both relatively low and high pH, while HAP is the main inorganic phase at pH 7.0. Additionally, the initial Ca-P concentration does not affect the kind of inorganic phase in the synthesized mineralized fibroin, but induce to different contents of inorganic mineral and different morphology of DCPD at pH 4.0 and pH 10.0.

The effect of Leucine on the crystal growth of calcium phosphate

Crystallization kinetics of hydroxyapatite, HAP, in the presence of Leucine, a natural amino acid with hydrophobic side groups, were investigated at conditions of sustained supersaturation, 37 degrees C, pH 7.40, ionic strength 0.15 M. In the presence of Leucine, the crystal growth rates of HAP decreased markedly. Tauhis action is due to adsorption and subsequent blocking of the active growth sites onto the surface of the HAP crystals. The kinetic results revealed that a Langmuir-type adsorption isotherm is followed and an affinity constants of k(aff )=20.26 x 10(2) L/mol for HAP crystal surface were calculated. The apparent order of the crystal growth reaction was found to be equal to two, suggesting a surface diffusion controlled mechanism.

Templated growth of calcium phosphate on tyrosine derived microtubules and their biocompatibility

Microtubular structures were self-assembled in aqueous media from a newly synthesized bolaamphiphile, bis( N-alpha-amido-tyrosyl-tyrosyl-tyrosine)-1,5-pentane dicarboxylate. In order to increase the biocompatibility of the microtubules, they were functionalized with the peptide sequence GRGDSP. Further, calcium phosphate nanocrystals were grown on the microtubules. In some cases, collagen was added in order to mimic the components of natural bone tissue. The biomaterials obtained were characterized via transmission electron microscopy (TEM), atomic force microscopy (AFM), IR, and energy dispersive X-ray spectroscopy (EDX) analyses. The biocompatibility of the calcium phosphate-coated microtubules was studied by conducting in vitro cell-attachment, cell-proliferation and cytotoxicity studies using mouse embryonic fibroblast (MEF) cells. The studies revealed that the biomaterials were found to be non-toxic and biocompatible. The functionalized tubular assemblies coated with calcium phosphate nanocrystals mimic the nanoscale composition of natural bone and may potentially support bone in-growth and osseointegration when used in orthopaedic or dental applications.

The nucleation and growth of calcium phosphate by amelogenin

The nucleation processes involved in calcium phosphate formation in tooth enamel are not well understood but are believed to involve proteins in the extracellular matrix. The ability of one enamel protein, amelogenin, to promote the nucleation and growth of calcium phosphate was studied in an in vitro system involving metastable supersaturated solutions. It was found that recombinant amelogenin (rM179 and rp(H)M180) promoted the nucleation of calcium phosphate compared to solutions without protein. The amount of calcium phosphate increased with increasing supersaturation of the solutions and increasing protein concentrations up to 6.5 μg/mL. At higher protein concentrations, the amount of calcium phosphate decreased. The kinetics of nucleation was studied in situ and in real time using a quartz crystal microbalance (QCM) and showed that the protein reduced the induction time for nucleation compared to solutions without protein. This work shows a nucleation role for amelogenin in vitro which may be promoted by the association of amelogenin into nanosphere templates, exposing charged functionality at the surface.

Growth of Calcium Phosphate on Poled Piezoelectric Poly-L-lactic Acid Membrane by a Biomimetic Method

The bioactivity of poled piezoelectric PLLA membrane was investigated by studying the calcium phosphate formation in vitro using a biomimetic method. Samples (φ10mm) were poled under DC electric field of 8~l0kV/cm at 70°C for 30 min followed by cooling under the electric field. Surface chemistry of the samples before and after poling treatment was studied by X-ray photoelectron spectroscopy (XPS). Poled/unpoled samples were immersed in supersaturated calcification solution (SCS) for periods up to 24 h (36.5°C). The surface morphology and composition of the soaked samples were evaluated by using scanning electron microscope (SEM) and X-ray diffraction analysis (XRD). Poled samples showed two different charged surfaces, negatively-charged surface (N-PLLA) and positively-charged surface (P-PLLA). On the N-PLLA surfaces, SEM together with XRD showed a gradually formed calcium phosphate (Ca-P), while no obvious Ca-P on either P-PLLA or unpoled samples was observed. This study demonstrated that poled piezoelectric PLLA substrates induce substantially higher level of Ca-P formation than electrically neutral substrates and only N-PLLA, however, can improve Ca-P formation after immersion in SCS.

Synthesis of Hydroxyapatite in Polymeric Solutions for Organic-Inorganic Nanocomposites

Synthesis of hydroxyapatite (HA) in organic solutions has received extensive attention in recent years with an attempt to obtain HA of a nanometer level. In this preliminary study, we demonstrated that organic-HA nanocomposites could also be achieved with one step method via in situ mineralization and subsequent crosslinking of organic species. This design was realized through in situ synthesis of hydroxyapatite in poly(vinyl alcohol) and acrylic acid aqueous solution as an organic template. The aforementioned organic-inorganic nanocomposites were analyzed by using X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electrical microscopy, thermal analysis. The comparative structural measurements were also conducted with the synthesized HA with absence of the organic template. The results indicated that the existence of organic species effectively inhibits the growth of calcium phosphate and that relatively pure HA can be obtained in sintered composite products. The present study provides a direct and versatile route for fabrication of nanocomposite biomaterials.

Functionalization of oligo(poly(ethylene glycol)fumarate) hydrogels with finely dispersed calcium phosphate nanocrystals for bone-substituting purposes

Biodegradable polymers that can be processed into injectable hydrogel matrices are promising candidates for bone-substituting purposes. Furthermore, by incorporating degradable calcium phosphate (CaP) particles and growth factors into these hydrogel matrices, a bone construct can be designed which stimulates the formation of new bone by the surrounding tissue, thereby compensating for the loss of structural integrity of the degrading synthetic bone-substitute. Generally, a major challenge in synthesis of nanoceramic-reinforced polymers is the achievement of a fine dispersion of nanoparticles throughout the polymer, since the unique properties of nanocomposites are lost when nanoparticles aggregate. In the current study, composite hydrogels consisting of oligo(poly(ethylene glycol)fumarate) (OPF) matrices and CaP dispersions of varying crystallinity were successfully developed using physical or chemical preparation strategies. Physical mixing of dried, micrometer-sized CaP powders resulted into formation of irreproducible composites with a highly heterogeneous dispersion of large and agglomerated CaP microparticles throughout the OPF matrix. On the contrary, reproducible and homogeneous hydrogels were fabricated using a chemical mixing strategy, whereby CaP crystals were formed in the presence of dissolved OPF macromers. This co-precipitation technique resulted into a much higher degree of dispersion of the CaP crystals, which can enable higher CaP contents in organic matrices such as OPF. By using these CaP suspensions instead of dried powders, the nanosized structure of separated CaP crystals was preserved, resulting into a higher reactivity of the CaP phase, as indicated by a reduced swelling behavior of these hydrogels. This effect was most likely caused by a physicochemical interaction between Ca2+ and unreacted COOH end-groups, thereby leading to increased physical cross-linking of the composite hydrogels.