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Enhancement of bone regeneration using resorbable ceramics and a polymer-ceramic composite material.

The aim of this experimental study was to evaluate the use of resorbable implants for the repair of nonloaded skeletal defects. Porous ceramic implants of alpha-TCP, of glass-ceramic, and of solid composite implants of glass-ceramic/polylactic acid 8 mm in diameter and 2 mm in thickness were fabricated and implanted pressfit into biparietal, full-thickness defects of the calvaria of 60 adult rats. Twenty rats received unfilled defects and served as controls. Fluorochrome labeling of bone formation was performed during the observation period. Five animals from each group were evaluated after 6, 13, 26, and 52 weeks. The control defects showed incomplete regeneration, with bone formation extending 1.66 mm, on average, into the defect after 52 weeks. In the group of alpha-TCP implants, histologic evaluation indicated that the bone formed during initial stages had undergone resorption later on, so that bone repair after 52 weeks was not significantly enhanced, with an average depth of 1.83 mm of bone ingrowth. The glass-ceramic implants exhibited extensive bone formation and nearly complete repair of the calvarial defect, with 3.90 mm of bone ingrowth into the implant pores. Degradation of the ceramic was nearly complete, with a few remaining particles surrounded by soft tissue. The composite implants showed a negligible bone ingrowth of 0.63 mm, on average. Soft tissue had invaded the polylactic acid implant body, but no bone formation had taken place at the surface of the embedded ceramic particles. Degradation of the polymer was not complete after 52 weeks. It is concluded that the balance between degradation and bone formation is delicate and that chemical events and cellular reaction during degradation may counteract complementary bone ingrowth.

Absorbable Implants↗

Effects of estrogen and androgen on the ultrastructure of secretory granules and intercellular junctions in regressed canine prostate.

Epithelial cells in the prostate of the castrated or hypophysectomized dog were studied by thin-section and freeze-fracture electron microscopy to determine in vivo responses to estradiol-17 beta 17-cyclopentylpropionate (ECP) and testosterone cyclopentylpropionate (TCP). Particular attention was given to changes in specific organelles and intercellular junctions that might reflect hormone action. The few secretory granules that remain in the regressed epithelium (vestigial granules) serve as markers of prior androgen responsiveness. Pharmacologic doses of ECP caused regressed glandular cells to acquire a novel phenotype. Characteristic features of these estrogen-modified glandular (EMG) cells are newly formed secretory granules and tonofilament bundles that coexist with vestigial granules, thus demonstrating bipotentiality of response. Glandular cell-tight junctions appear unaltered by the endocrine manipulations. Although EMG cells have squamous cell features, tight junctions remain intact. Desmosomes in the canine prostate are dimorphic and are classified 70F and 100F according to the width of the filaments that converge on the dense plaques. In intact dogs, 100F desmosomes are associated with basal-reserve cells, whereas only the 70F variety is found between glandular cells. TCP treatment does not alter this distribution. Following ECP administration, both 70F and 100F desmosomes are present between EMG cells. The coexistence of newly formed secretory granules and tonofilaments of 100F desmosomes in the same EMG cell represents estrogen-induced bidirectional differentiation. Our findings indicate that androgens and estrogens are individually capable of controlling the expression of secretory granules and desmosomes. In intact animals, male and female sex hormones may act in concert to direct epithelial cell differentiation of the prostate.

Androgens↗

Relationships between endothelial cells, pericytes, and osteoblasts during bone formation in the sheep femur following implantation of tricalciumphosphate-ceramic.

BACKGROUND: The origin of osteoblasts is still controversial. Whereas several authors consider the stromal fibroblast of the bone marrow as the osteoprogenitor cell, others propose that the osteoblasts can be derived from the "capillary system." The present study examines the replacement of tricalciumphosphate (TCP)-ceramic implanted into an artificial bone defect by newly formed bone. The results support the hypothesis that osteogenic cells can be derived from invading blood vessels. METHODS: The spongiosa of the trochanter major of sheep was removed and the defect was filled with TCP-ceramic. Two months after surgery the ceramic implants together with the surrounding bone were removed and processed for transmission electron microscopy. Serial ultrathin sections of three newly formed osteons were examined. RESULTS: The osteons contain one or two small sprouting capillaries, a peripheral layer of osteoblasts, and in between, a network of glycogen-rich cells. Some of the glycogen-rich cells are completely or partly surrounded by the endothelial basal lamina and are thereby characterized as pericytes. Weibel-Palade bodies, which are considered to be a marker of endothelial cells, were occasionally observed in glycogen-rich pericytes. CONCLUSIONS: Since pericytes differentiate into osteoblasts under in vivo and in vitro conditions, and have thus been regarded as osteoprogenitor cells, the presence of Weibel-Palade bodies in these cells suggest that osteogenic cells can be derived from endothelial cells.

Animals↗

Crystal populations in human synovial fluid. Identification of apatite, octacalcium phosphate, and tricalcium phosphate.

Defatted joint fluid pellets from 4 patients with the "Milwaukee shoulder" syndrome, an aspirate of milky fluid from subcutaneous tissue of a girl with dermatomyositis/calcinosis, and 2 rabbit synovial membranes calcified by calciphylaxis were studied by Fourier transform infrared analysis. This method permits sequential electronic subtraction ("stripping") of known patterns of highly characterized reference compounds from an unknown sample pattern. Collagen and hydroxyapatite, with carbonate partially (1-3%) substituted for phosphate, and to a lesser extent hydroxyl, were found in all samples. Octacalcium phosphate (OCP) was found in all samples except that from a patient with synovial chondromatosis which contained tricalcium phosphate (TCP). Sulfate was found in 2 of the shoulder joint fluid specimens. An unidentified organic phase was present in all samples. This study confirms recent observations by others that pathologic calcifications contain several mineral phases. These data raise the possibility that hydroxyapatite nucleation may be controlled by hydrolytic alteration of precursor calcium phosphate phases such as OCP or TCP. Studies of the biologic effects of synthetic calcium phosphate crystals must take into account the existence of several crystalline phases in natural pathologic calcifications.

Animals↗

Characterization of specific binding sites for [3H]-1,3-di-o-tolyl-guanidine (DTG) in the rat glioma cell line C6-BU-1.

The aim of the present study was to find out if a cell line of glial origin possesses sigma and/or phencyclidine (PCP) binding sites. Binding of [3H]1,3-di-o-tolyl-guanidine (DTG), a highly selective ligand for sigma binding sites, and of [3H]N-[1-(2-thienyl)cyclohexyl] piperidine ([3H]TCP), a radioligand specific for PCP receptors, to C6-BU-1 glioma cells was investigated. Binding of [3H]DTG to C6-BU-1 cell membranes was reversible, saturable (Bmax = 10.5 pmol/mg protein), and of high affinity (KD = 26 nM). C6-BU-1 cells do not possess PCP receptors as indicated by negligible specific binding of [3H]TCP to C6-BU-1 cell membranes. Specific binding of [3H]DTG was reduced in the presence of Ca2+ and to a lesser extent by Mg2+. The rank order of potency of various PCP and sigma ligands was DTG > (+)3-[(3-hydroxy-phenyl)-N-n-propyl-piperidine] [(+)3-PPP] > haloperidol > pentazocine > (-)3-PPP > PCP > metaphit > dextromethorphan > (-)butaclamol > (+)butaclamol > (-)N-allylnormetazocine [(-)SKF 10,047] > MK801 > (+)SKF 10,047 > ketamine. The drug specificity, confirmed by a reversed stereoselectivity for the benzomorphan opiate SKF 10,047, indicated that these sites correspond to a subtype of sigma binding sites, the so-called sigma 2 binding site. Thus, the C6-BU-1 cell line is the first glial cell line demonstrated to have sigma 2 binding sites.

Animals↗

Expression of Th1/Th2 cytokines in human blood after in vitro treatment with chlorpyrifos, and its metabolites, in combination with endotoxin LPS and allergen Der p1.

Exposure to organophosphate (OP) pesticides has been associated with respiratory symptoms and may be related to asthma; however, few studies have examined the molecular basis for these associations. Asthma and allergic disorders are characterized by elevated Th2 cytokines (IL-4, IL-5, IL-13), whereas the chronic inflammatory response in asthmatic airways is maintained by Th1 cytokine IFN-gamma. The goal of this in vitro study was to examine the effects of OP chlorpyrifos (CPF), and its metabolites chlorpyrifos-oxon (CPO) and 3,5,6-trichloro-2-pyridinol (TCP), singly, and in combination with endotoxin lipopolysaccharide (LPS) or house dust mite Dermatophagoides pteronyssinus (Der p1) allergen, on expression of IFN-gamma and IL-4, Th1 and Th2 signature cytokines, respectively. Cytokine expression was measured by ELISA and flow cytometry. Human blood cultures were treated with CPF/CPO/TCP (1-1000 microg ml(-1)) and LPS (1.5-2.5 microg ml(-1)) or Der p1 (200 AU ml(-1)) and supernatants were collected at 48 h. Pesticides CPF, CPO and TCP did not induce cytokine expression in vitro, while LPS and Der p1 induced IFN-gamma and IL-4 expression, respectively. Whole blood cultures treated with low doses of CPO (1 and 10 microg ml(-1)), in combination with LPS, expressed higher levels of IFN-gamma than LPS alone (P < 0.05). While CPO increased LPS-dependent induction of IFN-gamma, CPO treatment did not alter Der p1 induction of IL-4. The interaction between CPO and LPS, which results in an increased type 1 immune response, should be investigated further, particularly since the combination of OP pesticides and endotoxin is common in rural, agricultural communities.

Antigens, Dermatophagoides↗

In vivo investigations on composites made of resorbable ceramics and poly(lactide) used as bone graft substitutes.

Porous composites made of poly(L, DL-lactide) (PLA) and alpha-tricalcium phosphate (alpha-TCP) or the glass ceramic, GB14N, respectively, were investigated in a loaded implant model in sheep. Six, 12 and 24 months after implantation histological and biomechanical evaluation were performed and compared to autogenous bone transplants. No significant differences were observed between the composites. After 6 months, the interconnecting pores of the alpha-TCP-composite and the GB14N-composite were filled with newly formed bone (14 +/- 5% or 29 +/-15% of the implant, respectively) and soft tissue (30 +/-9% or 21 +/-12% of the implant, respectively). Only a mild inflammatory response was observed. The reaction was similar after 12 months. However, after 24 months a strong inflammatory reaction was seen. The newly formed bone was partly osteolytic. The adverse reaction occurred simultaneously to a significant reduction of the PLA component. The histological results were reflected by the biomechanical outcomes. Both composites showed compression strengths in the range of the autologous bone graft until 12 months of implantation. After 2 years, however, the strengths were significantly decreased. It is concluded that the new composites cannot yet be used for clinical application. An improvement in biocompatibility might be reached by a better coordination of the degradation times of the polymer and the ceramic component.

Absorbable Implants↗

Time-dependent mechanical properties of HA/TCP particles in relation to morsellized bone grafts for use in impaction grafting.

In reconstructive surgery human bone defects are sometimes filled with the use of the impaction bone grafting technique. Currently different types of biomaterial particles are being developed as bone-substitute materials. Before these biomaterials can be applied their mechanical and biological behavior should be characterized. In this study the time-dependent mechanical behavior of biomaterial particles with different tri-calcium-phosphate/hydroxy-apatite (TCP:HA) ratios, particle sizes, and porosities is determined and compared to the behavior of human bone grafts, the latter being the standard material currently used to augment bone defects. The mechanical properties were assessed with the use of dynamic confined compression creep tests with a loading and unloading phase. Different graft material groups were tested, consisting of 100% human bone grafts, 100% biomaterial particles, and 50:50 weight mixtures of human grafts and biomaterial particles. No damage to the particles was observed by the impaction in the test chamber or by the dynamic load. Relative to the human graft material, the biomaterial particles hardly deformed under loading, were much stiffer, and showed almost no viscoelastic behavior. The mixtures showed intermediate results. Particle size and porosity influenced the behavior of the biomaterial particles. TCP:HA ratio did not have a great effect. The conclusion is that the application of these particles should be done with great care, as their mechanical behavior is drastically different than that of the human graft material. Mixing it with human bone grafts gave the material some biphasic, viscoelastic behavior that may be important for its biological response.

Biocompatible Materials↗

Mineralization and calcium fixation within a porous apatitic ceramic material after implantation in the femur of rabbits.

Cylinders (0.8 cm long, 1.0 cm of diameter and with an axial hole), constituted, after firing, of a ceramic mixture of hydroxylapatite (HA) and beta-tricalciumphosphate (beta-TCP) in a 10:1 ratio, were implanted into mid-diaphyseal defects of one femur of 20 rabbits and stabilized with intramedullary rods. The implantation sites were checked radiographically every month, and after 3 months (3 animals) and 6 months (17 animals) the rabbits were sacrificed and the implants with the surrounding tissue were embedded in methylmethacrylate, cut to thick sections and analyzed by scanning electron microscopy (SEM). Porosimetric and x-rays diffraction analyses were carried out before and after implantation of the cylinders, and the state of mineralization at the bone-implant interface was determined by EDAX microprobe analysis. Bony callus formation started at 1 month at the osteotomy sites, as judged by radiography, but after 3 months a not-mineralized zone had still been demonstrated between bone and the implants. At 6 months, 13 implants showed themselves firmly fixed in their implantations beds, while 4 implants were only incorporated at their proximal ends. In bone contact zones, an enrichment of Ca2+ was displayed by microanalytical techniques in the outer zone of the implanted samples which may be explained by an apparent additional phase transformation of HA into TCP, thanks to the change of the Ca/P ratio, that takes place in vivo.

Animals↗

Cell responses to biomaterials. II: Endothelial cell adhesion and growth on perfluorosulfonic acid.

We report here the use of perfluorosulfonic acid (Nafion) as a substratum for the growth of bovine aortal endothelial cells. This support which can be generated in a number of forms is at least as efficient in maintaining the growth of endothelial and other cell types as tissue culture grade polystyrene (TCP) and represents an advance in this regard over polytetrafluoroethylene (Teflon). The mechanism underlying the different cell attachment capacities of these three polymers is not readily related to their different protein binding patterns. While Nafion adsorbs more total protein from serum than Teflon or TCP, it adsorbs relatively less of the major cell adhesive proteins, vitronectin and fibronectin, than does Teflon. Both Nafion and Teflon had comparable but low thrombogenic potential by in vitro tests. Teflon or expanded Teflon (Gore-tex) coated with a thin film of Nafion assumes the cell supportive characteristics of Nafion and hence the modification of these surfaces by the induction of a stable bond between Teflon (in various forms) and Nafion may provide a composite vascular graft material which has all the desirable qualities of both materials.

Animals↗

The influence of calcium phosphate ceramics and glass-ceramics on cultured cells and their surrounding media.

The cytotoxicity of hydroxyapatite (HA), tricalcium phosphate (TCP), and two types of apatite-containing glass-ceramics designated A.W-GC and A.W.CP-GC was evaluated by the direct contact method using V79 cells. Compatibility of the cells was assessed as a function of cellular attachment and subsequent growth using scanning electron microscopy. Although all of the tested materials have surfaces available for cellular attachment, cell growth was markedly inhibited by HA sintered at 600 degrees C and 900 degrees C, and slightly inhibited by HA sintered at 1200 degrees C, TCP and the glass-ceramics. In attempts to clarify the cause of this toxicity, the interactions of the ceramics with culture medium was assessed by measurement of calcium (Ca), inorganic phosphate (P), magnesium (Mg), pH and albumin content in the medium. The grade of toxicity corresponded to the degree of reduction of Ca, P, and albumin in the medium. Furthermore, surface reactions of the ceramics were demonstrated to vary in both mode and degree depending on their chemical structure and level of microporosity. Thus, the surface reactivity of ceramic materials should be always taken into account in discussing their biocompatibility in vivo.

Animals↗

Differences in ceramic-bone interface between surface-active ceramics and resorbable ceramics: a study by scanning and transmission electron microscopy.

The interface between bioactive ceramics and bone was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The materials were apatite-wollastonite-containing glass ceramic (A-W.GC) as a representative surface-active ceramic, and calcite and beta-tricalcium phosphate (beta-TCP) as resorbable ceramics. Particles of these materials, ranging between about 100 microns and 300 microns in diameter, were implanted into rat tibiae, and specimens were prepared for observation at 8 weeks after implantation. Both SEM and TEM demonstrated that A-W.GC was bonded to bone through a thin Ca-P-rich layer consisting of fine apatite crystals apparently different from those of bone in shape, size, and orientation. Collagen fibers of the bone reached the surface of this layer, and chemical bonding between A-W.GC and the bone was speculated. Calcite and beta-TCP, on the other hand, made direct contact with the bone, and no apatite layer was present at the interface. The surfaces of the implants became rough due to degradation, and bone grew into the finest surface irregularities. However, we were unable to demonstrate any continuity of crystals between the resorbable implants and bone by high-resolution TEM. Accordingly, the bonding strength was considered to be mainly attributable to mechanical interlocking.

Animals↗

Effect of bioactive ceramic composition and structure on in vitro behavior. III. Porous versus dense ceramics.

In vitro reaction kinetics of surface transformations of porous and dense bioactive ceramics were studied after immersion in simulated physiologic solution (SPS). Porous ceramics with identical macroporosity were commercially obtained: calcium carbonate marine coral (I-CC), coralline HA (I-HA), and a product, labeled as beta-tricalcium phosphate, which was partially transformed into beta-calcium pyrophosphate (I-beta-CP). Previously studied dense ceramics were used for comparison: Ca-deficient HA (CDHA), stoichiometric HA, both well and not well crystallized (wc and nwc s-HA), beta- and alpha-tricalcium phosphate (beta- and alpha-TCP). The induction time to precipitation increased as follows: CDHA, nwc HA < wc HA, I-HA < beta-TCP, I-CC << I-beta-CP. The reaction kinetics of ceramics with identical macroporosity varied substantially depending on composition, crystal structure, and ultrastructure. One of the porous ceramics, I-beta-CP, showed an inhibiting effect on precipitation reactions.

Biotransformation↗

Changes in phases and crystallinity of plasma-sprayed hydroxyapatite coatings under heat treatment: a quantitative study.

With three kinds of plasma-sprayed hydroxyapatite coatings (HACs) prepared, the objectives of this study were (1) to establish the calibration methods for quantitatively measuring the concentration of impurity phases and the degree of crystallinity of the HACs, and (2) to explore the effects of postheat treatments at various temperatures in vacuo on the changes of phases and crystallinity of the HACs. By the internal standard method used, the concentrations of impurity phases, such as alpha-TCP, beta-TCP, and TP, of the assprayed HACs were significantly higher than those measured by the direct intensity-ratio method, and the CaO phase was lower than the direct intensity-ratio method. When the HACs were heat treated in the temperature interval 630-850 degrees C, the concentrations of impurity phases obviously decreased, and the coating crystallinity apparently increased. After annealing at 850 degrees C, an HAC consisting of at least 95% crystallinity with few impurity phases was obtained. As the annealing temperatures in the interval 850-1000 degrees C were applied, however, the HA phase seriously decomposed, resulting in the appearance of a large number (higher than 20 wt%) of impurity phases in the HACs. This work suggests that the optimum heat treatment conditions in vacuo for maximizing crystallinity and minimizing impurity phases of the HACs do not occur at the same temperature.

Alloys↗

Osteoblast interactions with calcium phosphate ceramics modified by coating with type I collagen.

Complications associated with the use of autogenous bone in the repair or replacement of tissue lost through injury or disease have driven the search for alternative sources of graft material. Bioceramics containing hydroxyapatite (HA), tricalcium phosphate (TCP), or composites that combine the best properties of both of these materials are among the principal candidates. In this study, we have investigated the in vitro proliferation, morphology, and viability of an immortalized rat osteoblast cell line cultured on HA, TCP, and composites of the two in the ratios 75:25 (H75), 50:50 (H50), and 25:75 (H25) for 28 days. The biocompatibility of each material was examined in the presence and absence of a collagen coating. With the exception of H50, cell proliferation, quantified by carboxyfluorescein fluorescence, was enhanced by collagen coating of all materials for the first 14 days, although at later time points cell numbers were unaffected. It is notable that the collagen coating was least stable on H50, the only material not to show enhancement of cell growth on coating. Confocal laser scanning microscopy confirmed that cell growth was more extensive on coated materials over the first 7-14 days in culture, and the development of cell extensions and bridges across the pores in the materials was observed. Results indicate that collagen coating of calcium phosphate ceramics may also increase their compatibility and osseointegration in vivo.

Animals↗

Substratum roughness alters the growth, area, and focal adhesions of epithelial cells, and their proximity to titanium surfaces.

Epithelial (E) cells were cultured on smooth tissue culture plastic (TCP), TCP-Ti, polished Ti (P), and rough grit-blasted Ti (B), acid-etched Ti (AE), and grit-blasted and acid-etchedTi (SLA) surfaces and their growth, area, adhesion, and membrane-Ti proximity assessed. Rough surfaces decreased the growth of E cells compared to smooth surfaces in cultures up to 28 days. In general rough surfaces decreased the spreading of E cells as assessed by their area with the most pronounced affect for the SLA surface. On the other hand, the strength of E cells adhesion as inferred by immunofluorescence staining of vinculin in focal adhesions indicated that E cells formed more and larger focal adhesions on the smooth P surface compared to the rougher AE surface. As this finding indicates a stronger adhesion to smooth surfaces, it is likely that E cells on rough surfaces are more susceptible to mechanical removal. An immunogold labeling method was developed to visualize focal adhesions using back-scattered electron imaging with a scanning electron microscope (SEM). On rough surfaces focal adhesions were primarily localized on to the ridges rather than the valleys and the cells tended to bridge over the valleys. Transmission electron microscopy (TEM) measurements of membrane proximity to the Ti surface indicated that average distance of cell to the Ti increased as the Ti surface roughness increased. Therefore, the size and shape of surface features are important determinants of epithelial adhesive behavior and epithelial coverage of rough surfaces would be difficult to attain if such surfaces become exposed.

Cell Adhesion↗

Electrical polarization of plasma-spray-hydroxyapatite coatings for improvement of osteoconduction of implants.

We have established the electrical polarization method of hydroxyapatite (HA) coatings (HAC) for clinical use, such as dental and orthopedic implants. The HAC examined in the current study was prepared in titanium substrates by plasma spraying of beta-tricalcium phosphate (TCP) powders followed by hydrothermal treatment. The prepared HAC consisted of a single phase of HA, because the starting TCP phase was completely transformed to the HA phase during the posthydrothermal treatment. Polarization was carried out at the elevated temperature of 400 degrees C under a d.c. field of 1 kV . cm(-1). The electrical measurements showed that the stored charges of the polarized HAC were greater than the reported value of the sintered ceramic HA. The enhanced bioactivity of the polarized HAC was demonstrated using 1.5 simulated body fluid (SBF). The crystal growth from the SBF solution was accelerated on the negatively charged surface in comparison with the untreated HAC. Similar to the polarized ceramic HA, the current results confirmed that the bioactivity of HAC would be effective for improving the initial fixation by polarization.

Calcium Phosphates↗

Extensive H(+) release by bone substitutes affects biocompatibility in vitro testing.

Bone substitutes are widespread in orthopedic and trauma surgery to restore critical bony defects and/or promote local bone healing. Cell culture systems have been used for many years to screen biomaterials for their toxicity and biocompatibility. This study applies a human bone marrow cell culture system to evaluate the toxic in vitro effects of soluble components of different bone substitutes, which are already in clinical use. Different specimens of tricalcium phosphates (TCP) (Vitoss, Cerasorb), nondecalcified bovine bone (Lubboc), demineralized human bone matrices (DBM) (Grafton Flex/Putty), and collagen I/III matrix (ACI-Maix) were tested in Dulbecco's modified Eagle's medium (DMEM) and MesenCult culture solution and compared with a biomaterial-free cell culture. Biocompatibility parameters were cell viability evaluated by phase-contrast microscopy and laser flow cytometry, morphology, and the local H(+) release by bone substitutes. There were significant differences (p < 0.05) between the tested biomaterials and culture solutions. Collagen I/III, non-demineralized bovine bone, and TCP materials showed advantages for cell survival over other tested biomaterials (average values of vital cells/mL MesenCult/DMEM: Collagen I/III: 1090/1083; Vitoss: 893/483; Cerasorb: 471/523; Lubboc: 815/410; Grafton Putty: 61/44; Grafton Flex: 149/57). Especially the DBM materials lead to a significant decrease of pH, which is considered to be a major factor for cell death. DMEM culture solution supports cell survival for those bone substitutes that induce an alkaline reaction, whereas MesenCult media promotes cell vitality in biomaterials, which leads to an acidification of culture solution.

Animals↗