A. Ravaglioli

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.

Synthesis of hydroxyapatite-based powders by mechano-chemical method and their sintering

Abstract Ceramic hydroxyapatite (HA) is on the way to gaining credit as one of the most promising and diversified materials for employment in surgery, thanks to its good characteristics of biocompatibility and bioadaptability. The main and probably unique deficiency of these materials obtained by traditional methods consists in its weak mechanical resistance, that does not allow their use when even low loading is involved. To improve the mechanical properties of HA-based ceramic bodies additions are normally necessary. In this paper the authors propose a method to prepare powders and composite ceramic bodies with a matrix comprising HA. The powders are produced by the utilization of a simple and economic mechano-chemical method. The composite ceramic bodies are easily obtained by simple firing of the powders at a suitable temperature (1250 °C). The powders, after sintering, give products that show a flexural strength of more than 100 MPa in standard samples. This value is significantly higher than that usually attainable with present commercially available powders (60 MPa). The transformation in the components (HA and β-tricalcium phosphate) of the composite ceramic arises from the nature of the powders, consisting of highly defective HA. The mechano-chemical process is described together with the employed procedures specifically followed for reactive milling of starting powders. The preparation conditions of defective HA powders and properties acquired through such a method are reported. By this method an intimate mixing of β-tricalcium phosphate (β-TCP) inside the HA matrix is obtained that easily allows the preparation of ceramic bodies with reproducible properties, with no necessity for additions and mixing procedures, that could lead to inhomogeneity.

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.

Sol-gel derived 45S5 bioglass: synthesis, microstructural evolution and thermal behaviour

In this work, the 45S5 bioactive glass was synthesized through an aqueous sol–gel method. Characteristic functional groups were evidenced by Fourier transform infrared spectroscopy, the thermal behaviour was investigated by thermogravimetric and differential thermal analysis, crystallization kinetics and phase evolution were followed by X-ray diffraction measurements. The sintering behaviour of the sol–gel derived 45S5 was then studied by dilatometry and the microstructural evolution was followed step-by-step, interrupting the thermal cycle at different temperatures. In vitro dissolution tests were performed in order to assess the degradation behaviour of sol–gel derived 45S5 samples thermally treated at different temperatures. A relevant influence of the calcination conditions (namely, dwelling time and temperature) of the as-prepared powder on the phase appearance and its sintering behaviour as well as on the porosity features, in terms of pore dimension and interconnectivity, of the fired materials was stated.

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.

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.

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.

The effect of pulsed electromagnetic fields on the osteointegration of hydroxyapatite implants in cancellous bone: a morphologic and microstructural in vivo study

Effects of pulsed electromagnetic fields (PEMFs, 75 Hz, 1.6 mT) were investigated in 12 rabbits after placing hydroxyapatite (HA) implants in their femoral condyles. Six animals were stimulated with PEMFs for three consecutive weeks, 6 h/day, while the remaining animals were sham‐treated (Control Group). Rabbits were sacrificed at 3 and 6 weeks (after a 3‐week non‐stimulation period) for histomorphometric analysis and microhardness testing (at 200, 500, 1000, 2000 μm from the implant) around the implants around the implants. Histomorphometric analysis did not highlight any significant changes. On the contrary, there were statistically significant differences between the effects produced by PEMFs and Control Groups (F = 149.70, p < 0.0005) on the Affinity Index results, as well as by the experimental time of 6 and 3 weeks (F = 17.12, p = 0.001) on the same results. In PEMF‐stimulated animals the microhardness (HV) values measured in trabecular bone at a distance of 200 and 500 μm from the implants, were significantly higher with respect to controls. At 6 weeks, HV values at the bone‐implant interface in PEMF‐stimulated animals were not significantly different with respect to normal bone, while they remained significantly lower in control animals. Both morphological and structural results demonstrated a positive therapeutic effect of PEMFs in accelerating HA osteointegration in trabecular bone.

BIOMATERIALS FOR ORTHOPEDIC SURGERY IN OSTEOPOROTIC BONE : A COMPARATIVE STUDY IN OSTEOPENIC RATS

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.

Biocompatibility and osseointegration in osteoporotic bone: A PRELIMINARY IN VITRO AND IN VIVO STUDY

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.