Edward D. Eanes

Amorphous Calcium Phosphate-Based Bioactive Polymeric Composites for Mineralized Tissue Regeneration

Amorphous calcium phosphate (ACP), a postulated precursor in the formation of biological hydroxyapatite, has been evaluated as a filler phase in bioactive polymeric composites that utilize dental monomers to form the matrix phase on polymerization. In addition to excellent biocompatibility, these composites provided sustained release of calcium and phosphate ions into simulated saliva milieus. In an effort to enhance the physicochemical and mechanical properties and extend the utility of remineralizing ACP composites to a greater variety of dental applications, we have focused on: a) hybridizing ACP by introducing silica and/or zirconia, b) assessing the efficacy of potential coupling agents, c) investigating the effects of chemical structure and compositional variation of the resin matrices on the mechanical strength and ion-releasing properties of the composites, and d) improving the intrinsic adhesiveness of composites by using bifunctional monomers with an affinity for tooth structure in resin formulations. Si- and Zr-modified ACPs along with several monomer systems are found useful in formulating composites with improved mechanical and remineralizing properties. Structure-property studies have proven helpful in advancing our understanding of the remineralizing behavior of these bioactive composites. It is expected that this knowledge base will direct future research and lead to clinically valuable products, especially therapeutic materials appropriate for the healing or even regeneration of defective teeth and bone structures.

Behavior of a calcium phosphate cement in simulated blood plasma in vitro

OBJECTIVESnThe purpose of this study was to gain a better understanding of the integration of calcium phosphate cement (CPC) implants in biological tissue.nnnMETHODSnAn in vitro continuous flow system was employed to examine the protracted behavior of disc-shaped specimens of this bioactive material under sustained physiological-like solution conditions. Weight measurement, diameteral tensile strength measurement (DTS), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and powder x-ray diffraction (XRD) were used to characterize the CPC samples as a function of immersion time.nnnRESULTSnWhen CPC was immersed in simulated blood plasma in which the Ca (2.5 mmol/L) and inorganic phosphate (1.0 mmol/L) levels were kept constant, both the weight and DTS of the specimens steadily increased to about 1.5 times their original values over a period of 20 wk. SEM observations showed new precipitate formations in intimate contact with the original CPC surface. FTIR and XRD analyses revealed that the precipitate was a B-type carbonate hydroxyapatite (OHAp), the type of OHAp observed in bone and dentin. On the other hand, the interior of CPC discs did not show an increase in either bulk density or OHAp content. Thus, the increases in weight and DTS are attributable to the OHAp precipitation on the CPC surface.nnnSIGNIFICANCEnThe results suggest that under in vivo conditions, CPC implants would not dissolve in physiological fluids. OHAp coatings may form on the implants, which may enhance bonding of implants to bone by mechanically strengthening the interface between them.

The Hydrolysis of Anhydrous Dicalcium Phosphate into Hydroxyapatite

Hydrolytic conversion of dicalcium phosphate anhydrous (DCPA; CaHPO4) to non-stoichiometric hydroxyapatite (HAP; Ca10-x(HPO4)x(PO 4)6-x(OH)2-x) was investigated under controlled-solution conditions for identification of solution factors which influence the nucleation, growth, and crystal morphology of the apatitic product phase. The hydrolysis experiments (1 mmol to 7.5 mmol CaHPO4/250 mL) were carried out under CO2-free N2 at fixed pH in the presence of 25-100 mmol/L CaCl2 by standard pH-stat techniques at 37°C. The pH values of the experimental solutions ranged from 6.35 to 9.0. The crystal size and shape of the HAP product were established by x-ray diffraction, and by scanning and transmission electron microscopy. Generally, hydrolysis times and the size of the HAP crystals attained upon completion of the conversion were inversely related to pH. However, crystal size decreased sharply below pH 6.5. Crystal morphology was also strongly affected by pH, with the most equidimensionally-shaped crystals developing at pH 7.0. Although the HAP first appeared on the surface of the DCPA, the amount of DCPA used did not affect the final HAP crystal size or the time required for the hydrolysis to be completed. These data suggest that DCPA initiated HAP formation but that nucleation density (i.e., number of nuclei/unit area DCPA surface) and subsequent growth events were controlled by solution factors.

Mechanical properties of bioactive amorphous calcium phosphate/methacrylate composites

OBJECTIVESnThe aim of this study was to determine whether amorphous calcium phosphate (ACP)-containing composites, which have the ability to release mineralizing levels of Ca and PO4 ions, have appropriate mechanical properties for use as base and lining materials.nnnMETHODSnComposites of pyrophosphate-stabilized ACP particulates (mass fraction of 40%) and photo-activated methacrylate resins (mass fraction of 60%) were tested for biaxial flexure strength (BFS), diametral tensile strength (DTS), and compressive strength (CS). Hydroxyapatite (HAP; mass fraction of 40%), and micro-sized glass (mass fraction of 50%) composites as well as a commercial visible light curable base/liner were also tested. The significance between mean values was determined by Student-Newman-Keuls multiple comparisons (p < 0.05).nnnRESULTSnBFS of dry and wet (24 h at 37 degrees C in water) ACP composites (60.3 and 62.0 MPa, respectively) were significantly lower than those of the comparison materials (79.2-109.3 MPa). CS values were likewise lower (62.9 MPa dry and 67.6 MPa wet vs 80.6-196.8 MPa) except for the wet base/liner (58.5 MPa). DTS of the dry ACP composite (21.8 MPa) was comparable with that of the HAP (22.8 MPa) and glass (25.5 MPa) composites, but lower than that of the base/liner (36.2 MPa). DTS decreased significantly when the ACP composite was wet (17.8 MPa).nnnSIGNIFICANCEnThese results suggest that the remineralizing ACP polymeric composites, although mechanically weaker in some respects than other polymeric composites, have properties suitable for use as base and lining materials.

Membrane-mediated precipitation of calcium phosphate in model liposomes with matrix vesicle-like lipid composition☆

The present study examined calcium phosphate precipitation in aqueous suspensions of artificial liposomes which closely resembled matrix vesicles (MV) in membrane lipid composition. At 22 degrees C, the liposomes per se did not initiate precipitation in the suspending medium for up to 120 h when the latter was made supersaturated with respect to hydroxyapatite (2.25 mM Ca2+, 1.5 mM PO4, 240 mosmol, pH 7.4). Likewise, the suspending medium remained stable for up to 72 h when precipitation was induced within the aqueous interiors of the liposomes by encapsulating pH 7.4-buffered 50 mM PO4 solutions in the interior spaces and making the enclosing membranes permeable to external solution Ca2+ ions with the ionophore X-537A. However, extraliposomal precipitation readily occurred under these latter conditions when phosphatidylserine (PS) and sphingomyelin (Sph) were deleted from the MV-like lipid formulation used to prepare the liposomes. These results suggest that lipidic membrane constituents such as PS and Sph may have a controlling influence on MV-mediated calcification in vivo by affecting the release of intravesicularly formed mineral crystals into the extracellular matrix space where they can subsequently grow and proliferate.

The Effect of Supersaturation on Apatite Crystal Formation in Aqueous Solutions at Physiologic pH and Temperature

The importance of supersaturation in the dynamics of apatite precipitation from aqueous solutions is well-established. To determine whether this parameter has a comparable impact on the concomitant development of the textural properties of this phase, such as crystal size and shape, we investigated mineral accretion in synthetic solutions seeded with 0.67 g/L apatite over a range of supersaturations at pH 7.4 and 37°C. A dual specific-ion electrode-controlled titration method was used to maintain the seeded reactions under the following solution conditions: 1.0 to 1.8 mmol/L Ca2+, 0.67 to 1.2 mmol/L total phosphate (PO 4), Ca/PO4 (initial) = 1.5, 143 mmol/L KNO3, and 10 mmol/L HEPES. Samples were collected for chemical and textural analyses when the seed apatite was reduced by new accretions to 1/2,1/4,1/8,1/16, and 1/32 of the total solids in suspension. All new accretions were found to be apatitic. At the lowest supersaturation, accretion occurred primarily by growth of the seed crystals. However, at the highest supersaturation examined, the crystals at the end of the experiments were actually smaller, on average, than the original seeds, even though the total mass increased 32-fold. The results suggest that proliferation of new crystals supplanted growth of the seed crystals as supersaturation was increased. The results also suggest that differences in tissue fluid supersaturation may contribute to the large disparity in dimensions between dentin and enamel apatite crystals.

Mixed phospholipid liposome calcification

Synthetic lipid vesicle (liposome) suspensions have been used to experimentally model many of the calcium phosphate precipitation steps observed in matrix vesicle (MV) calcification. In particular, precipitate development in liposomes can be made to preferentially follow the progression seen in MV, i.e. to occur initially in intraliposomal spaces and then to expand into the surrounding suspending medium. This paper reviews results from studies by us which show that certain phospholipid (PL) constituents of the liposomal membrane can modulate this progression. Of greatest relevance to MV calcification is the observation that phosphatidylserine and sphingomyelin, two lipids selectively enriched in MV, slow the expansion of the precipitation from inside to outside the liposome.

Effect of different phospholipid-cholesterol membrane compositions on liposome-mediated formation of calcium phosphates

SummaryThe present report compares the effects of different membrane phospholipid (PL)-cholesterol compositions on the kinetics of liposome-mediated formation of calcium phosphates from metastable solutions (2.25 mM CaCl2; 1.5 mM KH2PO4) at 22°C, pH 7.4 and 240 mOsm. In most experiments, the liposomes were composed of 7:2:X mixtures of phosphatidylcholine (PC), neutral or acidic phospholipids, and cholesterol (Chol, X=0, 10, 35, or 50 mol%). The neutral phospholipids (NPL) examined, in addition to PC, were phosphatidylethanolamine (PE) and sphingomyelin (Sph), and the acidic phospholipids (APL) examined were dicetylphosphate (DCP), dioleolylphosphatidylglycerol (DOPG), dioleolylphosphatidic acid (DOPA), phosphatidylserine (PS) and phosphatidylinositol (PI). The 7:2:X liposomes did not initiate mineralization in metastable external solutions per se or, with the exception of DOPA, show extensive Ca-PL binding. However, solution Ca2+ losses due to precipitation occurred when the liposomes were encapsulated with 50 mM KH2PO4 and made permeable to external Ca2+ with X-537A. The extent of these Ca2+ losses was sensitive to both the phospholipid and Chol makeup of the membrane. Moderate-to-extensive intraliposomal precipitation occurred in all 7PC:2APL and 7PC:2NPL liposomes containing 0 or 10 mol% Chol. In contrast, at 50 mol% Chol, mineralization inside all liposomes was negligible. The only significant discriminating effect on internal mineralization among the different phospholipids was observed at 35 mol% Chol, where mineral accumulations ranged from negligible to moderate. At 0 or 10 mol% Chol, extraliposomal precipitation was extensive in all but DOPA- and PS-containing liposomes. However, onece intraliposomal yields declined at the higher Chol levels, external mineralization was either delayed or totally blocked in all liposome preparations. Other experiments showed that Sph substituted for PC in 7NPL:2DCP:1Chol liposomes totally blocked both intra- and extraliposomal precipitaiton. PE substituted in this manner, however, blocked only extraliposomal precipitation. The results of this study suggest that interference of the membrane transport processes controlling intraliposomal precipitation [15] by high (50 mol%) Chol levels is not significantly compromised by the specific APL or NPL incorporated in the membrane. Similarly, the data suggest that Chol does not directly affect the specfic interactions of the different membrane APLs with the mineral phase. On the other hand, the substitution of other NPLs for PC can affect the role of APLs such as DCP in liposome-mediated mineralization.

Effect of membrane cholesterol on calcium phosphate formation in aqueous suspensions of anionic liposomes

SummaryThe present study examined the effect of membrane cholesterol on liposome-mediated calcium phosphate precipitation in metastable aqueous solutions (2.25 mM Ca2+ and 1.5 mM inorganic phosphate) at 22°C, pH 7.4 and 240 mOsm. The liposomes were prepared from 7:2:X molar mixtures of phosphatidylcholine, dicetylphosphate, and cholesterol (x=0, 1, 5, or 9) and contained either 0 or 50 mM encapsulated phosphate. The membranes were made permeable to Ca2+ by addition of the cationophore, X-537A. Changes in external Ca2+ concentration were used as the principal monitor of the course of precipitation. Without encapsulated phosphate, 7:2:X liposomes (with or without ionophore) induced no precipitation. With 50 mM encapsulated phosphate and in the presence of ionophore, precipitation significantly depended on the cholesterol level in the membrane. At 0 and 10 mole% cholesterol, precipitate developed rapidly both within and outside the liposomes. At 35 and 50 mole% cholesterol, no observable intraliposomal precipitation occurred, and extraliposomal precipitation started only after an induction period of 24 hours. Delayed extraliposomal precipitation also took place in PO4-containing liposomes without added ionophore. In this latter case, however, cholesterol was essential for this precipitation to occur with the optimum level being around 10 mole%. Suppression of ionophore-mediated intraliposomal precipitation at higher cholesterol levels could be related to the inflexible cholesterol molecules making the membrane more rigid, thereby restricting Ca-ionophore transport. This restriction could be reversed with ethanol. Delayed extraliposomal precipitation in the absence of added ionophore (or at higher cholesterol levels in its presence) could be explained by seeding from low, unobserved levels of intraliposomal precipitate formed during slow, unfacilitate Ca2+ leakage into the liposomal interior.

In vitro inhibition of membrane-mediated calcification by novel phosphonates

The effects of a series of novel phosphonates on the kinetics of mineral development in an ionophore-primed 7:2:1 phosphatidylcholine (PC): dicetylphosphate (DCP): cholesterol (Chol) liposomal model system are reported. When present at 2.5 μmol/liter or 25 μmol/liter concentrations in the solution surrounding the liposomes, the investigated phosphonates did not significantly delay the initial formation of hydroxyapatite-like calcium phosphate salts (HAP) within the liposomes or the penetration of HAP crystals through the enclosing membranes. However, the phosphonates variably retarded the subsequent growth and proliferation of the HAP crystals once they became directly exposed to the phosphonate-containing solution. The effectiveness of phosphonates in inhibiting extraliposomal precipitation strongly depended on their structure. The inhibitory action on active surface growth sites of released intraliposomal crystals was found to be the most effective if the phosphonate molecule, contained two phosphonic groups linked to the same C atom. At a phosphonate concentration of 25 μmol/liter, the, following general order of effectiveness was established: geminal bisphosphonate≥geminal tetrakisphosphonate > bisacylphosphonates > monoacyl-phosphonate > bisalkylphosphonate. Within the bisacylphosphonate family, the highest inhibitory action was observed when four of five-CH2-groups separated the ketophosphonic groups.