Effects of low-intensity AC and/or DC electromagnetic fields on cell attachment and induction of apoptosis.

Rat tendon fibroblast (RTF) and rat bone marrow (RBM) osteoprogenitor cells were cultured and exposed to AC and/or DC magnetic fields in a triaxial Helmholtz coil in an incubator for up to 13 days. The AC fields were at 60 and 1000 Hz and up to 0.25 mT peak to peak, and the DC fields were up to 0.25 mT. At various combinations of field strengths and frequencies, AC and/or DC fields resulted in extensive detachment of preattached cells and prevented the normal attachment of cells not previously attached to substrates. In addition, the fields resulted in altered cell morphologies. When RTF and RBM cells were removed from the fields after several days of exposure, they partially reattached and assumed more normal morphologies. An additional set of experiments described in the Appendix corroborates these findings and also shows that low-frequency EMF also initiates apoptosis, i.e., programmed cell death, at the onset of cell detachment. Taken together, these results suggest that the electromagnetic fields result in significant alterations in cell metabolism and cytoskeleton structure. Further work is required to determine the relative effect of the electric and magnetic fields on these phenomena. The research has implications for understanding the role of fields in affecting bone healing in fracture nonunions, in cell detachment in cancer metastasis, and in the effect of EMF on organisms generally.

Protein denaturation induced by cyclic silicone.

While it has been demonstrated that such low-molecular-weight cyclic silicones as octamethylcyclotetrasiloxane (D4) exhibit adjuvant activity, the mechanism of immunological response to silicone is still not clear. This study therefore used fluorescence and circular dichroism (CD) spectroscopy to investigate the denaturation and conformational change of two proteins, fibronectin (Fn) and fibrinogen (Fbg), induced by D4 in vitro. Incubating D4 with Fbg or Fn at D4-to-protein ratios of > 100 or for > 10 h yielded white and mould-like precipitates of the proteins, indicating massive denaturation and aggregation. The fact that the decrease in fluorescence intensity of D4-treated Fn and Fbg was accompanied by a red shift in the maximum wavelength also indicated that denaturation of the proteins had taken place. These changes in fluorescence might result from exposure of tryptophan residuals in the proteins to polar water molecules inasmuch as the denaturation would lead to changes in the tertiary structures of the proteins and rearrangement of the tryptophan residues. The loss of the tertiary structure leads to protein denaturation and, consequently, precipitation. The difference in CD spectra between control Fbg (or Fn) and D4-treated Fbg (or Fn) indicated conformational changes of the proteins when incubated with D4. Thus it has been demonstrated that D4 can induce denaturation and conformational changes in Fbg and Fn and it can be expected that protein molecules that have undergone denaturation or conformational change induced by D4 may act as antigens and stimulate the immune system to generate antibodies, ultimately resulting in autoimmune disease.

Effect of annealing temperature on the degradation of reinforcing fibers for absorbable implants.

Calcium phosphate fibers designed for reinforcement of bioabsorbable fracture fixation devices were evaluated for their properties upon annealing. The composition of these fibers were 54% PO4, 27% Ca, 12% ZnO, 2.5% NaPO3, and 4.5% Fe2O3, and they were either not annealed, annealed at 250 degrees C, or annealed at 420 degrees C. Chemical degradation, mass loss, and morphology upon degradation were studied. Chemical degradation was performed in Tris-buffered HCl, while mass loss and morphologic studies were performed in both physiologic and nonphysiologic solutions. The results showed that degradation rates for fibers were inversely proportional to the annealing temperature. Mass loss analysis of fibers immersed in the two physiologic solutions (calf serum and simulated body fluid) revealed little change in fiber diameter up to 60 days. Morphologic examination revealed little change in fibers immersed in the two physiologic solutions until 60 days, after which thin shells were found to be peeling off the outer coating of the fiber. Samples in tris-buffered HCl revealed a dramatic difference in mode of degradation among the three fibers. Fibers not annealed and those annealed at lower temperatures underwent a delaminating type of degradation that appeared to destroy the overall integrity of the fiber, whereas fibers annealed at 420 degrees C underwent crater-like deterioration in which the overall alignment of the fiber remained intact. It is therefore concluded that annealing fibers at higher temperatures also undergo a mode of degradation that allows them to maintain their structural integrity. Although annealing fibers close to glass transition temperature may produce an initially weaker fiber, chemical and physical degradation occur much slower, making these fibers most suitable for reinforcement of biodegradable implants.

Determination of the mineral phases and structure of the bone-implant interface using Raman spectroscopy.

The bone-implant interface formed in a canine distal femur was examined by means of a Raman microprobe using an implant model designed to test calcium phosphate surface coatings. By using the 960 cm-1 band of calcium phosphate to characterize the interface and adjacent mineral, we obtained spatial and compositional information about the attachment of bone to the synthetic calcium phosphate coating on a titanium support. The interface between bone and the synthetic calcium phosphate is approximately 30-40 microns in width.

The effect of cadmium on the formation and properties of hydroxyapatite in vitro and its relation to cadmium toxicity in the skeletal system.

In order to understand the biological action of cadmium (Cd) in inducing bone pathologies, the effect of Cd on the formation, structure, and properties of hydroxyapatite (HA) in vitro was investigated using three biologically relevant test systems: (1) direct precipitation of HA with no precursor phase; (2) transformation of amorphous calcium phosphate (ACP) to crystalline HA; and (3) growth of HA seed crystals. Cd-containing HA was prepared by transforming ACP to HA in the presence of Cd at a pH of 10; Cd/Ca ratios of 0.05, 0.10, and 0.20 were obtained. Infrared and x-ray diffraction analyses were performed on the Cd-HA samples, and measurements were made of Cd adsorption on HA and of the dissolution characteristics of Cd-containing HA. Cd incorporation in HA introduced little strain in the lattice but resulted in a decreasing C-axis spacing and a corresponding crystal size decrease in the C-axis direction. Cd incorporation had a nominal effect on HA dissolution. Cd had an inhibitory effect on HA formation kinetics in all three test systems. Infrared spectroscopy of Cd-HA showed a complex series of small changes in the spectra as a function of Cd concentration resulting from some distortion in the crystal perfection and symmetry. The interference of Cd with mineralization can be partially explained by its inhibitory effect on HA nucleation and growth in addition to any cellular involvement. Furthermore, Cd probably has little effect on bone mineral dissolution. Our results explain the Cd incorporation reported in bone.

A new technique for quantitation of metal particulates and metal reaction products in tissues near implants.

Tissue specimens retrieved from four regions adjacent to hip implants during revision surgery were subjected to a novel treatment to make possible the quantitative separation of residual metal particulates and metal reaction products (metal ions and metal-protein complexes). The tissues were exposed to sodium hypochlorite solution that degraded and solubilized them, liberating metal reaction product and leaving behind metal wear particles, which were separated by centrifugation. Atomic absorption spectrophotometry was used to analyze the concentrations of the separated metal ions and wear particles. Co ion concentrations were 0.05 to 0.9 mM, Cr ion concentrations were 0.04 to 2.1 mM, and of Ti ion concentrations were 0.30 to 0.60 mM. The weight of Co metal particles was 0.1 to 4.9 mg/100 mg tissue, of Cr metal particles 0.07 to 2.2 mg/100 mg of tissue, and Ti particles 0.09 to 5.2 mg/100 mg tissue; one black tissue sample contained 3333 mg Ti/100 mg tissue. No correlation was found between the concentrations of these two entities in the samples examined, probably due to the complex and varied processes creating them. The procedures discussed here will result in data that can help elucidate the separate contributions of metal reaction products and metal particulates to implant loosening.

Evaluation of a low-temperature calcium phosphate particulate implant material: physical-chemical properties and in vivo bone response.

A study was conducted to evaluate the osteoconductive ability of a particulate, low-temperature hydroxylapatite (HA(LT)) material (OsteoGen; Impladent, Holliswood, NY). An implantable chamber model was used to determine the ability of this material to encourage bone ingrowth into channels lined with either rough-surfaced titanium or rough-surfaced plasma-sprayed hydroxylapatite. The HA(LT) material increased bone ingrowth into the titanium-lined channels comparable with that in plasma-sprayed hydroxylapatite-coated channels. It was incorporated into ingrowing bone without intervening soft tissue, with the bone bonding directly to the material surface in much the same fashion as it bonds at the plasma-sprayed hydroxylapatite surface. Mechanical testing of the ingrown bone showed no weakness because particles were incorporated. At 12 weeks, the particles began to show signs of dissolution. It was concluded that the HA(LT) material is a biocompatible, osteoconductive material that conducts bone ingrowth in much the same way as high-temperature particulate hydroxylapatite ceramics. This material has the additional desirable property of being slowly resorbable, a beneficial characteristic for many bone-filling applications.

Alkaline phosphatase and peptidase levels in invertebrate cartilage.

Cartilage is encountered in the skeletons of many advanced invertebrates, yet it never calcifies or is replaced by bone. In an attempt to account for the absence of bone in invertebrates, we tested a hypothesis proposing that absence or inadequate quantities of several enzymes associated with vertebrate osteogenesis may underlie the failure of the invertebrates to evolve bone. The enzymes examined were alkaline phosphatase, alanyl beta-naphthylamidase, and neutral protease. Their activities were measured in the gill cartilage of the Atlantic horseshoe crab, Limulus polyphemus, and the odontophore cartilage of the marine whelk, Busycon canaliculatum. Animals were collected from the Cape Cod area. Samples of cartilage of Limulus perichondrium, various non-skeletal tissues, and neonatal rat calvaria, the latter as a reference standard, were homogenized in 0.1 M phosphate buffer (pH 7.1) and analyzed for protein content and the above-mentioned enzyme activities. Alkaline phosphatase specific activity was readily detected in most tissues except the invertebrate cartilage specimens in which it was present only at near-trace levels. Naphthylamidase and protease activities were present in all tissues. In a single experiment, higher phosphatase values were recorded for Limulus cartilage retaining perichondrium, but in a subsequent trial assaying cartilage retaining perichondrium, denuded cartilage, and isolated perichondrium separately, it was demonstrated that phosphatase activity resided primarily within the perichondrium. Exposure of thick cryostat sections to p-nitrophenyl phosphate confirmed the suspicion that alkaline phosphatase activity was present principally in the perichondrium.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of hydroxyapatite formation by gelatin and type I collagen gels.

Calcium and phosphate were allowed to diffuse into gelatin and Type I collagen gels which were then cut into slices and analyzed for ion concentrations. Solutions of calcium and phosphate were then prepared, with ion concentrations equivalent to the highest levels in the slices, and mixed together, whereupon a rapid and copious precipitation of hydroxyapatite (HA) was observed. In contrast, HA bands were not visible in the gels until 1 to 2 1/2 days after analysis. These results indicate that Type I collagen exerts a considerable inhibitory effect on HA proliferation, probably by steric blockage of nuclei and crystal formation and growth. It thus appears that Type I collagen should be added to the list of agents that perform a regulatory role in bone mineral formation.

Molecular structure at the bone-implant interface: a vibrational spectroscopic characterization.

Test implant plates surgically retrieved from distal femurs of dogs were studied by Raman spectroscopy in order to characterize the bone-implant interface. The implant surface consisted of phosphate mineral, plasma sprayed on a titanium substrate. On the basis of its spectroscopic signature, the phosphate mineral of bone and the implant surface formed a mixed phase in the interface.

A new canine model to evaluate the biological response of intramedullary bone to implant materials and surfaces.

A new canine model utilizing an implantable chamber with multiple bone ingrowth channels has been used to study the response of intramedullary bone to various implant materials and surfaces. The first group of dogs received implants containing channels lined by smooth-surfaced coupons of titanium, titanium alloy, sputter-hydroxyapatite-coated (HA-coated) titanium alloy, and polyethylene. A pattern of early initial bone ingrowth by 2 weeks, becoming maximal at 6 to 12 weeks with remodeling to a more mature lamellar bone, and later resorption by 24 weeks was seen for all test groups, with fibrous tissue interfaces covering the smooth test coupons at all time points. Significantly increased bone ingrowth in the sputter-HA coated group was found only at 6 weeks. The second group of dogs received implants with channels lined by surface-roughened coupons of either titanium or plasma-HA-coated titanium, half of which were also packed with a crystalline-HA grouting at the time of surgery. At both 6 and 12 weeks, bone ingrowth was greatly enhanced by the presence of the plasma-HA coating or the crystalline-HA grouting as compared to the uncoated titanium channels. Histologically, bone was seen to bond directly to the plasma-HA coating and the crystalline-HA grouting. A thin fibrous tissue layer was noted between bone and the titanium in most areas, but evidence of direct bone contact to the metal surface was seen. Mechanical testing in tension of intact coupon-bone-coupon units revealed significant strength of the bone-plasma-HA bond, with failure initiating at the metal-HA interface with forces of 15.3 N at 6 weeks, increasing to 44.8 N at 12 weeks. Plasma-HA-lined channels with crystalline-HA packing required similar forces for failure. No significant adhesion strength was noted for the titanium channels at 6 weeks, and only the crystalline-HA-filled channels displayed measurable strength of the bone-titanium interface at 12 weeks, with a force of 9 N needed for failure.

A Raman and infrared spectroscopic investigation of biological hydroxyapatite.

Raman spectra were acquired on ox femur samples treated with hydrazine to remove the organic components of bone. A large increase in the signal-noise ratio of the mineral spectrum resulted from the exposure of the mineral surface and the removal of fluorescent components of the organic matrix. The effect of hydrazine treatment of the mineral matrix has been reinvestigated and shown to be slight on the basis of second derivative FTIR data. This is the first time that this high resolution technique has been applied to biological minerals.

A new canine model to evaluate the biological effects of implant materials and surface coatings on intramedullary bone ingrowth.

A new animal model for examining the intramedullary bone response to various implant materials and surfaces is presented, utilizing an implantable chamber with multiple bone ingrowth channels placed through a cortical defect in the lateral aspect of the distal femur. Twelve adult mongrel dogs received bilateral implants containing channels lined by smooth-surfaced coupons of titanium, titanium alloy, sputter-hydroxyapatite-coated titanium alloy, and UHMW polyethylene. A pattern was detected for all test groups of early initial bone ingrowth by two weeks, which became maximal at six to twelve weeks, followed by remodelling to a more mature lamellar bone and later resorption by 24 weeks, with fibrous tissue interfaces covering the smooth test coupons of all groups at all times. Significantly increased bone ingrowth in the sputter-HA coated group was found only at six weeks.

Mechanisms of inhibition of calcification.

Mineralization processes in the body are controlled by physicochemical and cellular regulation of hydroxyapatite (HA) nucleators and inhibitors. The chemical mechanism of action of HA inhibitors has been studied in vitro using solution pH-stat techniques or Types I and II collagen gel diffusion systems. Three biologically relevant systems are used with these methodologies: (1) transformation of amorphous calcium phosphate (ACP) to crystalline HA; (2) direct formation of HA; and (3) growth of HA crystals. Several different mechanisms have been identified for HA inhibition. Condensed phosphates (containing P-O-P linkages) and diphosphonates (containing P-C-P linkages) bind strongly to the surface of forming HA nuclei and crystals and poison growth sites at concentrations as low as 10(-6) M, blocking HA formation. From this in vitro work, diphosphonates have been developed for the treatment of Paget's disease. Proteoglycans, found in cartilage, delay HA formation by a steric effect whereby large volumes of solution become unavailable for HA formation and growth as these enormous macromolecules tumble about. Mg ions enter the structure of forming HA nuclei by replacing Ca, resulting in a distorted atomic structure that slows subsequent growth to HA. Al ions delay HA formation, not by entering the structure of forming HA nuclei, but by adsorbing on the surface of growing HA crystals. Serum proteins slow the transformation of ACP to HA by adsorbing on the ACP surface, which decreases its dissolution rate. Metal-citrate complexes can inhibit HA formation and growth at concentrations as low as 10(-5) to 10(-6) M. Phosphorylated molecules such as acidic proline-rich phosphoproteins and statherins found in saliva suppress HA crystal growth on tooth surfaces by adsorbing on active surface sites. Future research in this field lies in the study of interactions of HA inhibitors found together in calcifying tissues.

Effect of gallium on the in vitro formation, growth, and solubility of hydroxyapatite.

Gallium (Ga) is an effective treatment for the hypercalcemia of malignancy. The mechanism of action of the metal in blocking bone resorption in humans is not well understood. This paper examines the effect of Ga on the in vitro formation of hydroxyapatite (HA) in three test systems that have possible biological relevance in a pH-stat at pH 7.4, 37 degrees C, and 0.15 M NaCl: (1) the direct precipitation of HA; (2) the transformation of amorphous calcium phosphate to HA; and (3) the growth of HA seeds. In addition, the effect of Ga on HA solubility was measured at pH 5.0, the approximate pH of osteoclastic bone resorption. Ga decreased the HA formation and/or growth kinetics in a dose-related manner in all three test systems. In addition, the time to the onset of HA formation was increased in systems 1 and 2. Also, the adsorption of Ga on the surface of HA crystals was measured. Ga reduced the dissolution kinetics of HA compared with Ga-free control. The mechanism reported herein--the significant adsorption of Ga on forming and growing HA nuclei and on the surface of HA crystals--is believed to be responsible for the effects of the metal on HA proliferation and solubility. Accumulation of the metal on newly formed metaphyseal bone can now be explained by this adsorption of Ga. These in vitro results partly explain the in vivo action of Ga in treating hypercalcemia by decreasing bone apatite solubility.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of apatite formation by titanium and vanadium ions.

Ti4+ and V5+ ions were studied in two biologically relevant in vitro test systems to determine their effect on hydroxyapatite (HA) formation. System 1 involved direct HA precipitation from solution, and system 2 dealt with the growth of HA seed crystals. The experiments were carried out in a pH-stat by continuously recording NaOH uptake, which follows HA formation kinetics, at pH 7.4, 37 degrees C, and 0.15 M NaCl. In systems 1 and 2, Ti decreased HA formation kinetics in a dose-related manner without delaying the onset of HA formation. For V, the rate of HA proliferation decreased in system 1 in a dose-related manner, but the curves displayed a complicated shape. In system 2, V brought about a decrease in the HA seeded growth rate. Previous work has shown that Al blocks HA proliferation by adsorbing to active growth sites on the surface of HA crystals. By contrast, in this study V was found not to be adsorbed to the surface of HA. The mechanism of action of V probably involves the poorly understood hydrolysis and solution complex formation chemistry of the metal ion. We have shown here that V ions form V-PO4 complexes in solution in the HA formation systems; undoubtedly these are involved in the mechanism of V inhibition of HA formation. On the other hand, Ti was shown to bind to the surface of HA crystals in this study, which means that the ion may poison active crystal growth sites, as does aluminum. Ti-6Al-4V alloy is widely used in cementless total hip implants. Previous studies have shown that Ti concentrations 10 to 100 times higher than used here accumulate in osseous tissues around porous Ti implants in dogs 6 to 12 months after implantation. Ions leaching out over long periods of time into the implant interface could interfere with the normal osteoid mineralization and remodeling processes of bone in that region, which would result in subsequent loosening of the implant. This research suggests that further in vitro and animal studies should be carried out to determine the extent of Ti and V ion leaching from implants and their effect on tissue mineralization.

Hydroxyapatite ceramics and the nature of the bone-ceramic interface.

Hydroxyapatite ceramics have been used as bone implants in a number of experimental systems and clinical applications. We have developed a unique experimental model that allows study of the interface between bone and implant materials. A comparison of titanium and hydroxyapatite materials, using this model, has demonstrated the osteoconductive nature of hydroxyapatite and its ability to bond directly to bone.

The effect of aluminum and gallium ions on the mineralization process.

Metal ions have various and significant effects on the skeletal system. Aluminum accumulation in renal dialysis patients causes osteomalacia, while gallium is an effective therapeutic agent for treating the hypercalcemia accompanying certain malignancies. Using in-vitro systems that stimulate in-vivo mineralization, the authors have investigated the physical-chemical mechanisms of the actions of aluminum and gallium and report some of their findings.