The interaction of osteoblasts with bone-implant materials: 1. The effect of physicochemical surface properties of implant materials.

This comparative study of various surface treatments of commercially available implant materials is intended as guidance for orientation among particular surface treatment methods in term of the cell reaction of normal human osteoblasts and blood coagulation. The influence of physicochemical surface parameters such as roughness, surface free energy and wettability on the response of human osteoblasts in the immediate vicinity of implants and on the blood coagulation was studied. The osteoblast proliferation was monitored and the expression of tissue mediators (TNF-alpha, IL-8, MMP-1, bone alkaline phosphatase, VCAM-1, TGF-beta) was evaluated after the cell cultivation onto a wide range of commercially available materials (titanium and Ti6Al4V alloy with various surface treatments, CrCoMo alloy, zirconium oxide ceramics, polyethylene and carbon/carbon composite). The formation of a blood clot was investigated on the samples immersed in a freshly drawn whole rabbit blood using scanning electron microscope. The surfaces with an increased osteoblast proliferation exhibited particularly higher surface roughness (here R(a) 3.5 microm) followed by a high polar part of the surface free energy whereas the effect of wettability played a minor role. The surface roughness was also the main factor regulating the blood coagulation. The blood clot formation analysis showed a rapid coagulum formation on the rough titanium-based surfaces. The titanium with an etching treatment was considered as the most suitable candidate for healing into the bone tissue due to high osteoblast proliferation, the highest production of osteogenesis markers and low production of inflammatory cytokines and due to the most intensive blood clot formation.

Clinical correlations of potential activity markers in systemic sclerosis.

The objectives of the project were the following: (1) to establish a group of patients with a confirmed diagnosis of systemic sclerosis (Ssc), (2) to perform a detailed entrance examination of each patient, (3) to determine concentrations of potential activity markers, and (4) to make a comprehensive examination of each patient 1 year after inclusion into the study. A total of 49 patients were examined, 36 with a limited form of SSc, 9 with diffuse SSc, and 4 with other forms of SSc. We determined plasma or serum levels of the N-terminal propeptide of procollagen type III (NPIIIP), interleukin-6 (IL-6), soluble receptor for interleukin-2 (sIL-2r), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular adhesion molecule-1 (sVCAM-1), von Willebrand factor antigen (vWFAg), and big endothelin-1 (BET-1) using commercial kits, and urinary excretion of pyridinoline (PYR) and deoxypyridinoline (D-PYR) using high-performance liquid chromatography. Correlations of these markers with selected clinical data were calculated. The mean levels of all potential activity markers were increased compared with normal values, but differences were not significant. The levels of NPIIIP, D-PYR, and IL-6 were normal. The measured values after 1 year did not differ from the entry values. At entry, NPIIIP concentrations correlated with the finger-to-palm distance, and D-PYR corresponded with findings on a simplified health assessment questionnaire (FQ). IL-6 levels correlated with the leukocyte count, sIL-2r with the FQ, and ET-1 with the diffuse lung capacity for carbon monoxide. In general, we found only a few clinical correlates of potential activity markers. Our data confirmed the correlations of collagen metabolism markers with skin involvement and FQ, as was reported previously. Larger studies in this field are needed.

Biological properties of the intervertebral cages made of titanium containing a carbon-carbon composite covered with different polymers.

Intervertebral cages are used in orthopaedics for stabilization of injured lumbar parts of vertebral columns. Our study provides preliminary results of tests of the biological properties of titanium cages with a variously modified carbon/carbon composite (C/C) core. This core was produced from a C/C composite modified by hydrogel materials based on poly(2-hydroxyethyl methacrylate) (HEMA) enriched with 1% collagen or 35% methylmethacrylate or 30% terc-butylmethacrylamide. We evaluated the adhesion of the cells to the tested material coating using an in vitro study of the metabolic activity and cytokine production of the cells (TNF-alpha, IL-8). We studied the biocompatibility of intervertebral cages coated with different copolymers under in vivo condition and in an implantation experiment in the porcine femurs. Both in vitro and in vivo results revealed favourable biotolerance of the use system. Modification of the composite HEMA with the use of collagen seems to have a more positive effect on the new bone tissue formed around the implanted devices than HEMA copolymerized with methylmethacrylate or terc-butylmethacrylamide.

Autoantibodies can be prognostic markers of an erosive disease in early rheumatoid arthritis.

OBJECTIVE: To evaluate a contribution of selected laboratory parameters for a prediction of progressive and erosive development in patients with early rheumatoid arthritis (RA). METHODS: In a prospective study baseline levels of antibodies to cyclic citrullinated peptide (anti-CCP), IgM, IgA, and IgG rheumatoid factors (RFs) were measured by enzyme linked immunosorbent assay (ELISA) in 104 patients with RA with disease duration <2 years. Antikeratin antibodies (AKA) and antiperinuclear factor (APF) were detected by indirect immunofluorescence. Patients were divided into two groups based either on the presence or absence of erosions or according to progression of Larsen score at the end of the 24 months' follow up. RESULTS: Sixty seven (64%) patients developed radiographic erosions, 49 (47%) had progression in Larsen score, and 36 (35%) progressed by more than 10 Larsen units. Significant differences in erosions and progression between the two groups were detected for anti-CCP, AKA, APF, IgM RF, IgA RF, and IgG RF. Baseline Larsen score correlated significantly with anti-CCP, IgM RF, and IgA RF levels, and all measured antibodies correlated with the progression >10 units. The combination of anti-CCP and IgM RF increased the ability to predict erosive and progressive disease. CONCLUSION: The data confirmed that measurement of anti-CCP, AKA, APF, and individual isotypes of RFs was useful for prediction of structural damage early in the disease course. Combined analysis of anti-CCP and IgM RF provides the most accurate prediction.

Biological and biochemical properties of the carbon composite and polyethylene implant materials.

We studied the biocompatibility of the carbon composites and polyethylene materials with and without collagen or collagen and proteoglycan cover. We used the in vitro technology to study the adhesion of model cells evalution, their metabolic activity and the production of TNF-alpha as a cytokine model. Under in vivo condition, the biocompatibility of tested polymers were studied in the implantation experiment, subcutaneously in the interscapular region in the laboratory rat. We have found in the in vitro assay favorable proliferation and the smallest production of pro-inflammatory TNF-alpha cytokine in cells adherent to the hydrophobic polyethylene material coated with biological macromolecules. Using in vivo tests performed by the implantation of materials to the rat we demonstrated that the materials are not cytotoxic. The tissue capsule surrounding the implants was not significantly influenced by the type of the implant and the pre-treatment by the biological molecules. However, the foreign-body giant multinucleated cells were observed only in the vicinity of the collagen - covered hydrophobic polyethylene implant. Interestingly, while the collagen coating improved the biocompatibility of tested polymers in vitro, the inflammatory reaction against this covered materials was higher under in vivo conditions. The pre-treatment of carbon composites by both types of biological macromolecules reduced the occurrence of carbon debris in the implantation site. The tested carbon composites and polyethylene materials are not toxic. The pre-treatment of the materials by extracellular matrix components increased their biological tolerance in vitro and reduced implant wears in animal experiment, which can be important for the medical application.

[Relation between cytokines, adhesive immunoglobulins and matrix metalloproteinases in osteoarthritic joints]. / Vztahy mezi vybranými cytokiny, adhezivními imunoglobuliny a matrixovými metaloproteinázami v osteoartritickém kloubu.

BACKGROUND: The dysbalance of proteinase production and their inhibitors leads to destruction of tissues in many pathological processes. Recently it was shown that the expression of proteinases is regulated also by interactions of cells which is mediated by adhesive molecules. We wanted to find out whether this mechanism is involved also in the destruction of joints in osteoarthritis. METHODS AND RESULTS: Cartilage, synovial and subchondral bone tissues, and synovial fluids were obtained from 23 joints after total endoprosthesis surgery. The solid tissues were extracted by TRIS buffer. The investigated protein concentrations were assessed immunochemicaly. In all specimens gelatinase A, gelatinase B, stromelysin-1, TIMP-1, soluble adhesive molecules sICAM-1 and sVCAM-1 and cytokines TNF-alpha and IL-8 were found. In cartilage and synovial fluid the proteolytic potential of metalloproteinases was balanced with high concentrations of their inhibitor TIMP-1 (259.4 +/- 105.2 pmol/g protein vs 2343.8 +/- 637.5 pmol/g protein in synovial fluid, p < 0.00001, and 178.9 +/- 175.7 pmol/g dry weight vs 647.2 +/- 561.3 pmol/g dry weight in cartilage, p < 0.001) but in synovial tissues and pathological subchondral bones was not (257.4 +/- 617.2 pmol/g dry weight vs 171.3 +/- 170.8 pmol/g dry weight in synovium, p = 0.61716, and 17.4 +/- 15.4 pmol/g dry weight 33.6 +/- 33.3 pmol/g dry tissue in pathological subchondral bone, p = 0.16705). CONCLUSION: From correlation analysis ensues that the bond of ICAM-1 and VCAM-1 on chondrocytes with appropriate integrin ligands probably leads to up-regulation of gelatinase A and B and to down-regulation of TIMP-1. Moreover it is apparent that TNF-alpha up-regulates both investigated adhesive molecules followed and stromelysin-1 and TIMP-1.

Biomechanical and biological properties of the implant material carbon-carbon composite covered with pyrolytic carbon.

The aim of this study was to test C/C material (carbonized, graphitized or covered with pyrolytic carbon) designated for the use in orthopaedic and bone surgery. Using an in vitro assay we confirmed, that the cell proliferation was exhibited the mostly on the C/C composite coated with pyrolitic carbon and afterwards polished. The two latest of subsequent water extracts of this material had a slightly inhibiting effect on the cells metabolic activity. Biocompatibility test in vivo performed subcutaneously on rats did not show big differences between three tested implants (C/C composite, epoxy resine, titanium alloy), on the other hand the plates tested on pigs demonstrated foreign-body reaction induced by wear C/C composite material. Such debris were found both in the neighborhood of the implant as well as in the lymphatic node.

[Use of Bioimplants to Replace Cartilage Part I: Chondrocyte Cultivation in Three-dimensional Gel.]. / Vyuzití bioimplantátu k náhrade chrupavky I. cást: Kultivace chondrocytu v trojrozmerném gelu.

For the investigation pig chondrocytes were used which were cultivated in a medium containing cartilaginous collagen and aggregan. The substances used were identified by electrophoresis, chromatography and electron microscopy. Various conditions for cultivating chondrocytes were tested - a) a medium containing cartilaginous collagens only, b) with addition of aggregan, c) with addition of the tripeptide GHK, d) with addition of the tripeptide and aggregan. Under conditions of three-dimensional cultures the original phenotype of chondrocytes was preserved. In this respect the presence of aggregan was decisive. It proved possible to create a firm netlike formation which was subsequently used as a cartilaginous implant. Key words: cartilage, chondrocytes, cartilaginous chondrocytes, aggregan, three-dimensional gel, implant.

[Use of Bioimplants to Replace Cartilage Part II: Application of Implants in Animal Experiments.]. / Vyuzití bioimplantátu k náhrade chrupavky II. cást: Pouzití implantátu v pokuse na zvíreti.

The published paper pertains to a group of fullgrown minipigs where under anaesthesia with Narcamone after premedication with Rometar cartilage samples from the medial condyle of the femur in the area of the femoropatellar joint were taken. The collected cartilage samples served subsequently for the preparation of an autologous implant. After its preparation another operation was performed where by means of a bioptic trocar (diameter 3.5 mm) a cylindershaped defect was produced into which the implant was introduced. The animals were killed 8-12 weeks after the operation and part of the joint with the implant was subjected to further examination. The authors investigated three types of implants: 1. an implant based on three-dimensional cultivation of chondrocytes in a cultivation medium enriched with cartilaginous collagen and aggregan, 2. as sub 1 but the cultivation medium was enriched with the GHK tripeptide, 3. chondrocytes in the implant are added to gel prepared from cartilaginous collagens and aggregan. All three types of implants had a stimulating effect on the new formation of hyaline cartilage. When the tripeptide GHK is used, the newly formed tissue contains more cells. Simplest is the preparation of the third implant. Also the peroperative procedure is simplest. Key words: chondrocytes, cartilaginous collagens, cartilaginous implant, tripeptide gly-his-lys, aggregan, minipigs.

Effect of the tripeptide glycyl-L-histidyl-L-lysine on the proliferation and synthetic activity of chick embryo chondrocytes.

Under certain conditions chondrocytes form lattices with cartilage collagens, which may serve as cartilage implants. It is necessary to find the optimal conditions for culturing chondrocytes. Three different supports are compared: (a) plastic; (b) cartilage collagens; and (c) insoluble skin collagen solubilized under denaturing conditions (ISC-40). The effect of culture medium supplementation with the tripeptide (Gly-His-Lys)2.Cu.2H2O.2NaCl (GHK) on chondrocyte proliferation and synthetic activity is studied, with particular attention paid to collagen types I, II and III. The collagen supports stimulated chondrocyte proliferation, but on the ISC-40 support they started to dedifferentiate rather early. In the primary culture, chondrocytes on all three supports synthesized mainly collagen type II, and only small amounts of types I and III. In the first passage the synthesis of these two collagen types increased, relative to collagen type II, at least on the cartilage collagen support. Supplementation of culture medium with GHK stimulated chondrocyte proliferation in the primary structure mostly on the ISC-40 support. On the other two types of supports the stimulatory effect of GHK was expressed mostly in the first passages. The collagen synthetic rate was increased by GHK on both of the collagen supports; on the cartilage collagen support collagen type II was synthesized predominantly and on the ISC-40 support types I and III were mostly formed. It is suggested that supplementation of culture medium with GHK may be useful in the preparation of cartilage implants.

[The effect of aluminum on the structure and metabolism of collagen]. / Ovlivnení struktury a metabolismu kolagenu hliníkem.

BACKGROUND: Aluminium is considered to be the etiopathogenetic factor in various pathological conditions. It was demonstrated already previously that metals even under conditions in vivo link with collagen structures and influence the protein metabolism. Therefore the authors investigated the effect of aluminium (Al) on collagen and its metabolism. METHODS AND RESULTS: In the described trial aluminium was used as potassium alum, oxalate alum or aluminium chloride. The effect of aluminium was investigated in rats, chick embryos or fibroblast cultures. Using electron microscopy of collagen from the tail tendons, the development of transverse striation following 15 weeks of i.m. Al administration was revealed. At the same time also an increase of temperature of contraction of this collagen by 1.5 to 3.7 degrees C occurred, depending on the Al compound used. On fibroblast cultures an inhibitory effect of Al on their proliferation was found. In chick embryos Al caused a decline of the radioactive hydroxyproline concentration and thus also a reduced collagen synthesis practically in all investigated tissues. Similarly the negative effect of Al was manifested in the incorporation of radioactive glucosamine into proteoglycans of granulation tissue of rats. In cartilaginous collagen enhanced proline hydroxylation caused by Al was observed. CONCLUSIONS: Although it is not possible, with regard to Al chemistry, to make an unequivocal statement on the nature of the compound which causes a particular reaction in biological systems, it may be said, based on the achieved results, that Al has an effect on biological systems due to its bond with collagen structures and by influencing their metabolism.

The effect of different collagens and of proteoglycan on the retraction of collagen lattice.

The effect of various collagens and proteoglycan on the formation and retraction of collagen lattices was tested. The most rapid aggregation of collagen molecules was observed by the use of the least cross-linked collagen fractions (ie pepsin-digested calf skin collagen type I). Lattices formed with more cross-linked collagens (acid soluble collagen-ASC, type III) contracted slowly and less intensively. Unpurified pepsinized cartilage extract containing collagen types II, IX and XI, some glycoproteins and proteoglycans formed lattices rather well. On the contrary, purified collagen type II as well as polymeric collagen (solubilized by denaturing conditions) did not form lattices at all. The lattice formation and retraction was intensified by addition of proteoglycan into the culture medium. The authors suggest that the kinetics of the lattice formation and retraction depends on the amount of collagen cross-links and the concentration of proteoglycan in the culture medium.

[Report on the influence of some materials on the proliferation of cells.]. / Vliv nekterých implantacních materiálu na proliferaci bunek.

The authors have examined the biocompatibility of four materials used for the production of implants with the proliferation of cells. This applies mainly to Ti-6 AI-4 V, Ti- 5AI-2,5 Fe, AKV-Ultra 2 alloys and also to the carbon filament connected by polyamide. In their study the authors used diploid clone -human embryonal fibroblasts LEP and heteroploid lines - Vero cells. It has been found out that neither of the materials tested is of a toxic nature, however, under certain conditions there occurs the reduction of proliferation abilities of cells most probably due to the influence of agents which have passed to the cultivation medium. Key words: fibroblasts, DNA, implant materials: (C+polyamide, steel AKV-Ultra 2, Ti-6 AI-4 V, Ti-5 AI-2,5 Fe).

[The Influence of the Implanted Material (Glass Carbon) for the Proliferation of the Cell's.]. / Vliv implantacního materiálu (skelný uhlík) na proliferaci bunek.

The authors have examined the influence of the implanted material (glass carbon) for the proliferation of human embryonal fibroblasts. It has been found that the tested material did not inhibit the cell's proliferation and it performed a good biocompatibility. Key words: fibroblasts, implanted material, glass carbon.

The influence of some non-steroidal anti-inflammatory drugs on the retraction of collagen lattices.

The effects of some antirheumatics on the formation and retraction of collagen lattices seeded with fibroblasts have been studied. Among the antirheumatics, diclofenac was the most active inhibitor of lattice retraction, then tropesin and to a lesser extent indomethacin. Ibuprofen which is known as a very slight inhibitor of protein synthesis was able to significantly enhance lattice retraction when 10 micrograms/ml (48.5 microM) and 50 micrograms/ml (242 microM) were used.

Comparison of the influence of gelatine and collagen substrates on growth of chondrocytes.

The effect of culture substrates on adhesion and growth of chondrocytes and the influence of varying amounts of foetal calf serum on cell proliferation are compared. Skin and cartilage gelatines and type II collagen were used as substrates. The cells used in the experiments were the primary cultures of chondrocytes, frozen primary cultures of chondrocytes and chondrocytes after the 1st subculture. Cartilage gelatine had a more marked influence on cell proliferation than skin gelatine, especially in primary cultures. The differences were off in cells after the 1st subculture. Differences in the number of cells are evident also after decreasing the serum concentration in culture medium.

Determination of radiolabelled proline and hydroxyproline in collagen hydrolysates by high-performance liquid chromatography with on-line radiometric detection.

Radiolabelled proline and hydroxyproline were separated on a C8 column (10 cm X 4.6 mm I.D.) with 10.4 mM sodium dodecyl sulphate in water-n-propanol (88:12, v/v) (pH 2.6) as the mobile phase at a flow-rate of 0.6 ml/min. The retention times of hydroxyproline and proline were 5 and 8 min, respectively. On-line radiometric detection was performed either in a homogeneous mode (liquid scintillator was added to the column effluent in the ratio 3.33:1) or in a heterogeneous mode (the detection cell was packed with a solid scintillator and 0.1 M ammonia was mixed with the column effluent in the ratio 1:6 in order to prevent adsorption of amino acids on the cell packing). Detection limits were in the range 100-900 dpm for individual isotopes and detection modes and the reproducibilities were better than 10%. The application of the method to a collagen synthesis study is reported.

Effect of tolfenamic acid on the metabolism of the main connective tissue components in rats.

Tolfenamic acid (Clotam) has been used in the therapy of rheumatic diseases for some years. Regarding its chemical structure it belongs to the group of fenamates. The effect of tolfenamic acid on the synthesis of collagen and proteoglycans in granulation tissue, skin or cartilage of rat weanlings was tested and compared with the action of mefenamic acid. According to the results obtained, tolfenamic acid is a potent inhibitor of collagen as well as proteoglycan syntheses. The concentrations of the constituents of proteoglycans, i.e. protein core, link protein as well as glycosaminoglycans were decreased in the tissue after treatment with tolfenamic acid. In comparison with mefenamic acid, if the same doses were used (50 mg and 100 mg/kg of body weight/day in in vivo experiments and 10 mg/g wet tissue in in vitro experiments), tolfenamic acid exhibits more distinct inhibitory effect. A general inhibitory effect of tolfenamic acid on proteosynthesis is suggested.

Effect of glycosaminoglycan polysulphate on the metabolism of cartilage ribonucleic acid.

The effect of glycosaminoglycan polysulphate (GAGPS, Arteparon) on the metabolism of cartilage ribonucleic acid (RNA), collagen, and proteoglycan was studied. GAGPS stimulated the synthesis of collagen and the both components of proteoglycan. The effect of GAGPS on RNA was compared with those of chondroitin sulphate and heparin. All three investigated substances enhanced the RNA synthesis, GAGPS, however, had a comparable effect in concentrations ten times smaller.