Establishment of a three-dimensional culture system of gastric stem cells supporting mucous cell differentiation using microfibrous polycaprolactone scaffolds.

OBJECTIVES: To generate various polycaprolactone (PCL) scaffolds and test their suitability for growth and differentiation of immortalized mouse gastric stem (mGS) cells. MATERIALS AND METHODS: Non-porous, microporous and three-dimensional electrospun microfibrous PCL scaffolds were prepared and characterized for culture of mGS cells. First, growth of mGS cells was compared on these different scaffolds after 3 days culture, using viability assay and microscopy. Secondly, growth pattern of the cells on microfibrous scaffolds was studied after 3, 6, 9 and 12 days culture using DNA PicoGreen assay and scanning electron microscopy. Thirdly, differentiation of the cells grown on microfibrous scaffolds for 3 and 9 days was analysed using lectin/immunohistochemistry. RESULTS: The mGS cells grew preferentially on microfibrous scaffolds. From 3 to 6 days, there was increase in cell number, followed by reduction by days 9 and 12. To test whether the reduction in cell number was associated with cell differentiation, cryosections of cell-containing scaffolds cultured for 3 and 9 days were probed with gastric epithelial cell differentiation markers. On day 3, none of the markers examined bound to the cells. However by day 9, approximately, 50% of them bound to N-acetyl-d-glucosamine-specific lectin and anti-trefoil factor 2 antibodies, indicating their differentiation into glandular mucus-secreting cells. CONCLUSIONS: Microfibrous PCL scaffolds supported growth and differentiation of mGS cells into mucus-secreting cells. These data will help lay groundwork for future experiments to explore use of gastric stem cells and PCL scaffolds in stomach tissue engineering.

Formation and properties of composites comprised of calcium-deficient hydroxyapatites and ethyl alanate polyphosphazenes.

Composites comprised of calcium-deficient hydroxyapatite (HAp) and biodegradable polyphosphazenes were formed via cement-type reactions at physiologic temperature. The composite precursors were produced by blending particulate hydroxyapatite precursors with 10 wt% polymer using a solvent/non-solvent technique. HAp precursors having calcium-to-phosphate ratios of 1.5 (CDH) and 1.6 (CDS) were used. The polymeric constituents were poly[bis(ethyl alanato)phosphazene] (PNEA) and poly[(ethyl alanato)(1) (p-phenylphenoxy)(1) phosphazene] (PNEA(50)PhPh(50)). The effect of incorporating the phenyl phenoxy group was evaluated as a means of increasing the mechanical properties of the composites without retarding the rates of HAp formation. Reaction kinetics and mechanistic paths were characterized by pH determination, X-ray diffraction analyses, scanning electron microscopy, and infrared spectroscopy. The mechanical properties were analyzed by compression testing. These analyses indicated that the presence of the polymers slightly reduced the rate HAp formation. However, surface hydrolysis of polymer ester groups permitted the formation of calcium salt bridges that provide a mechanism for bonding with the HAp. The compressive strengths of the composites containing PNEA(50)PhPh(50) were superior to those containing PNEA, and were comparable to those of HAp produced in the absence of polymer. The current composites more closely match the structure of bone, and are thus strongly recommended to be used as bone cements where high loads are not expected.

Formation of hydroxyapatite-polyphosphazene polymer composites at physiologic temperature.

Aspects of the formation of bone analog composites at 37 degrees C are described. The composites are composed of hydroxyapatite (HAp) and the calcium salt of a biocompatible polymer and are capable of forming under in vivo conditions. Composite formation involves the formation of monolithic HAp from particulate calcium phosphate precursors while Ca ions liberated to the aqueous medium in which this reaction is occurring form crosslinks with the acidic polymer. The reactants are poly[bis(carboxylatophenoxy)phosphazene] (acid-PCPP), tetracalcium phosphate [Ca4(PO4)2O, TetCP], and anhydrous dicalcium phosphate (CaHPO4, DCPA). The effects of the proportion of polymer (5, 10, or 15 wt %) on the kinetics of HAp formation were studied. Compositional evolution of the solid calcium phosphates present was followed by X-ray diffraction and infrared spectroscopy analyses. HAp formation through a dissolution-precipitation process provided a mildly alkaline medium suitable for deprotonation of the acid-PCPP and for the formation of the calcium crosslinks, as monitored by infrared spectroscopy. Concurrence of crosslinking of the polymer and HAp formation was established, indicating true composite formation can be realized at physiologic temperature.

Composite formation from hydroxyapatite with sodium and potassium salts of polyphosphazene.

The low temperature synthesis of composites potentially suitable as bone substitutes which form in vivo, was investigated. The composites were comprised of stoichiometric hydroxyapatite (SHAp) and water-soluble poly phosphazenes. These constituents were selected because of their biocompatibility, and were mixed as powders with a phosphate buffer solution (PBS) to form the composites. The effects of poly[bis(sodium carboxylatophenoxy)phosphazene] (Na-PCPP) or poly[bis(potassium carboxylatophenoxy) phosphazene] (K-PCPP) on stoichiometric hydroxyapatite (SHAp) formation from tetracalcium phosphate and anhydrous dicalcium phosphate were assessed. The kinetics and reaction chemistries of composite formation were followed by isothermal calorimetry, X-ray diffraction, infrared spectroscopy and scanning electron microscopy. In the presence of 1% by weight of polyphosphazenes, composites comprised of SHAp and calcium cross-linked polymer salts were formed. Thus a mechanism for binding between polymer chains was established. Elevated proportions (5 and 10% by weight) of polyphosphazene, however, resulted in the inhibition of SHAp formation. This is attributed to the formation of viscous polymer solution coatings on the calcium phosphate precursors, retarding their reaction, and consequently inhibiting SHAp formation.

Low temperature formation of hydroxyapatite-poly(alkyl oxybenzoate)phosphazene composites for biomedical applications.

The formation of biodegradable composites which may be suitable as bone analogs is described. Polyphosphazene-hydroxyapatite (HAp) composites were produced via an acid-base reaction of tetracalcium phosphate and anhydrous dicalcium phosphate in the presence of polyphosphazenes bearing alkyl ester containing side-groups. The polyphosphazenes used were poly(ethyl oxybenzoate)phosphazene (PN-EOB) and poly(propyl oxybenzoate) phosphazene (PN-POB). The effects of temperature and the proportions of polymers, PN-EOB and PN-POB on the kinetics, reaction chemistry and phase evolution during the formation of stoichiometric HAp were studied. Kinetics, phase evolution and microstructural development were evaluated using isothermal calorimetry, X-ray diffraction and scanning electron microscopy, respectively. Analysis of solution chemistry revealed that the increases in the pH during the formation of SHAp, resulted in partial hydrolysis of the polymer surfaces, which led in turn to the formation of a calcium cross-linked polymer surface. The calcium cross-linked polymer surface appeared to facilitate the nucleation and growth of apatite deposits on the polymer. The current study illustrates the in situ formation of HAp in the presence of polyphosphazenes, where HAp is chemically bonded to the polymer.

Crystallization behavior of silica-calcium phosphate biocomposites: XRD and FTIR studies.

Silica and calcium phosphates (CaP) are the most important ingredients in bioactive materials that bond to bone and enhance bone tissue formation. In this study, silica-calcium phosphate (SiO2-CaP) composites were developed by powder metallurgy method, using silica (SiO2) and anhydrous dicalcium phosphate (CaHPO4) powders (CaP) in the ratios (wt%): 20/80, 40/60, 60/40 and 80/20. The effects of temperature and chemical composition on crystallization and phase transformation of the SiO2-CaP composites were evaluated by XRD and FTIR. Thermal treatment of the starting material suggested that CaHPO4 transforms into: gamma-Ca2P2O7 at 800 degrees C; beta-Ca2P2O7 at 1000 degrees C and alpha-Ca2P2O7 at 1200 degrees C. On the other hand, beta-quartz was the only detected phase after thermal treatment of silica in the temperature range 800-1200 degrees C. For all SiO2-CaP composites, SiO2 and CaP did not modify the crystallization behavior of each other when sintered in the temperature range 800-1000 degrees C. However, at 1200 degrees C, CaP promoted the transformation of gamma-quartz into alpha-cristobalite. Moreover, SiO2 stabilized beta-Ca2P2O7. The modifications in the crystallization behavior were related to ion substitution and formation of solid solutions.

Characterization of wollastonite-reinforced HAp--Ca polycarboxylate composites.

The effects of wollastonite on the mechanical properties and in vitro behavior of hydroxyapatite-Ca polyacrylate composites were studied. Powder mixtures of tetracalcium phosphate, poly(acrylic-co-itaconic), and wollastonite fibers (< or =75% by weight) were hot-pressed for 30 min at 300 degrees C and 60 kpsi. Tensile strengths, elastic moduli, and microstructures of the composites were investigated. The tensile strengths of these composites were improved by the addition of wollastonite fibers, whereas the elastic moduli decreased. The highest value of tensile strength (approximately 155 MPa) was achieved by the addition of 40% wollastonite. Composites were immersed in simulated body fluid (SBF) for up to 14 days and then in 1.5 SBF for a week. The changes in the concentrations of Ca, Si, and P ions and the pH of these solutions indicate bioactivity. An evaluation of the microstructures of the composites after SBF immersion indicated that apatite layers had formed on the surfaces of the composites.

Chemically formed HAp-Ca poly(vinyl phosphonate) composites.

The formation of biocompatible organic-inorganic composites by reactions between tetracalcium phosphate (Ca4(PO4)2O, TetCP) and the biomedical polymer poly(vinyl phosphonic acid) (PVPA) is described. Composites were prepared by hot pressing mixtures of these powders at 80 kpsi and 300 degrees C for 30 min. Composite formation was investigated depending on the proportions of reactants and the processing route used. Two inorganic phases were produced as a result of the acid-base reaction between TetCP and PVPA: hydroxyapatite (Ca10(PO4)6(OH)2, HAp) and anhydrous dicalcium phosphate (CaHPO4, DCPA). The later phase preferentially formed at lower TetCP/PVPA ratios while the amount of HAp increased with increasing TetCP/PVPA ratio. The reactions appear to start with the softening of the polymer when heated to T > Tg. The flowing polymer surrounds the TetCP grains permitting the TetCP to initially form DCPA crystallites in a matrix of the Ca salt of the polymer. When H2O is added prior to pressing, the DCPA produced reacts with the remaining TetCP forming HAp.

Preparation and characterization of calcium phosphate-poly(vinyl phosphonic acid) composites.

Composites of calcium phosphates and the calcium salt of a biomedical polymer were prepared in situ by hot-pressing particulate mixtures of poly (vinyl phosphonic acid) (PVPA) and tetracalcium phosphate Ca4(PO4)2O, or TetCP) at different temperatures, pressures, and time periods. The objective was to establish whether PVPA could react with TetCP (Ca/P ratio of 2.0) to form a calcium salt, and thereby decrease the available Ca/P ratio 1.67 to facilitate hydroxyapatite (Ca10(PO4)6(OH)2 or HAp) formation. The effects of varying the bulk composition, temperature (to 300 degrees C), pressure (to 690 MPa) and time (to 60 min) on the reaction between TetCP and PVPA were studied using X-ray diffraction, infra-red spectroscopy and scanning electron microscopy techniques. Results showed that the conversion of TetCP into HAp increased with compaction time as temperature and/or pressure were increased. Formation of anhydrous dicalcium phosphate (CaHPO4, or DCPA) was also observed. Complete conversion of TetCP to HAp was achieved in composites pressed at 250 degrees C and 415 MPa for 30 min.

Characterization of bioactive glass-reinforced HAP-polymer composites.

The effect of bioactive glass on the mechanical properties of hydroxyapatite-Ca polyacrylate composites was studied. Powder mixtures of tetracalcium phosphate (TetCP), poly(acrylic-co-itaconic) and bioactive glass (up to 50% by weight) were hot pressed for 30 min at 300 degrees C and 40 kpsi. Tensile strengths, elastic moduli, and microstructures of the composites produced were investigated. Results showed the mechanical properties of these composites were enhanced by the addition of bioactive glass. The highest values of tensile strength and elastic modulus were achieved with the addition of 10% bioactive glass. Composites were immersed in SBF for up to 10 days, then in 1.5 simulated body fluid (SBF) for a week. The changes in the concentrations of Ca, P, and Si ions of these solutions were measured. The microstructures of these composites after SBF immersion were also evaluated. Concentrations of Ca, P, and Si increased with the time of immersion in SBF owing to the formation of an apatite layer on their surfaces as found by SEM with energy-dispersive spectroscopy attachment.

An evaluation of mechanical property and microstructural development in HAP-Ca polycarboxylate biocomposites prepared by hot pressing.

A hot-pressing technique was used to prepare composites anticipated to be biocompatible. Ca(4)(PO(4))(2)O (TetCP) was reacted with an acrylic-itaconic copolymer (CoP) in the absence of a solvent to form composites comprised of Ca(10)(PO(4))(6)(OH)(2') (hydroxyapatite, or HAp) and the Ca polyalkenoate salt. The effect of temperature, pressure, and hot-pressing time on the mechanical properties and microstructure of the composites were studied. Results showed that both tensile strength and elastic modulus increased when temperature and time were increased. When the compaction pressure was increased, these properties initially increased but decreased at high pressures. These variations in the mechanical properties were correlated with the microstructure of these composites. The mechanism of the reaction was also studied. Reaction starts when the copolymer is heated to above its T(g) permitting it to flow and react with the TetCP grains. The COOH groups on the polymer are neutralized by Ca(2+) ions liberated from the TetCP. At the end of reaction, a network of the Ca polyalkenoate salt is formed in which HAp crystals are embedded.