The effects of alendronate in the treatment of experimental osteonecrosis of the hip in adult rabbits.

OBJECTIVE: Characterize the effects of alendronate (ALN) on the repair process of the osteonecrotic femoral head as well as the development of secondary osteoarthritis in the ipsilateral hip in an established experimental model of osteonecrosis. METHODS: Osteonecrosis of the femoral head was induced surgically in 60 adult, male New Zealand white rabbits. Animals were randomized in two placebo- (saline) and two treatment-groups (ALN 150 microg/kg/day S.C., 3x per wk) and were euthanized at 6 and 12 months post-operatively. Contralateral hip was used as control. Micro-Quantitative-CT (microQCT) analysis as well as histological assessment was performed in the femoral head and the acetabulum. Mankin Score was used to assess cartilage degeneration in the acetabulum. RESULTS: Repair in the osteonecrotic femoral head in the placebo group led to a significantly increased bone volume fraction (BVF) and volumetric bone mineral density (vBMD) in the trabecular region and to an increase in porosity in the cortical and subchondral region when compared to the normal femoral head on the contralateral side. ALN treatment significantly further increased BVF and vBMD in the trabecular region, and significantly reduced porosity and increased vBMD in the necrotic subchondral and cortical bone when compared to placebo. ALN led to a significant increase in vBMD in the subchondral region of the osteoarthritic acetabulum as well as to a significant reduction in articular cartilage degeneration. CONCLUSION: Inhibition of bone resorption by ALN treatment during repair of the osteonecrotic femoral head significantly increased bone mass in the trabecular region of the femoral head, inhibited subchondral resorption and reduced cartilage degeneration in the acetabulum.

Interactions of cisplatin with calcium phosphate nanoparticles: in vitro controlled adsorption and release.

A new system for the local delivery of chemotherapy to malignant solid tumors has been developed based on calcium phosphate (CaP) nanoparticles. The adsorption of the anti-neoplastic drug cis-diamminedichloroplatinum (cisplatin) was characterized on three types of apatitic CaP (poorly and well crystallized hydroxyapatite, and carbonated apatite). Adsorption isotherms obtained in chloride-free phosphate solutions at pH = 7.4 (24 and 37 degrees C) indicate that cisplatin adsorption increases with temperature and increases with decreasing crystallinity. Release studies in phosphate buffer saline (containing the chloride ion essential for release) showed that while the cumulative amount of released drug was the same for all apatites at 20 days (approximately 70% of the total bound), the least crystalline material released the drug more slowly. The drug release rate increased slightly with temperature. Cytotoxicity testing was conducted in a K8 clonal murine osteosarcoma cell line to verify that drug activity was retained after adsorption onto the apatite crystals. K8 cells were plated onto dried films of the apatite/cisplatin conjugates and after 24 h, viability was measured with tritiated uridine. The apatite/cisplatin formulations exhibited cytotoxic effects with a dose dependent diminishment of cell viability.

Phosphate ions in bone: identification of a calcium-organic phosphate complex by 31P solid-state NMR spectroscopy at early stages of mineralization.

Previous 31P cross-polarization and differential cross-polarization magic angle spinning (CP/MAS and DCP/MAS) solid-state NMR spectroscopy studies of native bone and of the isolated crystals of the calcified matrix synthesized by osteoblasts in cell culture identified and characterized the major PO(-3)(4) phosphate components of the mineral phase. The isotropic and anisotropic chemical shift parameters of the minor HPO(-2)(4) component in bone mineral and in mineral deposited in osteoblast cell cultures were found to differ significantly from those of brushite, octacalcium phosphate, and other synthetic calcium phosphates. However, because of in vivo and in vitro evidence that phosphoproteins may play a significant role in the nucleation of the solid mineral phase of calcium phosphate in bone and other vertebrate calcified tissues, the focus of the current solid-state 31P NMR experiments was to detect the possible presence of and characterize the phosphoryl groups of phosphoproteins in bone at the very earliest stages of bone mineralization, as well as the possible presence of calcium-phosphoprotein complexes. The present study demonstrates that by far the major phosphate components identified by solid-state 31P NMR in the very earliest stages of mineralization are protein phosphoryl groups which are not complexed with calcium. However, very small amounts of calcium-complexed protein phosphoryl groups as well as even smaller, trace amounts of apatite crystals were also present at the earliest phases of mineralization. These data support the hypothesis that phosphoproteins complexed with calcium play a significant role in the initiation of bone calcification.

Size and shape of mineralites in young bovine bone measured by atomic force microscopy.

Atomic force microscopy (AFM) was used to obtain three-dimensional images of isolated mineralites extracted from young postnatal bovine bone. The mean mineralite size is 9 nm x 6 nm x 2.0 nm, significantly shorter and thicker than the mineralites of mature bovine bone measured by the same technique. Mineralites of the young postnatal bone can be accommodated within the hole zone regions of a quasi-hexagonally packed collagen fibril in the fashion described by Hodge [9] in which laterally adjacent hole zone regions form continuous "channels" across the diameter of a fibril for a distance of at least 10 nm. Deposition of mineralites of the size noted above in this void volume of the fibrils would result in little or no distortion of the collagen molecules or supramolecular structure of the collagen fibril. The new AFM data supporting this claim is consistent with findings obtained by electron microscopy and low-angle x-ray and neutron diffraction that mineralites formed within collagen fibrils during initial stages of calcification occur within the hole zone region. However, the deposition of additional mineralites in the intermolecular spaces between collagen molecules in the overlap region of the fibrils would significantly distort the fibrils since the space available between adjacent molecules is considerably less than even the smallest dimension of the mineralites.

Shape and size of isolated bone mineralites measured using atomic force microscopy.

The inorganic phase of bone is comprised primarily of very small mineralites. The size and shape of these mineralites play fundamental roles in maintaining ionic homeostasis and in the biomechanical function of bone. Using atomic force microscopy, we have obtained direct three-dimensional visual evidence of the size and shape of native protein-free mineralites isolated from mature bovine bone. Approximately 98% of the mineralites are less than 2 nm thick displaying a plate-like habit. Distributions of both thickness and width show single peaks. The distribution of lengths may be multimodal with distinct peaks separated by approximately 6 nm. Application of our results is expected to be of use in the design of novel orthopaedic biomaterials. In addition, they provide more accurate inputs to molecular-scale models aimed at predicting the physiological and mechanical behavior of bone.

Characterization of demineralized bone matrix-induced osteogenesis in rat calvarial bone defects: III. Gene and protein expression.

Our previous studies of rat cranial defect repairs after the implantation of demineralized bone matrix (DBM) have demonstrated that healing occurs initially and principally by the direct induction and proliferation of osteoblasts derived principally from resident mesenchymal stem cells of the dura, and to a lesser extent by resident mesenchymal stem cells of the connective tissues beneath the skin flap. A small amount of cartilage is also synthesized after the direct process of ossification occurs. To further confirm the molecular phenotypes of the repair cells in rat cranial defects, the present study evaluated mRNA expression and synthesis of collagens I, II, and X and osteocalcin in the DBM-induced repair tissue by Northern blot analyses, autoradiography after in vivo (3)H-proline labeling of collagen, and immunohistochemistry. The results demonstrated that osteocalcin mRNA appeared in small amounts by day 4 and continued to increase over the experimental period. Much lesser quantities of collagen types II and X mRNAs appeared by day 6 and day 8, respectively. Collagen type I mRNA was present at all times examined but its expression significantly increased by day 5. Autoradiographic and immunohistochemical studies showed that type II collagen was not detected whereas type I collagen was synthesized on days 3-5. The data provide definitive molecular evidence confirming that the initial and by far the major pathway of cranial defects repair induced by implantation of DBM is by the direct induction of resident mesenchymal stem cells to osteoblasts and the direct formation of bone, which is spatially and temporarily distinct from the later formation of cartilage.

Structure, composition, and maturation of newly deposited calcium-phosphate crystals in chicken osteoblast cell cultures.

Characterization of the very early calcium phosphate (CaP) crystals deposited in bone or in osteoblast cell cultures has been hampered by the overwhelming presence of organic matrix components and cells that obscure spectral analyses. We have overcome this problem using isolated protein-free crystals and have obtained new data including 31P nuclear magnetic resonance (NMR) spectra for the first time from mineral crystals deposited during osteoblast calcification in culture. Crystals were isolated from cultures at two time points: (a) at first calcium accumulation (day 8-10) and (b) after 60 days of culture, to assess maturational changes. The analyses show that the chemical composition overall and short range order of the early and mature crystals are characteristic of the apatite crystals found in young embryonic chick bone in vivo. No mineral phase other than apatite was detected by any of the methods used. 31P NMR spectroscopy identified the HPO4 groups as those present in bone apatite. Similar to bone apatites, no OH groups were detected by Fourier transform infrared (FTIR) spectroscopy. The temporal maturational changes in composition and structure of the mineral phase were difficult to assess because of the continuous deposition of crystals throughout culturing. The pathway of the maturational changes observed were similar to those occurring in chick bone in vivo and synthetic apatite crystals in vitro although to a much smaller extent.

Evidence of hydroxyl-ion deficiency in bone apatites: an inelastic neutron-scattering study.

The novelty of very large neutron-scattering intensity from the nuclear-spin incoherence in hydrogen has permitted the determination of atomic motion of hydrogen in synthetic hydroxyapatite and in deproteinated isolated apatite crystals of bovine and rat bone without the interference of vibrational modes from other structural units. From an inelastic neutron-scattering experiment, we found no sharp excitations characteristic of the vibrational mode and stretch vibrations of OH ions around 80 and 450 meV (645 and 3630 cm(-1)), respectively, in the isolated, deproteinated crystals of bone apatites; such salient features were clearly seen in micron- and nanometer-size crystals of pure hydroxyapatite powders. Thus, the data provide additional definitive evidence for the lack of OH(-) ions in the crystals of bone apatite. Weak features at 160-180 and 376 meV, which are clearly observed in the apatite crystals of rat bone and possibly in adult mature bovine bone, but to a much lesser degree, but not in the synthetic hydroxyapatite, are assigned to the deformation and stretch modes of OH ions belonging to HPO(4)-like species.

Thin film of low-crystalline calcium phosphate apatite formed at low temperature.

Surface modification of biomaterials to improve biocompatibility without changing their bulk properties is desired for many clinical applications and has become an emerging technology in biomaterial research and industry. In the present study, a simple method of coating the solid surfaces of metals, organic tissue matrices, glasses, inorganic ceramics as well as organic polymers with a thin film of low-crystalline apatite crystals (LCA) was developed. Acidic solution containing calcium and phosphate ions was neutralized with alkaline solution to form calcium phosphate precipitates at low temperature. Precipitates of solid calcium phosphate particles were, then, removed by filtration. Concentration of free ions in the filtered ion solution which were not involved in the formation of calcium phosphate precipitate was high enough to induce the heterogeneous nucleation on the solid surfaces at low temperature. Thin layers of calcium phosphate crystals were formed on the surfaces of metals, glasses, inorganic ceramics, organic polymers including hydrophobic ones, and biological tissue matrices with this solution. The thin layer of crystals consisted of poorly crystalline calcium phosphate apatite crystals which contain high amount of labile ions like bone crystals and did not dissolve in the physiologic solutions. Various cells attached to this crystal layer and proliferated well.

Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity.

Cell-mediated (type-1) immunity is necessary for immune protection against most intracellular pathogens and, when excessive, can mediate organ-specific autoimmune destruction. Mice deficient in Eta-1 (also called osteopontin) gene expression have severely impaired type-1 immunity to viral infection [herpes simplex virus-type 1 (KOS strain)] and bacterial infection (Listeria monocytogenes) and do not develop sarcoid-type granulomas. Interleukin-12 (IL-12) and interferon-gamma production is diminished, and IL-10 production is increased. A phosphorylation-dependent interaction between the amino-terminal portion of Eta-1 and its integrin receptor stimulated IL-12 expression, whereas a phosphorylation-independent interaction with CD44 inhibited IL-10 expression. These findings identify Eta-1 as a key cytokine that sets the stage for efficient type-1 immune responses through differential regulation of macrophage IL-12 and IL-10 cytokine expression.

The nuclear factor of activated T cells (NFAT) transcription factor NFATp (NFATc2) is a repressor of chondrogenesis.

Nuclear factor of activated T cells (NFAT) transcription factors regulate gene expression in lymphocytes and control cardiac valve formation. Here, we report that NFATp regulates chondrogenesis in the adult animal. In mice lacking NFATp, resident cells in the extraarticular connective tissues spontaneously differentiate to cartilage. These cartilage cells progressively differentiate and the tissue undergoes endochondral ossification, recapitulating the development of endochondral bone. Proliferation of already existing articular cartilage cells also occurs in some older animals. At both sites, neoplastic changes in the cartilage cells occur. Consistent with these data, NFATp expression is regulated in mesenchymal stem cells induced to differentiate along a chondrogenic pathway. Lack of NFATp in articular cartilage cells results in increased expression of cartilage markers, whereas overexpression of NFATp in cartilage cell lines extinguishes the cartilage phenotype. Thus, NFATp is a repressor of cartilage cell growth and differentiation and also has the properties of a tumor suppressor.

Characterization of matrix-induced osteogenesis in rat calvarial bone defects: II. Origins of bone-forming cells.

Two experimental models that separated demineralized bone matrix (DBM) implants from the host bone were utilized to identify the origins of bone-forming cells in the repair of calvarial defects in rats. Rat DBM, Guanadine-HCl (Gdn-HCl) extracted insoluble residue of DBM, and Gdn-HCl extracted insoluble DBM to which the dialyzed Gdn-HCl extract was added back, were implanted in the two models which prevented cells of the adjacent host bone from participating in the repair. In addition, cells in the dura and in the subcutaneous tissue overlying the calvarial defect were locally labeled with (3)H-thymidine to identify the origins of those cells that were stimulated to divide and differentiate to osteoblasts. Histological studies of the temporal events that occurred during the healing process in these defect models, combined with (3)H-thymidine labeling demonstrated that the osteoblasts induced by DBM were initially derived from undifferentiated mesenchymal stem cells of the dura and later augmented by cells in the overlying connective tissue covering the defect, and not from cells in the cranial bone surrounding the circular defect. The cells of both dura and subcutaneous tissue were stimulated to proliferate and differentiate principally to osteoblasts and to a very much lesser extent to chondroblasts by DBM and by reconstituted components of DBM after Gdn-HCl extraction. Gdn-HCl-extracted insoluble DBM failed to induce bone or cartilage. These results indicate that the cytokines or other factors present in DBM are required to induce bone-forming cells derived from the dura and the overlying connective tissue for the repair of the calvarial defect.

Characterization of matrix-induced osteogenesis in rat calvarial bone defects: I. Differences in the cellular response to demineralized bone matrix implanted in calvarial defects and in subcutaneous sites.

The cellular and biochemical sequences of osteogenesis induced by implanting demineralized bone matrix (DBM) in rat cranial defects and in subcutaneous sites have been studied by histological, histochemical, and biochemical techniques from days 2 to 28 after implantation. In subcutaneous sites, allogenic DBM induced cartilage cells and matrix for approximately the first 10 days which were subsequently resorbed and replaced by bone with little evidence for the classical endochondral sequence of ossification. In sharp contrast, the first cells that differentiated from the mesenchymal stem cells in the cranial defects were alkaline phosphatase (ALP) positively stained osteoblasts that appeared 3 days after implantation followed by synthesis of bone matrix which calcified shortly thereafter. A few clusters of cartilage cells were observed beginning at days 6-7 which were spatially distinct from the new bone and later resorbed. By day 28 the tissue induced in both the subcutaneous and cranial sites consisted almost solely of bone; however, the total amount of new bone in the subcutaneous implants was significantly less than the mass of bone formed in the calvarial defects. Bovine DBM induced bone formation in rat cranial defects to a very much lesser extent than allogenic DBM. A few cartilage cells were induced by bovine DBM in subcutaneous sites and rapidly resorbed and not replaced with bone. These results clearly indicate that the cellular sequence induced by allogenic and xenogenic DBM and the repair tissues synthesized are distinctly different in the cranial defects from those induced in the subcutaneous sites.

Multinuclear solid-state three-dimensional MRI of bone and synthetic calcium phosphates.

Multinuclear three-dimensional solid-state MRI of bone, tooth, and synthetic calcium phosphates is demonstrated in vitro and in vivo with a projection reconstruction technique based on acquisition of free induction decays in the presence of fixed amplitude magnetic field gradients. Phosphorus-31 solid-state MRI provides direct images of the calcium phosphate constituents of bone substance and is a quantitative measurement of the true volumetric bone mineral density of the bone. Proton solid-state MRI shows the density of bone matrix including its organic constituents, which consist principally of collagen. These solid-state MRI methods promise to yield a biological picture of bone richer in information concerning the bone composition and short range-crystalline order than the fluid-state images provided by conventional proton MRI or the density images produced by radiologic imaging techniques. Three-dimensional solid-state projection reconstruction MRI should be readily adaptable to both human clinical use and nonmedical applications for a variety of solids in materials science.

Evaluation of bone mineral density using three-dimensional solid state phosphorus-31 NMR projection imaging.

A solid state magnetic resonance imaging technique is used to measure true three-dimensional mineral density of synthetic hydroxyapatite phantoms and specimens of bone ex vivo. The phosphorus-31 free induction decay at 2.0 T magnetic field strength is sampled following application of a short, hard radiofrequency excitation pulse in the presence of a fixed amplitude magnetic field gradient. Multiple gradient directions covering the unit sphere are used in an efficient spherical polar to Cartesian interpolation and Fourier transform projection reconstruction scheme to image the three-dimensional distribution of phosphorus within the specimen. Using 3-6 Gauss/cm magnetic field gradients, a spatial resolution of 0.2 cm over a field of view of 10 cm is achieved in an imaging time of 20-35 minutes. Comparison of solid state magnetic resonance imaging with dual energy X-ray absorptiometry (DXA), gravimetric analysis, and chemical analysis of calcium and phosphorus demonstrates good quantitative accuracy. Direct measurement of bone mineral by solid state magnetic resonance opens up the possibility of imaging variations in mineral composition as well as density. Advantages of the solid state magnetic resonance technique include avoidance of ionizing radiation; direct measurement of a constituent of the mineral without reliance on assumptions about, or models of, tissue composition; the absence of shielding, beam hardening, or multiple scattering artifacts; and its three-dimensional character. Disadvantages include longer measurement times and lower spatial resolution than DXA and computed tomography, and the inability to scan large areas of the body in a single measurement, although spatial resolution is sufficient to resolve cortical from trabecular bone for the purpose of measuring bone mineral density.

Expression of bone microsomal casein kinase II, bone sialoprotein, and osteopontin during the repair of calvarial defects.

The temporal expression of bone microsomal casein kinase II, osteopontin, bone sialoprotein, alkaline phosphatase, and the accumulation of a solid calcium-inorganic orthophosphate mineral phase, have been charted from day 2 to day 21 during the repair of calvarial defects in rats induced by the implantation of decalcified rat bone matrix. Unlike the sequence of events that occur when the same decalcified bone matrix is implanted subcutaneously or intramuscularly, in which cases the first tissue to form in response to the implant is cartilage that subsequently calcifies and is later resorbed and replaced by bone, the repair of cranial defects is quite different. In the latter case, the first cells induced are undifferentiated mesenchymal cells and early fibroblasts followed by osteoblastic direct bone formation. Somewhat later a few small islands of cartilage are formed, widely separated and spatially distinct from the newly formed bone matrix. All of the cartilage and most of the implanted decalcified bone matrix are later resorbed and replaced by new bone by day 21. This in vivo model of the repair of a bone defect by direct bone formation has provided an excellent system to follow specific biochemical and physicochemical events. The total accumulation and rate of accumulation of the mineral and the two noncollagenous phosphoproteins (bone sialoprotein and osteopontin), as well as the activities of alkaline phosphatase, and for the first time either in vivo or in cell culture, the activity of microsomal casein kinase II, the major enzyme that phosphorylates the bone phosphoproteins, have been determined as a function of healing time in vivo. The overall general pattern of accumulation of the phosphoproteins and calcium-phosphate mineral phase and their relationships are similar to those reported in osteoblast cell cultures also monitored as a function of time.

Isolation of a novel bone glycosylated phosphoprotein with disulphide cross-links to osteonectin.

An 80 kDa protein was purified from calf bone by HCl-demineralization followed by 0.5 M EDTA/1.0 M NaCl extraction and sequential chromatography on DE-52, hydroxyapatite, and TSK-gel G3000SW HPLC columns. From the DE-52 column the protein was eluted at three different fractions, of which one further separated into two fractions on the hydroxyapatite column, indicating that the protein is present in four different molecular forms designated as 80 k-I-1, k-I-2, k-II, k-III. The N-terminal sequence analysis of all four forms gave the same sequence, SEQYNQEPNNV. Several tryptic internal peptides were also generated, purified and sequenced, leading to the identification of several repeat sequences, IFLGXXEI. Homology searching of the N-terminal and internal sequences indicates that this is a novel protein. Both 80 k-I-2 and k-III had similar amino acid composition with high contents of Asx, Glx and Leu and contained 7 and 16 phosphoserines per 1000 total amino acids, respectively. The 80 k-I-1 and 80 k-II forms were stained with Rhodamine B specific for phosphoproteins. The four forms contained different contents of neutral sugars ranging from 5.5 to 26% (w/w protein) and approximately 1.7% sialic acid. These data indicated that the 80 kDa protein exists in four isomeric forms, at least based on the different post-translational modifications. The evaluation of the 80 kDa glycosylated phosphoprotein under alkylating, reducing and non-reducing conditions indicated that this protein undergoes polymerization through intermolecular disulphide bonds. Furthermore, the 80 kDa protein and osteonectin (ON), both of which are cysteine-rich proteins, can cross-link with each other via disulphide bonds, and this process can be induced to take place in vitro under experimental conditions. The occurrence of such a phenomenon in vivo was confirmed from the presence of similar high Mr components containing both 80 kDa and ON in the same SDS/PAGE bands, detected by the respective antibody reactions in crude bone extracts which were extracted in the presence of alkylating agent.

Enamel specific protein kinases and state of phosphorylation of purified amelogenins.

Ameloblastic tissue samples from unerupted bone molars were used to prepare subcellular enamel protein kinase preparations, nuclear + plasma membrane, cytosolic and microsomal, and used in in vitro phosphorylation of purified 20 kDa bovine amelogenin in the presence of 32P-ATP. Both cytosolic and microsomal preparations can phosphorylate purified native amelogenins, the addition of Ca2+ slightly increased the microsomal enzyme activity or at least did not inhibit the activity, whereas the presence of Ca2+ substantially decreased the cytosolic kinase activity towards phosphorylation of amelogenins. A comparative analysis using the enamel microsomal kinase against osteopontin, dephosphorylated casein and bone sialoprotein showed no phosphorylation of the first two proteins, and only minor phosphorylation of the bone sialoprotein. Overall, the present work demonstrates for the first time that the protein kinase responsible for the phosphorylation of amelogenins is a novel kinase, which is not inhibited by Ca2+, unlike the microsomal protein kinase (casein kinase type-II) of bone which phosphorylates secretory proteins osteopontin and bone sialoprotein and is strongly CaZ+ inhibited. The direct phosphoserine analysis on the purified bovine 20 kDa amelogenin indicated the presence of 0.8 moles of phosphoserine/mole protein naturally occurring, consistent with the quantitative analysis of 14C-radiolabeling of phosphoserines by conversion to dehydroalanine and in situ reaction with the thiol agent, 14C-mercaptoethanol, 0.64 moles 14C-incorporated/mole 20 kDa amelogenin. The purified low Mramelogenins 5.3 kDa E4 (TRAP) and 7.2 kDa E3 (LRAP), were also derivatized by 14C-mercaptoethanol, providing 0.46 and 0.88 moles 14C-incorporated/mole respectively. Further studies of the 14C-radiolabeled E4 amelogenin by sequence analysis confirmed one site of label to be at position 16 from the N-terminal and hence provided a direct evidence for the naturally occurring phosphoserine residue at this position.