Composites between hydroxyapatite and poly(epsilon-caprolactone) synthesized in open system at room temperature.

The hydroxyls present on the surface of hydroxyapatite (HA) granules, annealed at 700 composite function, 900 composite function and 1,100 composite function C, are able to initiate the polymerization of epsilon-caprolactone (CL), not only at 185 composite function C under vacuum, but also at room temperature in open system. A polymer layer ionically linked to the substrate is formed on HA surface, enhancing the compatibility between the organic phase and the inorganic one in composite biomaterials. We studied the characteristics of the polymer, produced by the reaction carried out at room temperature in open system, as well as the percentages of the poly(epsilon-caprolactone) (PCL) ionically bonded to the HA structure and of the "free" one. Both percentages appear very dependent on the annealing temperature; in particular, HA annealed for 1 h at 1,100 composite function C is the most efficient initiator of the reaction leading to ionically bonded PCL. The percentages of "free" polymer are much higher than at 185 composite function C under vacuum. Its formation is attributed to the role of water in opening the CL rings, and to the presence of CO(3) (2-) and HPO(4) (2-) ions in the HA annealed at lower temperatures. The presence of water appears to be the limiting factor for the production of PCL not bonded to the HA structure.

Comparison between the in vitro surface transformations of AP40 and RKKP bioactive glasses.

Two bioactive silica-phosphate glasses, AP40 and RKKP, were compared in their behaviour in simulated biological environment. Their chemical composition is practically identical, except that RKKP contains small amounts of amphoteric network-former oxides Ta2O5 and La2O3 (composition in wt% for AP40: beta-Ca3(PO4)2 24.50, SiO2 44.30, CaO 18.60, Na2O 4.60, K2O 0.19, MgO 2.82, CaF2 4.99; RKKP: beta-Ca3(PO4)2 24.23, SiO2 43.82, CaO 18.40, Na2O 4.55, K2O 0.19, MgO 2.79, CaF2 4.94, Ta2O5 0.99, La2O3 0.09). Previous investigations showed a better performance in osteopenic bone for RKKP. To gain more insight into these differences in biological behaviour, the in vitro bioactivity of the glasses was studied by treatment with a continuously replenished Hanks' Balanced Salt Solution (HBSS). The glasses were examined before and after HBSS treatment for 20 and 40 days by X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), X-ray Energy Dispersion (EDX), Raman and IR vibrational spectroscopies. Some slight but notable differences between the two glasses were observed after HBSS treatment. IR and EDX analyses showed that deposits formed on both glasses were composed of a calcium deficient carbonate-apatite; however, the layer formed on RKKP glass was found to be slightly more calcium deficient and thinner. EDX analysis evidenced the presence of a small percentage of F- ions only in the layers formed on the RKKP samples. The differences disclosed, although slight, can contribute to the understanding of the different biological behaviour previously observed.

Osteointegration of bioactive glass-coated and uncoated zirconia in osteopenic bone: an in vivo experimental study.

In elderly and osteoporotic patients an age-related loss of osteoinductivity could be the biological cause of implant failure regardless of the high quality of the implanted device. yttria stabilized tetragonal zirconia (YSTZ), either coated with the bioactive glass named RKKP bioglaze (RKKP) or uncoated, was implanted in the distal femurs of sham-operated and ovariectomized female rats. Animals were sacrificed at 30 and 60 days. Histomorphometry and microhardness tests were performed to assess osteointegration rate as well as bone quality around the implants. Significant decreases (p < 0.0005) in trabecular bone volume, BV/TV (41%), trabecular bone surface BS/TV (33%), trabecular thickness Tb.Th (20%), and trabecular number Tb.N (32%), together with a significant increase in trabecular separation Tb.Sp (184%), were found for the osteopenic rats compared with the sham-operated rats. At both experimental times the RKKP coating ensured a better osteointegration rate with higher AI values than the uncoated YSTZ, even when osteopenic rats were used (48% at 30 days and 12% at 60 days). No differences were observed at the bone-biomaterial interfaces for either material when comparing sham-operated with osteopenic rats. The present results demonstrate that the RKKP bioactive glass used as a coating ensures a high osteointegration rate even in osteoporotic bone, which is already visible from postoperative day 30 and is still apparent on day 60.

Protein adsorption onto two bioactive glass-ceramics.

Recent research suggests that the biocompatibility of an implant is to a large extent determined by selective adsorption of proteins from surrounding body fluids. Protein adsorption from human plasma onto two bioactive glass-ceramics (RKKP and AP40) which differ in La and Ta content, was studied by means of chromatography and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The quantitative analysis showed that the glass-ceramics have good protein binding capacities indicating multilayer formation. A correlation between chemical composition and the amount of adsorbed proteins was observed. The presence of La and Ta decreased the protein adsorption, so AP40 bound significantly more protein per surface unit then did RKKP. Preferential adsorption of apolipoprotein J, fibrinogen and fibronectin was observed.

Osteointegration of bioactive glass-coated zirconia in healthy bone: an in vivo evaluation.

Osteointegration of yttria stabilised tetragonal zirconia (YSTZ), either coated with bioactive glass named RKKP bioglaze (RKKP) or uncoated, was evaluated in an animal model. RKKP-coated and uncoated (controls) YSTZ cylinders were implanted in the distal femoral epiphyses of 14 Sprague Dawley rats under general anaesthesia. At the experimental times of 30 and 60 days after sacrifice, histomorphometry and SEM microanalysis were performed on methylmethacrylate-embedded undecalcified sections to determine the osteointegration rate. At 30 days, a significantly higher affinity index was demonstrated in vivo by histomorphometric evaluation in RKKP-coated versus uncoated YSTZ implants p < 0.05); at 60 days, the coated implants behaved better than controls (affinity index of + 32%), but the difference observed lay within the statistical uncertainty. SEM analysis demonstrated better bone adhesion to the material in RKKP-coated YSTZ at both 30 and 60 days. These findings suggest that YSTZ coated with the bioactive glass named RKKP enhances osteointegration of ceramics.

Improvement in zirconia osseointegration by means of a biological glass coating: An in vitro and in vivo investigation.

The biocompatibility and osseointegration of zirconia (ZrO(2)), either coated with RKKP bioglazeor uncoated, were evaluated in vitro and in vivo. The in vitro test was performed in human osteoblasts, whereas maximal sensitization was performed in 23 Dunkin Hurtley guinea pigs. RKKP bioglaze-coated and uncoated (controls) ZrO(2) cylinders were implanted in the distal femoral epiphyses of 14 Sprague-Dawley rats under general anesthesia, and animals were sacrificed at 30 and 60 days. Lactate dehydrogenase, alkaline phosphatase, and Thiazolyl Blue (MTT) were tested in vitro. A graded score was used for evaluating the results of the sensitization test. Histomorphometry and microhardness testing were performed to quantify the osseointegration rate, as well as bone quality around the implants. Neither in vitro cytotoxicity nor sensitization were observed. Histomorphometry demonstrated that at 30 days, the affinity index was significantly higher in coated implants than in uncoated ones (p < 0.05); at 60 days, the behavior of coated implants was better than that of uncoated ones, but differences were not significant. Significant increases in bone microhardness were found at 1000 microm from the interface area for both uncoated (p < 0.0005) and RKKP bioglaze-coated (p < 0.0005) ZrO(2), and also within 200 microm from the interface (p = 0.014) but only for coated ZrO(2.) These results suggest that RKKP bioglaze-coated ZrO(2) permits biocompatible devices with improved osseointegration properties to be manufactured.

Plasma protein adsorption pattern on characterized ceramic biomaterials.

The protein/biomaterial interactions of three biomaterials used in hard tissue surgery were studied in vitro. A dynamic flow system and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) were used to investigate the adsorption of proteins from diluted human plasma on hydroxyapatite, alumina and zirconia, with regard to total protein binding capacity, relative binding capacity for specific proteins and flow-through and desorption patterns. The ceramics were characterized regarding physicochemical properties; namely, chemical composition by elementary analyses and specific surface, pore volume and pore size distribution using the BET-method and Hg-porosimetry. The materials were found to adsorb a surprisingly low amount of plasma proteins, leaving more than 70% of the surface free. The cellular response will therefore be highly affected by the physico-chemical properties of the material, in contrast to a surface fully covered with proteins. Regarding the adsorption of proteins, most proteins exhibited similar flow-through patterns on the three adsorbents. The exceptions with different flow-through patterns were apolipoprotein D (Apo D), apolipoprotein J (Apo J), complement factor C1s (C1s), complement factor C3 (C3), ceruloplasmin, fibrinogen, alpha1 B glycoprotein and alpha2 HS glycoprotein and serum retinal-binding protein (SRBP). The role of these proteins on acceptance or rejection of implants has to be investigated.

Biological glass coating on ceramic materials: in vitro evaluation using primary osteoblast cultures from healthy and osteopenic rat bone.

ZrO2 and Al2O3 substrates were successfully coated by a double layer of a silica-based glass named RKKP, using a low-cost firing technique. RKKP is a glass well known for its bioactivity; therefore, a RKKP coating on Al2O3 or ZrO2, allows to combine the excellent mechanical properties of these strong ceramic substrates with its bioactivity. ZrO2 samples were easily coated using a double layer of RKKP by a simple enamelling technique. To accommodate the thermal expansion coefficient mismatch between Al2O3 and RK K P, this substrate was coated using a multilayered composite approach. All of the coatings were characterised from a morphological and compositional point of view, and an extensive biological evaluation was performed using fresh rat osteoblasts. Osteoblast primary cultures were derived from the trabecular bone of femoral condyles harvested from intact (NB) and osteopenic (OB) rats. After characterisation of their phenotype, osteoblasts were seeded on material samples of ZrO2 or Al2O3 coated with RKKP, and cultured for 7 days. Cell proliferation (MTT test) and cell differentiation (alkaline phosphatase activity) were evaluated at the end of the experiment, to assess osteoblast behaviour in the presence of biomaterials and determine if the results were related to the host bone quality. Results of both materials showed a good level of biocompatibility. In particular, MTT significant higher values were detected in NB cultures on ZrO2-RKKP samples; ALP activity significantly increased in NB cultures on Al2O3-RKKP and in OB cultures on both coated samples.

In vitro characterisation of zirconia coated by bioactive glass.

An in vitro evaluation of a biomedical device, which combines the mechanical properties of zirconia substrates with the bioactivity of two different glass layers (AP40 and RKKP), was performed. In this work, data on different kinds of analysis were reported both on as-sintered zirconia samples and on RKKP- and AP40-coated zirconia substrates. Structure, composition and morphology of the apatite layer growth on the coated samples after 30 days of soaking in an acellular simulated body fluid, serum protein adsorption, fibroblasts and human osteoblast-like cells adhesion, growth, morphology and biochemical aspects were studied. Results of soaking test in SBF, revealed the growth of an apatite layer on the surface of the glass-coated samples. Proteins adsorbed to the materials were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and results evidenced that the two glass-coated materials bound a higher amount of total protein than did the zirconia substrate. Fibroblasts and osteoblast-like cells cultured on RKKP- and AP40-coated zirconia showed a higher proliferation rate, leading to confluent cultures with higher cell density and a generally better expression of osteoblast alkaline phosphatase activity in comparison with zirconia substrate. In conclusion, our results indicate that the surface chemical characteristics of the two glass coatings AP40 and RKKP, with no great differences between them, substantially enhance zirconia integration with bone cells at least in vitro. This effect may be of significance in the stability of glass-coated zirconia orthopaedic and dental implants.

Biocompatibility and osseointegration in osteoporotic bone.

We implanted nails made of titanium (Ti6Al4V) and of two types of glass ceramic material (RKKP and AP40) into healthy and osteopenic rats. After two months, a histomorphometric analysis was performed and the affinity index calculated. In addition, osteoblasts from normal and osteopenic bone were cultured and the biomaterials were evaluated in vitro. In normal bone the rate of osseointegration was similar for all materials tested (p < 0.5) while in osteopenic bone AP40 did not osseointegrate (p < 0.0005). In vitro, no differences were observed for all biomaterials when cultured in normal bone-derived cells whereas in osteopenic-bone-derived cells there was a significant difference in some of the tested parameters when using AP40. Our findings suggest that osteopenic models may be used in vivo in the preclinical evaluation of orthopaedic biomaterials. We suggest that primary cell cultures from pathological models could be used as an experimental model in vitro.

Coatings on zirconia for medical applications.

In order to combine the mechanical properties of a high-strength inert ceramic (yttria-stabilised zirconia, ZrO2-3%Y2O3, defined as zirconia in the text) with the specific properties of bioactive materials, some zirconia samples were coated by two bioactive phosphosilicate glasses and glass-ceramics: RKKP and AP40. Coatings of about 200-300 microm thickness were prepared by a simple and low-cost firing method. They were characterised by optical and scanning electron microscopy (SEM) and compositional analysis (EDS). The adhesion of the coatings on zirconia was tested by shear tests. Vickers indentations at the coating/zirconia interface were performed in order to observe the crack propagation path. The reactivity of glasses and glass-ceramics coatings towards a simulated body fluid (SBF), having the same ion concentration as that of human plasma, was evaluated and compared to that of the bulk glass and glass-ceramics, by examining the morphology of the reaction layer formed on the surface of the coated zirconia after one month of soaking in the SBF at 37 degrees C.

Porous ceramic bodies for drug delivery.

An approach to the production of ceramic drug delivery devices is proposed. Two examples of possible ceramics are dealt with: hydroxyapatite weakly modifiable by living tissue and the bioinert alumina. The possibility to control the formed porosity was taken into consideration for both materials. The ratio between the acquired porosity and the quantity and quality of the agents inducing porosity is also described and discussed. A test on the role of porosity was performed on the obtained porous ceramic bodies and a study was made on the release of a substance with pharmacological activity from previously impregnated porous ceramic bodies. This paper is preliminary to a planned work targeted to the preparation of ceramic drug delivery systems.

The effect of osteopenia on the osteointegration of different biomaterials: histomorphometric study in rats.

The osteointegration of Hydroxyapatite (HA), Titanium (Ti-6Al-4V: Ti), Zirconia (ZrO2), Alumina (Al2O3) and 2 biological glasses (AP40 and RKKP) was comparatively investigated in normal and osteopenic rats by means of histomorphometry. Thirty-six Sprague Dawley female rats were left intact (Group C) while 36 were ovariectomized (Group OVX). Group C and OVX were further divided into 6 subgroups. After 16 weeks all animals were submitted to the femoral implant of nails made of the above-mentioned materials. Eight weeks after implantation the animals were euthanized, the femurs were harvested for histomorphometric analysis. The data showed that: (1) all the tested materials were biocompatible in vitro; (2) no significant differences existed in Affinity Index (AI) of Group C; and (3) results from paired comparison applied to the AI showed significant differences among the Groups C and OVX. The AI did not significantly change among intact groups, while it significantly decreased when some materials were implanted in OVX subgroups (AP40, ZrO2 and Ti-6Al-4V: p < 0.0005, p < 0.05 and p < 0.01). It is confirmed that bone mineral density is a strong predictor of the osteointegration of an orthopedic implant and that the use of pathological animal models is necessary to completely characterize biomaterials.

In vitro and in vivo assessment of bone-implant interface: a comparative study.

The present in vitro and in vivo comparison of three bioactive (HA, AP40, RKKP) and three bioinert (Ti6-Al4-V, Al2O3, ZrO2) materials was undertaken to identify which of them provide(s) the most suitable coating for prostheses implanted in patients with altered metabolic status. The experimental design included in vitro tests with human osteoblasts and morphological observations by scanning electron microscopy. For the in vivo evaluation, the materials were implanted in the femoral condyle of ovariectomised and intact female rats, and two months after surgery an X-ray microanalytical study was performed. The in vitro study showed good biocompatibility with all materials. Microanalysis evidenced a similar behaviour with all materials except the two biological glasses. The differences in Ca and P content observed between intact and ovariectomised rats can be explained by the intrinsic capability of biological glasses to undergo surface modifications in the presence of alterations of the bone metabolism. Thus, their use seems to be indicated in recipients with osteoporotic pathologies.

Periodontal membranes from composites of hydroxyapatite and bioresorbable block copolymers.

Biomembranes are frequently proposed as devices for "guided bone regeneration." Such membranes consist generally of a thin sheet of polymeric material, mostly textured from polymeric yarns or clots, which all have a diffuse very fine winding porosity. The cross-section size of the holes of such porosity is nanometric (diameter < 0.1 microm); thus these holes can be indicated as nanoholes. Whatever the method of production, the surface density of nanoholes (number per square centimeter) has to be as high as possible. It is important also that no variation of this density occurs. The fine dimension of these microholes allows the crossing of small molecules (O2, CO2, H2O, sugars, many nutritional organic compounds and even some simple proteins) but not other larger molecules and particulates, including cells of any kind. These biomembranes have, consequently, a semipermeable behavior, providing the functional role which is the interposition of a barrier for the cells, separating the bone from the surrounding soft tissues. The kinetic of proliferation of osteoblasts is lower than that of fibroblasts. Most membranes of this kind are not resorbable. The main problem for the resorbable ones is the speed of size increase of the holes during the time. Their diameter must not exceed a threshold value until the reconstruction of bone is complete, otherwise soft tissue cells will invade the growing bone tissue with formation of undesirable mixed tissue. The present paper deals with a resorbable membrane made with a composite polymer/ceramic. A poly(epsilon-caprolactone)-block-poly(oxyethylene)-block-poly(epsilon-caprolactone) copolymer is the polymeric matrix which contains dispersed ceramic hydroxyapatite microgranules, a stiff filling additive. The main possible use is that of periodontal membranes. The copolymer, obtained by thermal polymerization of epsilon-caprolactone onto poly(ethylene-glycol), presents good biological tolerance, is resorbable under physiological conditions and can promote cell growth. Histological tests, performed 6 months after implantation, showed that the polymeric matrix is almost totally resorbed. New-formed bone colonizes even the innermost parts of the membrane, with bone trabeculae closely surrounding the hydroxyapatite granules.

New composites of hydroxyapatite and bioresorbable macromolecular material.

Composite materials were prepared by mixing in different proportions of hydroxyapatite (HA) and poly(epsilon-caprolactone-oxyethylene-epsilon-caprolactone) block copolymer (PCL-POE-PCL) to produce a new resorbable material for biomedical applications. This material has proved to be very interesting for production of periodontal membranes. Mechanical properties are linearly proportional to the amount of HA introduced. Fourier transform infrared (FTIR) investigations have pointed out that HA is able to influence some close epsilon-caprolactone molecules to start its homopolymerization giving PCL with an end chain ionic bonding. HA grains are therefore surrounded by a film of PCL which grants close connection of HA grains within copolymeric matrix. This interface bond with PCL is, however, an interesting occurrence for preparations of HA/PCL composites.

Bioactivity modulation of bioactive materials in view of their application in osteoporotic patients.

The application of bioactive ceramic coatings to prostheses confers strength to a material (ceramic or biological glass) that exerts beneficial effects on bone-tissue growth but that itself lacks the toughness and stability required of an implant device. The rate of bioactivity is related to the chemical reactivity of the material and causes interface dissolution, precipitation and ion-exchange reactions. Ceramics may differ in sintering temperature and thus exhibit differences in their in vitro dissolution features and in vivo performance. To test these effects, in vitro and in vivo studies were carried out on two biocompatible biological glasses and a ceramic of proven bioactivity in view of their potential utilization as covering materials. In addition, a modified chitosan was adsorbed on the surface of a series of hydroxyapatite (HA) samples. Human fibroblasts and/or osteoblasts were used for the in vitro tests, and normal (INT) and osteoporotic (OVX) rats, normal rabbits and sheep for the in vivo studies. Similar chemical changes were observed in both glasses, suggesting that these materials underwent modifications directly dependent on their biological environment. The in vivo tests point to the possibility of improving the bioactivity of ceramic substrates with chitosan. However, the different behaviour of the materials in vitro and in vivo suggests that these tests should be conducted in parallel.

Coating of ZrO2 supports with a biological glass.

The possibility of obtaining a good bioactive coating on biomedical devices made with zirconia ceramic was considered. Yttria partially stabilized zirconia was used to produce suitable substrates. The obtained adhesion was good (66+/-13 MPa) and encouraging for biomedical applications. Small microcrystals formed inside and on the surface of the glass layer. These microcrystals transformed the glass into a glass-ceramic. An accurate investigation of the nature of this microcrystal formation was carried out to verify its compatibility with the planned biomedical applications.

Cell growth on cordierite: an approach to the identification of reliable supports for continuous-flow solid-bed reactors.

This study aimed to assess the biocompatibility of two cordierite ceramics (DF and Cord 1014), with similar chemical composition and different porosity, as a potential support for cell growth in a continuous-flow, solid-bed reactor. The Chinese hamster ovary (CHO) cell line transfected with HBV-DHFR recombinant plasmid was seeded on cordierite or polystyrene dishes and evaluated for cell growth and production of recombinant hepatitis B surface antigen. Proliferation of the CHO cells, in terms of cell number, was generally similar in polystyrene and Cord 1014 and always lower in DF. Flow cytometric analysis showed no difference in cell cycle distribution for cells grown on different supports, and showed a two-fold increase in percentage of debris for cells grown on DF than for those grown on Cord 1014 and polystyrene culture dishes. Moreover, the morphology of cells grown on Cord 1014 did not change during the experiment, and cells were well spread and organized. Finally, total recombinant hepatitis B surface antigen production was higher on Cord 1014 than on polystyrene and DF samples. Such evidence suggests that Cord 1014 could be a promising support for growing cells in a continuous-flow, solid-bed reactor.

Biomaterials for orthopedic surgery in osteoporotic bone: a comparative study in osteopenic rats.

UNLABELLED: To evaluate orthopedic devices in pathological bone, an experimental study was performed by implanting Titanium (Ti) and Hydroxyapatite (HA) rods in normal and osteopenic bone. Twenty-four rats were used: 12 were left intact ( CONTROL: C) while the other 12 were ovariectomized (OVX). After 4 months all the animals were submitted to the implant of Ti or HA in the left femoral condyle (Ti-C, HA-C, Ti-OVX, HA-OVX). Two months later the animals were sacrificed for histomorphometric, ultrastructural and microanalytic studies. Our results show a significant difference between the Affinity Index (A.I.) of HA-C and Ti-C (77.0 +/- 7.4 vs 61.2 +/- 9.7) (p < 0.05). No significant differences were observed between the osteointegration of Ti-C and Ti-OVX (61.2 +/- 9.7 vs 48.2 +/- 6.7). Significant differences also exist between the osteointegration of HA-C and HA-OVX (77.0 +/- 7.4 vs 57.6 +/- 11.5) (p < 0.01). Microanalysis shows some modifications in Sulphur (S) concentration at the bone/biomaterial interface of the Ti-OVX group. Therefore our results confirmed the importance of biomaterials characteristics and of bone quality in osteointegration processes.