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[Chromatography of nucleic acids, proteins and certain phages on granulated hydroxyapatite].

A modification of the classical method of hydroxyapatite synthesis is proposed. The essence of the modification is hydroxyapatite synthesis in the presence of an additional component silicic acid particles. The subsequent steps of the method are modified so, as to retain the intactness of crystals at all the stages of preparation and use of the adsorbent. The final product consists of large spherical agregates (200-250 mu in diameter) and contains about 1% of tightly bound silicic acid. It slightly differs from usual hydroxyapatite in its chromatographic properties. Granulated hydroxyapatite obtained has a high specific capacity and can be repeatedly used in experiments (up to 50 chromatographic cycles). Native high-polymeric T2 phage DNA was practically quantitatively eluated from the column. Conditions for chromatography of some proteins (lysozyme, RNase, DNase) are described. Fractionation and purification of T2 and T3 bacteriophages and TMV are carried out by means of chromatography on granulated hydroxyapatite.

Chromatography↗
PubMed1992

Induction of bone in composites of osteogenin and porous hydroxyapatite in baboons.

A major goal of the combined effort of basic scientists and plastic and reconstructive surgeons is the development of novel bone substitutes based on osteogenic growth and differentiation factors with optimal delivery systems for skeletal repair. Osteogenin is a protein initiator of bone differentiation. The present study examined the osteogenic potential of osteogenin in combination with porous hydroxyapatite replicas obtained after hydrothermal conversion of calcium carbonate exoskeletons of corals. Bovine osteogenin, with an apparent molecular weight of 28 to 42 kDa, purified by hydroxyapatite-Ultrogel adsorption chromatography, heparin-Sepharose affinity chromatography, and HR Sephacryl S-200 molecular sieve chromatography, was delivered into rods of nonresorbable and resorbable hydroxyapatite replicas with an average porosity of 600 microns. A total of 48 rods were bioassayed for osteogenic activity by intramuscular implantation into eight adult baboons (Papio ursinus) as a prerequisite for clinical trials in humans. Bovine osteogenin fractions reconstituted with baboon insoluble collagenous bone matrix were implanted in an additional four adult baboons. Specimens were harvested at 30 and 90 days after implantation and subjected to histomorphometry and alkaline phosphatase activity determination. Differentiation of bone occurred in nonresorbable hydroxyapatite rods, both osteogenin-treated and controls. However, no bone formation was observed in resorbable rods, even in the presence of osteogenin. These results demonstrate that the surface and chemical characteristics of the substratum, independent of the osteogenic stimulus, have a profound influence on the morphogenesis of bone. The demonstration of bone induction in nonhuman primates with porous nonresorbable hydroxyapatite replicas and baboon insoluble collagenous bone matrix reconstituted with bovine osteogenin establishes the therapeutic potential of the principle of bone induction in craniofacial, periodontal, and orthopedic reconstructive surgery.

Analysis of Variance↗
PubMed1991

Works in progress #6. Total hip arthroplasty using a hydroxyapatite-coated acetabular and femoral component.

The development of alternative methods of implant fixation in primary and revision total hip arthroplasty is ongoing. Porous-coated implants abound; however, concerns surrounding their use still exist. Hydroxyapatite is a calcium phosphate ceramic, which has demonstrated properties in animal studies that make it an attractive alternative for implant fixation. We describe the early clinical and roentgenographic results of a prospective study that utilized a hydroxyapatite-coated stem and cup in 52 patients (58 hips). The average follow-up was 10.4 months (minimum, six months). At one year, 96.2% of the hips had no or slight pain, 95.1% could ambulate with no external support, 82.9% could ambulate unlimited distances, and 87.8% could walk without a limp. The roentgenographic analysis of the hydroxyapatite-coated stems (58) and cups (17) at one year revealed no radiolucencies or radiodense lines adjacent to the hydroxyapatite-coated portion of the prosthesis. None of these stems or cups migrated. We believe that these hydroxyapatite-coated components will provide rapid and enhanced prosthetic fixation and are optimistic that they will help to achieve an excellent clinical result.

Gait↗
PubMed1989

[Implants with plasma spray coatings of hydroxyapatite: an experimental study in alveolar bone].

Sintered hydroxyapatite ceramic can be coated to a titanium core by means of a high temperature plasma spray procedure. The biological response of alveolar bone to an implant with such a coating of hydroxyapatite was evaluated in an animal experimental study. The results indicated that the plasma spray coating gave a very strong and direct bonding to cortical alveolar bone. The shear strength between bone and ceramic was greater than the shear strength between ceramic and titanium. From a radiological, macroscopic and microscopic point of view the conclusion could be drawn that the biological properties of a plasma spray coating of hydroxyapatite are the same as the properties of bulk hydroxyapatite ceramic. The outermost layer of the hydroxyapatite coating appeared to be unstable under the experimental conditions.

Animals↗
PubMed1994

Bone formation in the presence of phagocytosable hydroxyapatite particles.

Small particles of biomaterials used in orthopaedic surgery have been shown to induce the resorption of bone. The purpose of this study was to determine whether phagocytosable particles of hydroxyapatite had an adverse effect on bone ingrowth. Bone harvest chambers were implanted bilaterally in the proximal tibial metaphyses of 13 mature rabbits. The bone harvest chamber has a transverse 1-mm wide pore, providing a continuous canal through the chamber for tissue ingrowth. After an initial 6-week period for osseointegration of the bone harvest chambers, the contents of the canal were harvested at 3-or 6-week intervals. Hydroxyapatite particles (diameter, 5 mu) were mixed with a carrier solution, 1% sodium hyaluronate, and implanted in the canal of one chamber in each animal. The contralateral chamber was implanted with the carrier only and served as a control. Histological sections from the tissue harvested from the chambers were evaluated by light microscopy and histomorphometry, and the area of bone ingrowth was measured as a percentage of total area in each section. At 3 weeks there was more bone in the hydroxyapatite sections than in controls; at 6 weeks there was no difference. Hydroxyapatite particles were incorporated within the matrix of new ingrown bone at both time periods. There was no evidence of granuloma formation or inflammation. Previous studies have shown that particles of high density polyethylene and bone cement adversely affect bone ingrowth in this model. The present results suggest that hydroxyapatite particles, small enough to be phagocytosed by macrophages, did not have such effects.

Animals↗
PubMed1996

Basic fibroblast growth factor promotes bone ingrowth in porous hydroxyapatite.

The effect of basic fibroblast growth factor on tissue ingrowth and differentiation in porous hydroxyapatite of coralline origin was studied in a bone chamber model. The hydroxyapatite with or without basic fibroblast growth factor was placed in 22 mm3 titanium bone conduction chambers implanted bilaterally in rat tibiae. Ingrowing bone could enter the cylindrical interior of the chamber only at 1 end. It then penetrated the porous hydroxyapatite inside the chamber. The distance that the ingrown tissue had reached into the material then was measured on histologic slides. Because fibrous tissue always reached further into the material than did bone, both total tissue ingrowth and bone ingrowth distances were measured. In implants supplemented with 0.04 microg basic fibroblast growth factor in a hyaluronate gel carrier, the bone ingrowth distance was increased by 70% at 6 weeks, as compared with paired controls in the contralateral leg. The total tissue ingrowth distance also was increased by 58%. When the dose of basic fibroblast growth factor was increased to 1.0 microg, still using the hyaluronate carrier, there was no difference in bone ingrowth compared with controls, but this dose still increased the total tissue ingrowth. In hydroxyapatite with 1.5 microg basic fibroblast growth factor without hyaluronate gel at 4 weeks, no increase in bone ingrowth was shown, but total tissue ingrowth was increased. At 6 weeks, bone ingrowth and total tissue ingrowth were increased by 41% and 33%, respectively. With a lower dose of 0.15 microg without carrier, only the total ingrowth distance was increased. The results suggest that basic fibroblast growth factor may promote tissue ingrowth into porous hydroxyapatite and that bone ingrowth may be increased by appropriate doses. The hyaluronate gel carrier reduced the optimal dose.

Animals↗
PubMed1997

Hydroxyapatite coated implants. Total hip arthroplasty in the young patient and patients with avascular necrosis.

Two high risk groups for total hip arthroplasty, 136 patients (155 hips) younger than 50 years of age (average age, 38 years) and 44 patients (53 hips) with the diagnosis of avascular necrosis, have a minimum followup of 5 years and a mean followup of 6.8 years. The average Harris Hip Score at last followup totaled 93 and 90, respectively, and thigh pain was reported in 1.3% and 3.8%, respectively. All patients in both groups received the same hydroxyapatite coated femoral stem and the mechanical failure was 0%. No stem was revised for aseptic loosening, 100% of stems were bony stable by radiographic criteria, new bone formation was progressive about the femoral stem, and 0% endosteal lysis was found. The acetabular components had a mechanical failure rate of 10% and 7.5%, respectively, without osteolysis, and an additional 7% and 7.5% failure as a direct result of progressive osteolysis. The results with the porous press fit and hydroxyapatite threaded sockets were far superior to that of the smooth hydroxyapatite press fit sockets and socket failure was associated with thin polyethylene liners and the use of 32-mm head diameters. These findings show a high success rate with a nonporous hydroxyapatite coated titanium femoral stem. However, hydroxyapatite on a smooth acetabular component yielded less predictable results indicating that for long term fixation of the acetabulum an interlock of bone is preferred.

Adolescent↗
PubMed1998

BMP-induced osteogenesis on the surface of hydroxyapatite with geometrically feasible and nonfeasible structures: topology of osteogenesis.

Bone morphogenetic protein (BMP) is known to require a suitable carrier to induce ectopic bone formation in vivo. Hydroxyapatite ceramics have been reported to be effective in some forms but ineffective in others as a carrier of BMP-induced bone formation. In this study we compare three geometrically different forms of hydroxyapatite to examine their functions as carriers of BMP-induced bone formation. A fraction containing all the active BMPs (BMP cocktail) was partially purified from a 4M guanidine extract from bovine bone by a three-step chromatographic procedure. The BMP cocktail was combined with each of three forms of hydroxyapatite--solid particles (SPHAP), porous particles (PPHAP), and coral-replicated porous tablets (coral-HAP)--and implanted subcutaneously into rats. Both the PPHAP and coral-HAP systems induced osteogenesis 2 weeks after implantation, as evidenced by morphological and biochemical observations. Details of the osteogenetic process were followed by double-fluorescence labeling in the coral-HAP system to confirm bone formation on the surface of hydroxyapatite. However, there was no evidence of osteogenesis or chondrogenesis in the SPHAP system. The results indicate that the geometry of the interconnected porous structure in PPHAP and coral-HAP create spaces for vasculature that lead to osteogenesis while the smooth structure and close contact of particles in SPHAP inhibit vascular formation and proliferation of mesenchymal cells, preventing bone and cartilage formation. It was concluded that the geometrical structure in hydroxyapatite ceramics that induces vasculature is crucial as a carrier for BMP-induced bone formation.

Alkaline Phosphatase↗
PubMed1998

Hydrothermal-electrochemical deposition of hydroxyapatite.

Hydroxyapatite crystals were formed on a titanium electrode using the hydrothermal-electrochemical method in an autoclave with two electrodes. The electrolyte dissolving NaCl, K2HPO4, CaCl2.2H2O, trishydroxyaminomethane, and hydrochloric acid was maintained at 80 degrees - 200 degrees C. After loading of a constant current at 12.5 mA/cm2 for 1 h, the deposited amounts were measured through the weight gain of the electrode. The deposits were characterized by X-ray diffractometry, Fourier transform infrared spectroscopy, field emission-type scanning electron microscopy, field emission-type transmission electron microscopy, and energy dispersive X-ray spectroscopy. The deposited amount increased with electrolyte temperatures up to 150 degrees C and slightly decreased above that temperature. The deposits were identified as hydroxyapatite crystal rods grown along the c axis and perpendicular to the substrate. The crystallinity of the deposited hydroxyapatite increased continuously with the electrolyte temperature and closed to stoichiometric hydroxyapatite. At 150 degrees - 160 degrees C, the hydroxyapatite rod grew very homogeneously perpendicular to the substrate, and the edge of the needle had a flat hexagonal plane.

Durapatite↗
PubMed2001

Effect of hydroxyapatite sintering temperature on intracellular ionic concentrations of monocytes: a TEM-cryo-X-ray microanalysis study.

Hydroxyapatite used as bone replacement can lead to particle release in the implantation site. These particles interact with monocytes, which are the first immune cells to colonize the implant and an inflammatory site. Thanks to cryo-X-ray microanalysis, we can observe cells in a state close to the physiological one and we have access to diffusible ions. We paid particular attention to the potassium-to-sodium ratio, which is one of the best viability criteria. We used this method to study the interaction between three hydroxyapatite particles treated at three different temperatures (not treated, treated at 600 degrees C and 1180 degrees C), and monocytes. In the culture condition, the hydroxyapatite treated at 1180 degrees C underwent the least dissolution. We demonstrate that monocytes were altered by the three hydroxyapatite particles. The hydroxyapatite particules treated at 600 degrees C were found to be more toxic.

Bone Substitutes↗
PubMed2001

Enhanced osteobonding by negative surface charges of electrically polarized hydroxyapatite.

Negative surface charges of electrically polarized hydroxyapatite ceramics have been proven to enhance osteobonding in canine bone tissues. Even in a wide gap of 0.2 mm between hydroxyapatite and bone, the negatively charged hydroxyapatite conducted formation of unidirectionally oriented bone layer into direct contact with the hydroxyapatite surface 7 days after implantation. By day 14, the gap was filled with maturing bones linked to each other while conventional hydroxyapatite required at least 28 days for direct bone-to-bone contact. The electrostatic force was deduced to have affected both the activation of bone formation by myeloid cells and the orientation of the bone domain on negatively charged surfaces.

Animals↗
PubMed2003

Study of the toughening mechanisms in bone and biomimetic hydroxyapatite materials using Raman microprobe spectroscopy.

A Raman microprobe spectroscopy characterization of microscopic fracture mechanisms is presented for a natural hydroxyapatite material (cortical bovine femur) and two synthetic hydroxyapatite-based materials with biomimetic structures-a hydroxyapatite skeleton interpenetrated with a metallic (silver) or a polymeric (nylon-6) phase. In both the natural and synthetic materials, a conspicuous amount of toughening arose from a microscopic crack-bridging mechanism operated by elasto-plastic stretching of unbroken second-phase ligaments along the crack wake. This mechanism led to a rising R-curve behavior. An additional micromechanism, responsible for stress relaxation at the crack tip, was recognized in the natural bone material and was partly mimicked in the hydroxyapatite/silver composite. This crack-tip mechanism conspicuously enhanced the cortical bone material resistance to fracture initiation. A piezo-spectroscopic technique, based on a microprobe measurement of 980 cm(-1) Raman line of hydroxyapatite, enabled us to quantitatively assess in situ the microscopic stress fields developed during fracture both at the crack tip and along the crack wake. Using the Raman piezo-spectroscopy technique, toughening mechanisms were assessed quantitatively and rationally related to the macroscopic fracture characteristics of hydroxyapatite-based materials.

Biocompatible Materials↗
PubMed2005

The role of bivalent metals in hydroxyapatite structures as revealed by molecular modeling with the HyperChem software.

Hydroxyapatite, the major component of bone, demonstrates significant reactivity with metals. Knowledge of spatial structure and energy data of the molecule helps understand the binding of metals by hydroxyapatite and elucidate the chemical and physical properties of such complexes. We used HyperChem software (Hypercube Inc.) to analyze the structure of hydroxyapatite when the central calcium atom is replaced by one of the metal ions (Mg, Cu, Zn, Fe, Cr, Mn) marked by us in bone. Our results show that hetero-ionic exchange affects composition and leads to deformation of hydroxyapatite crystals. Replacement was accompanied by changes in bond lengths between oxygen and calcium atoms in the hydroxyapatite molecule and by displacement of groups of atoms surrounding the central calcium atom. The use of molecular modeling as a computational tool enabled a preliminary and theoretical understanding of chemical structure without the need for laboratory tests.

Durapatite↗
PubMed1999

Effect of serum proteins on osteoblast adhesion to surface-modified bioactive glass and hydroxyapatite.

Previous studies indicate that modification of the surface of porous bioactive glass promotes osteoblast function. We hypothesize that bone formation on treated bioactive glass is due to the selective adsorption of serum attachment proteins. To test this hypothesis, we examined the profile of proteins adsorbed to treated bioactive glass and compared these proteins with those adsorbed to untreated bioactive glass and porous hydroxyapatite. Porous bioactive glass was treated with Tris-buffered electrolyte solution to generate a calcium phosphate-rich surface layer and then immersed in tissue-culture medium containing 10% serum. Proteins adsorbed to the ceramic surfaces were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Porous hydroxyapatite bound a higher amount of total protein than did the other substrates. However, surface-modified porous bioactive glass adsorbed more fibronectin than did hydroxyapatite. The effect of serum-protein adsorption on osteoblast adhesion to bioactive glass and hydroxyapatite was also evaluated. Cell adhesion to porous bioactive glass that was surface-modified and serum-treated was significantly greater than to porous bioactive glass that was either surface-modified or serum-treated. Furthermore, cell adhesion to porous bioactive glass treated to form the dual layer of calcium phosphate and serum protein was significantly higher than adhesion to porous hydroxyapatite with adsorbed serum protein. Results of the study strongly suggest that adsorption of serum fibronectin to the surface of modified porous bioactive glass coated with calcium phosphate may be responsible for enhanced osteoblast adhesion.

Absorption↗
PubMed1997

Preparation and Characterization of Barium-Strontium Hydroxyapatites

Barium-strontium hydroxyapatite solid solutions with different molar ratio Ba/(Ba + Sr) were synthesized by a wet method and characterized by various means. The solid solution particles could be prepared at molar ratios ranging from 0 to 1; however, Ba2+ ions were more difficult to be incorporated into hydroxyapatite crystals compared to Sr2+ ions. With increasing Ba2+ content, the particles grew and finally turned into pure rod-shaped barium hydroxyapatite particles with a size of ca. 0.2 x 2 &mgr;m. The resulting particles were agglomerates consisted of primary fine particles except for strontium hydroxyapatite. The molar ratios (Ba + Sr)/P of all the particles were larger than the stoichiometric ratio of 1.67, suggesting that CO32- ions, OH- ions, and/or H2O molecules substitute for PO43- ions in the crystal lattices. The amount of CO2 adsorbed irreversibly on the particles increased with increasing (Ba + Sr)/P except for strontium hydroxyapatite and fitted a curve with a minimum at a cation/P ratio of ca. 1.56 as well as other HAPs.

Journal Article↗
PubMed1996

The contributions and limitations of hydroxyapatite coatings to implant fixation: A histomorphometric study of load bearing implants in dogs.

The contribution and limitation of hydroxyapatite coatings to implant fixation are discussed following a 5-year histomorphological study of the bony incorporation of macroporous stemless hemiarthroplasties in dogs. Implants without coating, with a pure titanium coating and with a hydroxyapatite coating were compared. The mean extent of direct contact between bone and hydroxyapatite-coated implants was 66% compared with 0 to 5% in the other implants. Areas of resorption of the coating were seen in every case associated with direct bone apposition on the metal surface. When the initial fixation is stable, osteointegration of hydroxyapatite-coated implants is obtained consistently, despite design features known to preclude bone incorporation. Implant fixation must depend on a mechanical interlock with bone because of the resorbability and mechanical properties of hydroxyapatite coatings.

Animals↗
PubMed2000

Improved extraction torque of hydroxyapatite-coated pedicle screws.

Loosening of the screws is a problem in instrumentation with pedicle screws. Coating with hydroxyapatite improves the holding characteristics for metal implants, but the possible effects on the anchorage of pedicle screws have not been described. In this study, seven patients were operated on with spinal instrumentation using four stainless steel pedicle screws. Hydroxyapatite-coated screws were used in either the upper or the lower of the instrumented levels. The insertion torque was measured. In four cases the screws were removed after 10-22 months and the extraction torque was measured. The mean insertion torque was found to be significantly greater in the hydroxyapatite-coated screws (107 Ncm) than in the standard screws (76 Ncm). In three cases, the extraction torque for the hydroxyapatite-coated screws exceeded the range for the torque wrench (600 Ncm), while the conventional screws were loose (< 5 Ncm). In one case, the extraction torque was 475 and 550 Ncm for the coated screws, and 5 and 25 Ncm for the conventional screws. The difference in extraction torque was significant. Hydroxyapatite coating was shown to have improved the purchase of pedicle screws very effectively. By using fully coated screws, as in the present study, extraction was extremely difficult compared to extraction of conventional stainless steel screws, which were regularly loose. By reducing the area of the screws that is coated, it may be possible to achieve an enhanced purchase while extraction will be easier when compared to fully coated screws.

Adolescent↗
PubMed2006

Hydrothermal synthesis and nanostructure of carbonated calcium hydroxyapatite.

The influence of precursor concentration, pressure, temperature and time of hydrothermal synthesis on the development of calcium hydroxyapatite structure has been analyzed. The obtained results show that it is possible to adjust the conditions of hydrothermal synthesis from solutions of relatively high concentrations to obtain calcium hydroxyapatite nanopowders of well-defined structure. The relationship between the synthesis and the lattice parameters, as well as the crystallite size and the microstructure of synthesized hydroxyapatite has been established. The synthesized powders are preferentially carbonated hydroxyapatite of the B type in the form of agglomerates that accommodate two-modal size pores of 1.5-10 and 50-200 nm. The structure of calcium hydroxyapatite particles consists of crystallites 8-22 nm in size, bound within prime particles, which size is between 10 and 63 nm, that in turn form bigger agglomerates 200 nm in size, which further cluster building up agglomerates 5-20 microm in size.

Biocompatible Materials↗