The formation of hydroxyapatite–calcium polyacrylate composites

Tetracalcium phosphate (TetCP, Ca₄(PO₄)₂O) reacts rapidly with polyacrylic acid (PAA). Complete reaction results in the formation of hydroxyapatite (HAp) and calcium polyacrylate. Consequently, this combination of reactants can react to form a dental cement. However, reaction occurs so rapidly that it would be difficult to achieve a homogeneous mixture of reactants suitable for use in restorations. In order to explore extending the working time, the effects of prehydrating the TetCP to form surface layers of HAp on the TetCP particles was explored. Prehydration was found to be an effective means of allowing workability. Therefore, the effects of the proportions of TetCP and PAA, with and without HAp filler, on cement properties were investigated. The extents of the reactions were investigated by X-ray diffraction analysis; the extents of PAA neutralization were studied by Fourier transform infra-red spectroscopy (FTIR); pore structures were determined by mercury intrusion porosimetry; microstructures were observed by scanning microscopy, and compressive strengths were determined. After curing for 17 days at room temperature PAA neutralization was almost complete; however, residual TetCP could be detected by X-ray diffraction and PAA by FTIR. As expected, the compressive strengths of the cements showed a dependence on the liquid (water+polymer)-to-solid (TetCP+HAp filler) used. The presence of HAp filler caused a significant decrease in compressive strength and increasing the proportion of HAp filler resulted in a decrease in the compressive strength. The characteristics of the load–deflection curves showed a dependence on the presence of HAp filler. In the absence of filler, two slopes were observed in the curves whereas a linear curve, typical of a ceramic, was observed when HAp filler was present. Mercury intrusion porosimetry (MIP) indicated the majority of the porosity was present in pores larger than 0.1 m. Porosity increased with increasing liquid-to-solids ratio and with an increasing proportion of HAp filler at a constant liquid-to-solids ratio. Microstructural observations indicated the effect of HAp filler on increasing porosity was the result of porosity present in the filler itself. Thus, poorly consolidated HAp filler contributed to increased porosity and reduced compressive strength. © 1999 Kluwer Academic Publishers     

A PLA/calcium phosphate degradable composite material for bone tissue engineering: an in vitro study

Biodegradable polymers reinforced with an inorganic phase such as calcium phosphate glasses may be a promising approach to fulfil the challenging requirements presented by 3D porous scaffolds for tissue engineering. Scaffolds’ success depends mainly on their biological behaviour. This work is aimed to the in vitro study of polylactic acid (PLA)/CaP glass 3D porous constructs for bone regeneration. The scaffolds were elaborated using two different techniques, namely solvent-casting and phase-separation. The effect of scaffolds’ micro and macrostructure on the biological response of these scaffolds was assayed. Cell proliferation, differentiation and morphology within the scaffolds were studied. Furthermore, polymer/glass scaffolds were seeded under dynamic conditions in a custom-made perfusion bioreactor. Results indicate that the final architecture of the solvent-cast or phase separated scaffolds have a significant effect on cells’ behaviour. Solvent-cast scaffolds seem to be the best candidates for bone tissue engineering. Besides, dynamic seeding yielded a higher seeding efficiency in comparison with the static method.     

Pressureless sintering of dense hydroxyapatite–zirconia composites

Hydroxyapatite (HA)–TZP (2.5 mol% Y₂O₃) containing 2, 5, 7.5 and 10 wt% TZP were prepared using calcium nitrate, diammonium hydrogen orthophosphate, zirconium oxychloride and yttrium nitrate. The composite powder was prepared by a reverse strike precipitation method at a pH of 10.5. The precipitates after aging and washing were calcined at 850°C to yield fine crystallites of HA and TZP. TEM study of the calcined powder revealed that while HA particles had both spherical and cuboidal morphology (∼50–100 nm) the TZP particles were only of spherical nature (∼50 nm). X-ray analysis showed that the calcined powder of all the four composition had only HA and t-ZrO₂. Uniaxially compacted samples were sintered in air in the temperature range 1,150–1,250°C. High sintered density (>95% of theoretical) was obtained for composites containing 2 and 5 wt% TZP, while it was 92% for 7.5 wt% and 90% for 10 wt% TZP compositions. X-ray analysis of sintered samples shows that with 2 wt% TZP, the retained phases were only HA and t-ZrO₂. However, for 5, 7.5 and 10 wt% TZP addition both TCP and CaZrO₃ were also observed along with HA and t-ZrO₂. Bending strength was measured by three point bending as well by diametral compression test. While in three point bending, the highest strength was 72 MPa, it was 35.5 MPa for diametral compression. The strength shows a decreasing trend at higher ZrO₂ content. SEM pictures show near uniform distribution of ZrO₂ in HA matrix. The reduction in sintered density at higher ZrO₂ content could be related to difference in the sintering behaviour of HA and ZrO₂.     

Factors influencing the compressive strength of an injectable calcium sulfate–hydroxyapatite cement

A biphasic injectable bone substitute, suitable for filling bone defects, that sets in the body, based on calcium sulfate and hydroxyapatite (HA), is presented. For applications in bone defects the compressive strength is important to assure support of the defect site during loading when the patient is weight bearing. To control the strength, the influence of four different factors; the liquid-to-powder (L/P) ratio, the HA particle morphology, the HA content and the amount of accelerator, were investigated. -Calcium sulfate hemihydrate (CSH) and four different HA powders (three sintered and one spray-dried) were used. All differed in size and morphology. CSH and each HA powder were mixed together with distilled water to form the bone substitute. An accelerator, in form of calcium sulfate dihydrate, was added to the powder phase to obtain an adequate setting time. Cylindrical specimens were compression tested. A lower L/P-ratio gave stronger cement, but was more difficult to inject. The shape and the morphology of the HA particles influenced the strength, and reducing the amount of HA increased the strength. The amount of accelerator (calcium sulfate dihydrate) had no influence.     

In vitro bioactivity of sol–gel-derived hydroxyapatite particulate nanofiber modified titanium

A chemically-etched titanium surface was modified by electrospinning a sol–gel-derived hydroxyapatite (HAp) that was subjected to calcination within the temperature range of 200–1400°C in the normative atmospheric condition. After heat treatment, crystal structures of the filmed titanium oxide and HAp on the titanium’s surface were identified using wide-angle X-ray diffraction. A highly porous layer of HAp was found to have formed on the oxidized titanium surfaces. The surfaces of three different samples; (1) electrospun HAp, (2) HAp calcined at 600°C, and (3) HAp calcined at 800°C, were investigated for their ability to foster promotion, proliferation, and differentiation of human osteoblasts (HOB) (in the 9th passage) in vitro up to 6 days. Among the three samples, cells cultured on the HAp calcined at 800°C titanium surfaces displayed the best results with regard to adhesion, growth, and proliferation of HOB. This novel method for fabrication of titanium substrates would provide a promising improvement for titanium-based medical devices over the current standards, which lack such substrates. These titanium substrates explicitly provide enhanced HOB proliferation in terms of both desired surface properties and their produced bulk quantity.     

Formation of calcium deficient HAp/collagen composites by hydrolysis of α-TCP

Bone-like composites containing calcium deficient hydroxyapatite (CDHAp) were formed by the hydrolysis of alpha-tricalcium phosphate (α-TCP) in the presence of type I collagen. CDHAp-collagen composites were synthesized using two techniques. In one technique α-TCP was mixed with non-milled (as-received) collagen prior to the addition of the aqueous solution. In the second, the collagen was milled with α-TCP in heptane at room temperature prior to its conversion to CDHAp. The effect of milling strongly facilitates the formation of CDHAp at physiological temperature. The proportion of milled collagen between 5 and 20 wt% present in the α-TCP/collagen composites has no significant effect on the rate of CDHAp formation. Variations in pH and in calcium and phosphate concentrations were determined as a function of collagen processing and variations specific to the presence of collagen were discerned. Compared to CDHAp or to composites containing non-milled collagen, diametrical and compressive strengths of CDHAp increased in the presence of milled collagen. Lack of collagen dispersion and incomplete formation of CDHAp during 48 h were the bases for reduced strengths of composites containing non-milled collagen.     

Toughening characterization in alumina platelet–hydroxyapatite matrix composites

Fracture toughness of Al₂O₃ platelet-reinforced hydroxyapatite (HAP) ceramics was investigated using the Vickers' indentation technique. The geometrical anisotropy of alumina platelets induces an anisotropic toughening. The efficiency of reinforcing mechanisms remains maximum for a crack propagating with an angular deviation inferior to 30° around the direction perpendicular to alumina disc faces. This is assumed to result from a crack deflection mechanism which induces a favorable contribution of mode II failure. A small effect of hydroxyapatite grain size becomes noticeable in the direction parallel to alumina disc faces. The toughening depends on the size and volume content of alumina platelets. Large size platelets provoke a spontaneous microcracking of the HAP matrix which is detrimental to the mechanical reliability, whereas small platelets lead to a strong toughening. The results relate to the intensity of thermoelastic residual stresses within the matrix around alumina inclusions. © 1999 Kluwer Academic Publishers     

In vitro study of hydroxyapatite/polycaprolactone (HA/PCL) nanocomposite synthesized by an in situ sol–gel process

Hydroxyapatite (HA) is the most substantial mineral constituent of a bone which has been extensively used in medicine as implantable materials, owing to its good biocompatibility, bioactivity high osteoconductive, and/or osteoinductive properties. Nevertheless, its mechanical property is not utmost appropriate for a bone substitution. Therefore, a composite consist of HA and a biodegradable polymer is usually prepared to generate an apt bone scaffold. In the present work polycaprolactone (PCL), a newly remarkable biocompatible and biodegradable polymer, was employed as a matrix and hydroxyapatite nanoparticles were used as a reinforcement element of the composite. HA/PCL nanocomposites were synthesized by a new in situ sol–gel process using calcium hydroxide and phosphoric acid precursors in the presence of Tetrahydrofuran (THF) as a solvent. Chemical and physical characteristics of the nanocomposite were studied by X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM) and Fourier transform infrared (FTIR) analyses. The results indicated that pure HA nanoparticles were well-incorporated and homogenously dispersed in the PCL matrix. It was found that the mechanical property of PCL was improved by addition of 20 wt.% HA nanoparticles. Furthermore, the biological property of nanocomposites was investigated under in vitro condition. For this purpose, HA/PCL scaffolds were prepared through a salt leaching process and immersed in a saturated simulated body fluid (SBF) after 3 and 7 days. It was found that a uniform layer of biomimetic HA could be deposited on the surface of HA/PCL scaffolds. Therefore, the prepared HA/PCL scaffolds showed good potential for bone tissue engineering and could be used for many clinical applications in orthopedic and maxillofacial surgery.     

Characterisation of the bioactive behaviour of sol–gel hydroxyapatite–CaO and hydroxyapatite–CaO–bioactive glass composites

The fabrication and characterization of sol–gel derived hydroxyapatite–calcium oxide (HAp–CaO) material is investigated focusing on the effect of the addition of a bioactive glass on the material bioactive behaviour through the fabrication of a novel HAp–CaO (70 wt.%)–bioactive glass (30 wt.%) composite material. The bioactive behaviour of the materials was assessed by immersion studies in Simulated Body Fluid (SBF) and the alterations of the materials surfaces after soaking periods in SBF were characterized by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). A brittle and weakly crystalline carbonate hydroxyapatite (HCAp) layer was found to develop on the surface of all samples, few hours after immersion in SBF, confirming the high bioactivity of the material. Alterations of the morphology of the developed HCAp layer, which led to a more compact structure, were observed on the surface of composite samples after 7 days of immersion in SBF. The presence of the CaO phase seems to accelerate the formation of HCAp, while the bioactive glass affects both the morphology and cohesion of the developed layer.     

Investigations on the in vitro bioactivity of swift heavy oxygen ion irradiated hydroxyapatite

Poly(propylene fumarate) (PPF) is an ultraviolet-curable and biodegradable polymer with potential applications for bone regeneration. In this study, we designed and fabricated three-dimensional (3D) porous scaffolds based on a PPF polymer network using micro-stereolithography (MSTL). The 3D scaffold was well fabricated with a highly interconnected porous structure and porosity of 65%. These results provide a new scaffold fabrication method for tissue engineering. Surface modification is a commonly used and effective method for improving the surface characteristics of biomaterials without altering their bulk properties that avoids the expense and long time associated with the development of new biomaterials. Therefore, we examined surface modification of 3D scaffolds by applying accelerated biomimetic apatite and arginine-glycine-aspartic acid (RGD) peptide coating to promote cell behavior. The apatite coating uniformly covered the scaffold surface after immersion for 24 h in 5-fold simulated body fluid (5SBF) and then the RGD peptide was applied. Finally, the coated 3D scaffolds were seeded with MC3T3-E1 pre-osteoblasts and their biologic properties were evaluated using an MTS assay and histologic staining. We found that 3D PPF/diethyl fumarate (DEF) scaffolds fabricated with MSTL and biomimetic apatite coating can be potentially used in bone tissue engineering.     

Physico-chemical and in vitro biological evaluation of strontium/calcium silicophosphate glass

Strontium is known to reduce bone resorption and stimulate bone formation. Incorporation of strontium into calcium phosphate bioceramics has been widely reported. In this work, calcium and calcium/strontium silicophosphate glasses were synthesized from the sol–gel process and their rheological, thermal, and in vitro biological properties were studied and compared to each other. The results showed that the gel viscosity and thus the rate of gel formation increased by using strontium in glass composition and by increasing aging temperature. In strontium-containing glass, the crystallization temperature increased and the type of the crystallized phase was different to that of strontium-free glass. Both glasses favored precipitation of calcium phosphate layer when they were soaked in simulated body fluid; however strontium seemed to retard the rate of precipitation slightly. The in vitro biodegradation rate of the strontium/calcium silicophosphate glass was higher than that of strontium-free one. The cell culture experiments carried out using rat calvaria osteoblasts showed that the incorporation of strontium into the glass composition stimulated proliferation of the cells and enhanced their alkaline phosphatase activity, depending on cell culture period.     

Preparation and characterization of hydroxyapatite/polycaprolactone–chitosan composites

Hydroxyapatite (HA)/polycaprolactone (PCL)–chitosan (CS) composites were prepared by melt-blending. For the composites, the amount of HA was varied from 0% to 30% by weight. The morphology, structure and component of the composites were evaluated using environmental scanning electron microscope, X-ray diffraction and Fourier transform infrared spectroscope. The tensile properties were evaluated by tensile test. The bioactivity and degradation property were investigated after immersing in simulated body fluid (SBF) and physiological saline, respectively. The results show that the addition of HA to PCL–CS matrix tends to suppress the crystallization of PCL but improves the hydrophilicity. Adding HA to the composites decreases the tensile strength and elongation at break but increases the tensile modulus. After immersing in SBF for 14 days, the surface of HA/PCL–CS composites are covered by a coating of carbonated hydroxyapatite with low crystallinity, indicating the excellent bioactivity of the composites. Soaking in the physiological saline for 28 days, the molecular weight of PCL decreases while the mass loss of the composites and pH of physiological saline increase to 5.86% and 9.54, respectively, implying a good degradation property of the composites.     

In vitro and in vivo biocompatibility of graded hydroxyapatite–zirconia composite bioceramic

To obtain bioceramics with good osteoinductive ability and mechanical strength, graded hydroxyapatite–zirconia (HA–ZrO₂) composite bioceramics were prepared in this work. The biocompatibility of the bioceramics was investigated in vitro based on acute toxicity and cytotoxicity tests and hemolysis assay. Results showed the studied graded HA–ZrO₂ had little toxicity to mouse and L929 mouse fibroblasts. Also, hemolysis assay indicated a good blood compatibility of the bioceramics. Based on the results of in vitro tests, animal experiments were performed on white New Zealand rabbits by implantation into hip muscles and femur. It was found that the graded HA–ZrO₂ composite bioceramics exhibited superior osteoinductive ability, which may be a promising bioceramics implant.     

In vitro study on the influence of strontium-doped calcium polyphosphate on the angiogenesis-related behaviors of HUVECs

Angiogenesis is of great importance in bone tissue engineering, and has gained large attention in the past decade. Strontium-doped calcium polyphosphate (SCPP) is a novel biodegradable material which has been proved to be able to promote in vivo angiogenesis during bone regeneration. An in vitro culture system was developed in the present work to examine its influence on angiogenesis-related behaviors of human umbilical vein endothelial cells (HUVECs), including cell adhesion, spreading, proliferation and migration. The effects of microtopography, chemical property and the ingredients in the degradation fluid (DF) on cell behaviors were discussed. The results showed that cells attached and spread better on SCPP scaffold than on calcium polyphosphate (CPP), which might partially result from the less rough surface of SCPP scaffold and the less hydrogel formed on the surface. In addition, cell proliferation was significantly improved when treated with SCPP DF compared with the treatment with CPP DF. Statistical analysis indicated that Sr(2+) in SCPP DF might be the main reason for the improved cell proliferation. Moreover, cell migration, another important step during angiogenesis, was evidently stimulated by SCPP DF. The improved in vivo angiogenesis by SCPP might be assigned to its better surface properties and strontium in the DF. This work also provides a new method for in vitro evaluation of biodegradable materials' potential effects on angiogenesis.     

Biocompatibility of polyhydroxybutyrate microspheres: in vitro and in vivo evaluation

Microspheres have been prepared from the resorbable linear polyester of β-hydroxybutyric acid (polyhydroxybutyrate, PHB) by the solvent evaporation technique and investigated in vitro and in vivo. Biocompatibility of the microspheres has been proved in tests in the culture of mouse fibroblast cell line NIH 3T3 and in experiments on intramuscular implantation of the microspheres to Wistar rats for 3 months. Tissue response to the implantation of polymeric microspheres has been found to consist in a mild inflammatory reaction, pronounced macrophage infiltration that increases over time, involving mono- and poly-nuclear foreign body giant cells that resorb the polymeric matrix. No fibrous capsules were formed around polymeric microparticles; neither necrosis nor any other adverse morphological changes and tissue transformation in response to the implantation of the PHB microparticles were recorded. The results of the study suggest that polyhydroxybutyrate is a good candidate for fabricating prolonged-action drugs in the form of microparticles intended for intramuscular injection.     

In vitro biocompatibility assessment of PHBV/Wollastonite composites

Biodegradable and biocompatible materials are the basis for tissue engineering. As an initial step for developing bone tissue engineering scaffolds, the in vitro biocompatibility of degradable and bioactive composites consisting of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and wollastonite (W) was studied by culturing osteoblasts on the PHBV/W substrates, and the cell adhesion, morphology, proliferation, and alkaline phosphatase (ALP) activity were evaluated. The results showed that the incorporation of wollastonite benefited osteoblasts adhesion and the osteoblasts cultured on the PHBV/W composite substrates spread better as compared to those on the pure PHBV after culturing for 3 h. In the prolonged incubation time, the osteoblasts cultured on the PHBV/W composite substrates revealed a higher proliferation and differentiation rate than those on the pure PHBV substrates. In addition, an increase of proliferation and differentiation rate was observed when the wollastonite content in the PHBV/W composites increased from 10 to 20 wt%. All of the results showed that the addition of wollastonite into PHBV could stimulate osteoblasts to proliferate and differentiate and the PHBV/W composites with wollastonite up to 20 wt% were more compatible than the pure PHBV materials for bone repair and bone tissue engineering.     

Hydrogen peroxide versus water synthesis of bioglass–nanocrystalline hydroxyapatite composites

This article reports a comparison of the structural and textural properties of bioglass–hydroxyapatite (HA) composites obtained in the SiO₂–CaO–P₂O₅ system by sol–gel method, with different amounts of hydrogen peroxide (3% H₂O₂) or water (H₂O). X-ray diffraction, Raman, and FT-IR spectroscopy reveal the presence of nanocrystalline HA. Scanning electron microscopy images illustrate that the HA phase is mainly distributed on the glass surface. The results point out that the sintering at 550 °C of a sol–gel derived SiO₂–CaO–P₂O₅ bioglass leads to a single crystalline phase of HA, and validate a new processing method for obtaining bioglass–HA composites. Structural analyses of the investigated composites indicate the existence of a silicate network built up from Q₃ and Q₂ units. The replacement of water with hydrogen peroxide has as consequence the increase of depolymerization degree of silica network. Textural properties were investigated with N₂-adsorption technique. The composites prepared with hydrogen peroxide exhibit a more uniform and narrow mesoporous distribution that recommends them for drug uptake and release applications. It was found that the specific surface area and pore volume are clearly influenced by the H₂O₂(H₂O):TEOS molar ratio.     

In vitro and in vivo evaluation of the effects of demineralized bone matrix or calcium sulfate addition to polycaprolactone–bioglass composites

The objective of this study was to improve the efficacy of polycaprolactone/bioglass (PCL/BG) bone substitute using demineralized bone matrix (DBM) or calcium sulfate (CS) as a third component. Composite discs involving either DBM or CS were prepared by compression moulding. Bioactivity of discs was evaluated by energy dispersive X-ray spectroscopy (ESCA) and scanning electron microscopy (SEM) following simulated body fluid incubation. The closest Calcium/Phosphate ratio to that of hydroxyl carbonate apatite crystals was observed for PCL/BG/DBM group (1.53) after 15 day incubation. Addition of fillers increased microhardness and compressive modulus of discs. However, after 4 and 6-week PBS incubations, PCL/BG/DBM discs showed significant decrease in modulus (from 266.23 to 54.04 and 33.45 MPa, respectively) in parallel with its highest water uptakes (36.3 and 34.7%). Discs preserved their integrity with only considerable weight loss (7.5–14.5%) in PCL/BG/DBM group. In vitro cytotoxicity tests showed that all discs were biocompatible. Composites were implanted to defects on rabbit humeri. After 7 weeks, new tissue formation and mineralization at bone-implant interface were observed for all implants. Bone mineral densities at interface were higher than that of implant site and negative controls (defects left empty) but lower than healthy bone level. However, microhardness of implant sites was higher than in vitro results indicating in vivo mineralization of implants. Addition of DBM or CS resulted with higher microhardness values at interface region (ca. 650 μm from implant) compared to PCL/BG and negative control. Histological studies revealed that addition of DBM enhanced bone formation around and into implant while CS provided cartilage tissue formation around the implant. From these results, addition of DBM or CS could be suggested to improve bone healing efficacy of PCL/BG composites.     

In vitro study of the SBF and osteoblast-like cells on hydroxyapatite/chitosan–silica nanocomposite

Hydroxyapatite/chitosan–silica (HApCSi) nanocomposites were synthesized by co-precipitated method and their potential application as filler materials for bone regeneration were investigated in simulated body fluid (SBF). To study their biocompatibility, they were cultured with rat osteoblast-like UMR-106 cells for 3, 7, 14, and 21 days. Studies of the silica contents in chitosan matrix showed the presence of silinol (Si–OH) groups in CSi hybrid and their decrease after being composited with calcium phosphate (CaP) which is desirable for the formation of the apatite. XRD and TEM studies showed that the HAp formed in the CSi matrix were nanometer (20–40 nm) in size. Nanocomposites of HApCSi20 processed with 20%v/v silica whisker showed a micro hardness of 84.7 ± 3.3 MPa. Mineralization study in SBF showed the formation of apatite crystals on the HApCSi surface after being incubated for 7 days. In vitro biocompatibility, cell morphology, proliferation, and cell adhesion tests confirmed the osteoblast attachment and growth on the HApCSi20 surface. The density of cells and the production of calcium nodules on the substrate were seen to increase with increasing cultured time. The mechanical evaluation and in vitro experiment suggested that the use of HApCSi composite will lead to the formation of new apatite particles and thus be a potential implant material.     

Effect of polymerization degree of calcium polyphosphate on its microstructure and in vitro degradation performance

Preparation, characterization and in vitro study of a series of calcium polyphosphate (CPP) with different polymerization degree were reported. A series of CPP with different polymerization degree were prepared by controlling calcining time. Average polymerization degree was analyzed by liquid state ³¹P nuclear magnetic resonance (NMR). The microstructure was observed by scanning electric microscope (SEM). X-ray diffraction (XRD) analysis was used to demonstrate that polymerization degree would not affect the crystal system and space group of CPP. The results showed that polymerization degree increased with the increase of calcining time. Degradation studies were performed during 32 days in physiological saline solution (aqueous solution, 0.9 wt.%NaCl) to assess the effect of polymerization degree on the degradation velocity of the samples. It was also shown that the degradation velocity of CPP (polymerization degree = 13) doubles than another two samples (polymerization degree = 9,19). The results in the present study may be able to provide some fundamental data for controlling CPP degradation.