Showing papers in "Journal of Biomedical Materials Research in 1996"

Preparation of bioactive Ti and its alloys via simple chemical surface treatment.

Abstract: A simple chemical method was established for inducing bioactivity of Ti and its alloys. When pure Ti, Ti-6A1-4V, Ti-6A1-2Nb-Ta, and Ti-15Mo-5Zr-3A1 substrates were treated with 10M NaOH aqueous solution and subsequently heat-treated at 600 degrees C, a thin sodium titanate layer was formed on their surfaces. Thus, treated substrates formed a dense and uniform bonelike apatite layer on their surfaces in simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma. This indicates that the alkali- and heat-treated metals bond to living bone through the bonelike apatite layer formed on their surfaces in the body. The apatite formation on the surfaces of Ti and its alloys was assumed to be induced by a hydrated titania which was formed by an ion exchange of the alkali ion in the alkali titanate layer and the hydronium ion in SBF. The resultant surface structure changed gradually from the outermost apatite layer to the inner Ti and its alloys through a hydrated titania and titanium oxide layers. This provides not only the strong bonding of the apatite layer to the substrates but also a uniform gradient of stress transfer from bone to the implants. The present chemical surface modification is therefore expected to allow the use the bioactive Ti and its alloys as artificial bones even under load-bearing conditions.

Surface roughness modulates the local production of growth factors and cytokines by osteoblast-like MG-63 cells.

Abstract: Titanium (Ti) surface roughness affects proliferation, differentiation, and matrix production of MG-63 osteoblast-like cells. Cytokines and growth factors produced in the milieu surrounding an implant may also be influenced by its surface, thereby modulating the healing process. This study examined the effect of surface roughness on the production of two factors known to have potent effects on bone, prostaglandin E 2 (PGE 2 ) and transforming growth factor β 1 (TGF-β 1 ). MG-63 cells were cultured on Ti disks of varying roughness. The surfaces were ranked from smoothest to roughest : electropolished (EP), pretreated with hydrofluoric acid-nitric acid (PT), fine sand-blasted, etched with HCl and H 2 SO 4 , and washed (EA), coarse sand-blasted, etched with HCl and H 2 SO 4 , and washed (CA), and Ti plasma-sprayed (TPS). Cells were cultured in 24-well polystyrene (plastic) dishes as controls and to determine when confluence was achieved. Media were collected and cell number determined 24 h postconfluence. PGE 2 and TGF-β 1 levels in the conditioned media were determined using commercial radioimmunoassay and enzyme-linked immunosorbent assay kits, respectively. There was an inverse relationship between cell number and Ti surface roughness. Total PGE 2 content in the media of cultures grown on the three roughest surfaces (FA, CA, and TPS) was significantly increased 1.5-4.0 times over that found in media of cultures grown on plastic or smooth surfaces. When PGE 2 production was expressed per cell number, CA and TPS cultures exhibited six- to eightfold increases compared to cultures on plastic and smooth surfaces. There was a direct relationship between TGF-β 1 production and surface roughness, both in terms of total TGF-β 1 per culture and when normalized for cell number. TGF-β 1 production on rough surfaces (CA and TPS) was three to five times higher than on plastic. These studies indicate that substrate surface roughness affects cytokine and growth factor production by MG-63 cells, suggesting that surface roughness may modulate the activity of cells interacting with an implant, and thereby affect tissue healing and implant success.

Preliminary biological evaluation of polyamidoamine (PAMAM) StarburstTM dendrimers

Abstract: PAMAM Starburst™ dendrimers are spherical macromolecules composed of repeating polyamidoamino units. They can be produced in successive “generations,” each with a defined size, molecular weight, and number of terminal amino groups. Because of these well-defined characteristics, PAMAMs are finding utility in a variety of applications, many of which are biological in nature. Little is known, however, about the biological behavior of the PAMAMs, which is critical to their use in vivo. Generation 3 (G3; MW = 5,147; 24 terminal amines), 5 (G5; MW = 21,563; 96 amines), and 7 (G7; MW = 87,227; 384 amines) PAMAMs were studied in V79 cells or in Swiss-Webster, mice for a number of biological properties, including (1) in vitro toxicity, (2) in vivo toxicity, (3) immunogenicity, and (4) biodistribution. Potential biological complications were observed only with G7 at the highest level tested. No evidence of immunogenicity was seen. The biodistribution properties of the Starburst™ dendrimers were rather unusual. G3 showed the highest accumulation in kidney tissue (∼15% ID/g over 48 h); G5 and G7 appeared to preferentially localize in the pancreas (peak levels ∼32% ID/g at 24 h, and ∼20% ID/g at 2 h, respectively). In addition, G7 showed extremely high urinary excretion, with values of 46 and 74% ID/g at 2 and 4 h, respectively. In general, the dendrimers did not exhibit properties that would preclude their use in biological applications. Depending on the situation (desired endpoint, dose, and generation used), however, the biodistribution of biological preparations should be carefully studied. © 1996 John Wiley & Sons, Inc.

Effect of crystallization on apatite-layer formation of bioactive glass 45S5.

Abstract: The bioactive glass 45S5 was crystallized to 8-100 vol % of crystals by thermal treatments from 550-680 degrees C. The micro-structure of the glass-ceramics had a very uniform crystal size, ranging from 8 to 20 microns. Fourier-transform infrared (FTIR) spectroscopy was used to determine the rate of hydroxycarbonate apatite (HCA) formation that occurs on bioactive glass and glass-ceramic implants when exposed to simulated body fluid (SBF) solutions. Crystallization did not inhibit development of a crystalline HCA layer, but the onset time of crystallization increased from 10 h for the parent glass to 22 h for 100% crystallized glass-ceramic. The rate of surface reactions was slower when the percentage of crystallization was > or = 60%.

Studies on the biocompatibility of materials: fibroblast reorganization of substratum-bound fibronectin on surfaces varying in wettability.

Abstract: The ability of human fibroblasts to remove and reorganize fibronectin (FN) bound on material surfaces was studied as a novel feature of material surface biocompatibility. Other traditional parameters of biocompatibility analyzed included cell spreading, clustering of fibronectin receptors into focal adhesions, development of stress fibers, and cell growth. Five different materials with surface wettability ranging from hydrophilic (underwater contact angle 25 degrees) to hydrophobic (underwater contact angle 111 degrees) were used, i.e., clean glass (GLASS), aminopropylsilane (APS), octadecylsilane (ODS), polylactate (PL), and silicone (SI). When cells were cultured on these materials in serum-containing medium, formation of FN receptor-rich focal adhesions and actin stress fibers were more evident on the hydrophilic surfaces (GLASS and APS) compared to the hydrophobic ones (PL, ODS, and SI). Cell growth showed a similar pattern, that is, increased cell proliferation with increasing material surface wettability. Preadsorption of FN on the material surfaces increased subsequent cell spreading and cytoskeletal reorganization on hydrophobic surfaces except SI. Removal and reorganization of FN from the material surfaces into extracellular matrixlike structures occurred on GLASS but not on less wettable surfaces, suggesting that this removal/reorganization process may be more sensitive to changes in surface wettability than other parameters of biocompatibility.

Preparation of stable aqueous solution of keratins, and physiochemical and biodegradational properties of films.

Abstract: A stable aqueous solution of reduced keratins was prepared by extracting the proteins from wool (Corriedale) with a mixture of urea, mercaptanol, surfactant, and water at 40-60 degrees C. Sodium dodecyl sulfate was especially effective as a surfactant, not only in promoting extraction but also in stabilizing the aqueous protein solution. The proteins had the following constants: MW, 52,000-69,000 daltons; cysteine content, 8-9 mol%; pl about 6.7. A clear film was readily prepared from a keratin solution containing glycerol. The film was insoluble in water and organic solvents including dimethyl sulfoxide. The keratin film was permeable to glucose, urea, and sodium chloride. The keratin film was degraded in vitro (by trypsin) and in vivo (by subcutaneous embedding in mice).

Polylactide Macroporous Biodegradable Implants for Cell Transplantation. Ii. Preparation of Polylactide Foams by Liquid-Liquid Phase Separation

Abstract: Potential of thermally induced phase separation as a porogen technique has been studied in an effort to produce a surgical implant suitable for cell transplantation. Emphasis has been placed on the liquid-liquid phase separation of solutions of amorphous poly DL-lactide and semicrystalline poly L-lactide in an 87/13 dioxane/water mixture. The related temperature/composition phase diagrams have been set up by turbidimetry, and the possible occurrence of a gel has been discussed. Freeze-drying of some phase-separated polylactide solutions can produce flexible and tough foams with an isotropic morphology. Interconnected pores of 1-10 microns in diameter are expected to result from the spinodal decomposition of the polylactide solutions with formation of co-continuous phases. Thermodynamics of the polymer/solvent pair has a decisive effect on the final macroporous foams, as shown by the dependence of their porosity, density, porous morphology, and mechanical behavior on molecular weight and crystallinity of polylactide and concentration of the original solutions. On the basis of the foam characteristics, potential of the liquid-liquid phase separation (spinodal decomposition) has been compared with the solid/liquid phase separation (solvent crystallization) as a porogen technique.

Collagen-based devices for soft tissue repair

Abstract: The intrinsic biological and physiochemical characteristics of collagen have been exploited to prepare a variety of commercially available medical products spanning many medical specialties. The properties of purified collagen can be modified to obtain forms which comply to specific applications. In particular, collagen materials in the form of gels, matrices, and films have been applied, either alone or in combination with other agents, to address soft tissue repair. This review describes how the versatile properties of collagen have made it one of the most useful biomaterials for wound care applications.

Torque and histomorphometric evaluation of c.p. titanium screws blasted with 25‐ and 75‐μm‐sized particles of Al2O3

Abstract: A comparison was made between screw-shaped c.p. titanium implants blasted with either 25- or 75-microns particles of Al2O3. The implant surfaces were investigated with respect to topography and composition before implantation in rabbit bone. Grit blasting with 25- or 75-microns particles produced two different surface roughnesses, but no significant difference in the surface composition for the two surfaces. After 12 weeks insertion time in the rabbit tibia and femur, a higher removal torque and more bone-to-metal contact was found for the implants blasted with 75-microns particles compared with the 25-microns-blasted ones.

Immediate bone forming capability of prefabricated osteogenic hydroxyapatite

Abstract: We employed culture technology, which provides bone tissue in vitro, to expand and promote the osteogenic ability of marrow cells in porous hydroxyapatite (HA). Marrow cells were obtained from rat femur and cultured in a standard medium for 10 days, then trypsinized to make composites of HA and the cells. An additional 2-week culture (subculture) was done for the composite in a standard medium with or without the addition of dexamethasone (Dex). The 2-week subcultured composites were implanted into subcutaneous sites of syngeneic rats. These implants were harvested and prepared for the biochemical analysis of alkaline phosphatase activity and bone Gla protein content, as well as histological analysis of decalcified and undecalcified sections. In Dex-treated composites, high alkaline phosphatase activity could be detected 1 week after implantation and was maintained until 8 weeks after implantation. The bone Gla protein content could also be detected 1 week after implantation, followed by a steady increase with the passage of time until 8 weeks after implantation. The histological analysis showed active bone formation even 1 week after implantation. The bone formation was evidenced by active osteoblast lining and the appearance of calcein labeling following calcein injection 1 week after implantation. Thus, Dex-treated subcultured marrow cells in pore regions of HA showed a high osteogenic response immediately after transplantation. In contrast, Dex-untreated composite did not show bone formation and contained traces of these biochemical parameters. These results indicate that the inherent osteogenic ability of marrow stromal stem cells in pore regions of HA can be stimulated using tissue culture technology; and thus, formed osteogenic HA can show immediate osteoblastic activity in in vivo situations, suggesting the applicability of the HA in clinical situations.

Controlled release of antibiotics from coated orthopedic implants.

Abstract: Chronic osteomyelitis is one of the most serious complications of orthopedic open fracture treatment. The objective of this study was to develop a biodegradable implant coating with impregnated antibiotics as an adjunct to current therapy. We used a polylactic-co-glycolic acid copolymer (PLGA) as the biodegradable carrier and gentamicin as the antibiotic. Our objectives were to establish elution characteristics of the antibiotic from the polymer, and determine if the coated orthopedic implants would inhibit bacterial growth in vitro. In the elution study, coated implants were incubated in phosphate buffered saline (PBS) at 37 degrees C and sampled daily for gentamicin levels. The in vitro model consisted of test tubes containing Mueller-Hinton culture broth inoculated with 5 x 10(6) cfu of Staphylococcus aureus and incubated at 37 degrees C. The implants were switched to a new set of inoculated tubes each day. Tubes were sampled for colony counting to determine bactericidal effects. Implant coatings consisted of 40 mg of gentamicin as a 20% mixture with PLGA. The elution curve showed an average level of 138 micrograms/mL over 15 days. This local concentration would be more than adequate to kill susceptible organisms. The in vitro study showed a significant reduction in bacterial growth in the test tubes containing coated implants. Control tubes averaged 2.5 x 10(8) cfu/mL of S.aureus over 24 days. Coated implant tubes averaged 0.9 cfu/mL. This was a reduction of greater than 99.999% (p < 0.0001). This study showed that a thin biodegradable implant coating can be developed with bactericidal activity against the organisms frequently associated with osteomyelitis in cases of open fractures.

In vitro bone formation by rat marrow cell culture.

Abstract: Fresh marrow cells were obtained from the femora Fischer rats and cultured in a medium containing 15% fetal calf serum (FCS) to leach confluent. After trypsinization, cells were subcultured at a cell density of 100 × 103/35 mm well in the presence of FCS, 10 mM β-glycerophosphate, 82 μg/mL ascorbic acid phosphate, and 10-8M dexamethasone (Dex). Osteoblastic cells and microscopic mineralized nodules began to appear at about 1 week after the subculture, and at 2 weeks many macroscopic nodules that showed high alkaline phosphatase activity (ALP) and appearance of bone Gla protein (BGP) mRNA were evident. As demonstrated by in situ hybridization, the mRNA was manifested by cuboid-shaped cells (osteoblastic cells). X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR) showed the mineralization of fine crystals of hydroxyapatite comparable to natural rat bone mineral. In contrast to these findings, subculture done under the same conditions except for the lack of Dex did not show mineralized nodules, nor did they show the osteoblastic phenotype expression. These analyses indicate that Dex-induced mineralization using rat bone marrow cell culture is an in vitro counterpart of bone formed in vivo. Such a culture is useful for investigating materials/osteogenic cells interactions. © 1996 John Wiley & Sons, Inc.

Resorption of, and bone formation from, new β‐tricalcium phosphate‐monocalcium phosphate cements: An in vivo study

Abstract: Hard cylinders (4.7 x 10 mm) of two kinds of beta-tricalcium phosphate-monocalcium phosphate monohydrate-calcium sulfate hemihydrate (beta-TCP-MCPM-CSH) cements with and without beta-TCP granules (500-1000 microns) were implanted into holes drilled in rabbit femoral condyles for up to 16 weeks. Empty cavities were used as control. Cement resorption and new bone formation in the cylinders were evaluated with contact microradiography and quantified through an automatic image analysis system. At 4 weeks, both kinds of cement cylinders were surrounded by new bone. At 8 weeks, except for beta-TCP granules, both cement cylinders were almost completely resorbed and replaced by bone tissue. At 16 weeks the bone in the cavities of both cements recovered a trabecular pattern, but only the bone trabeculae in the initial cavity of the cement with beta-TCP granules became thick and mature. However, the cavities of the empty control were still empty and large. These results show that the beta-TCP-MCPM-CSH cements stimulate bone formation and are rapidly replaced by bone tissue. When added with nonresorbable beta-TCP granules, this cement maintains bone formation for a longer time.

Resorbable polyesters in cartilage engineering: Affinity and biocompatibility of polymer fiber structures to chondrocytes

Abstract: The resorbable polymers polyglycolic acid (PGA) and polylactic acid (PLA) are gaining increasing importance in tissue engineering and cell transplantation. The present investigation was focused on the biocompatibility and cell retaining behavior of PGA/poly-L-lactide (PLLA) (90/10) and PLLA nonwoven structures for the in vitro development of chondrocyte-polymer constructs. The effect of the relevant monomers to chondrocytes was analyzed. Type II collagen and poly-L-lysine were compared to improve loading of PGA/PLLA and PLLA polymer nonwovens with chondrocytes. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) test was applied for quantifications. At concentrations above 2 mg/mL, glycolic acid was more cytotoxic than lactic acid. As shown by pH equilibration, the cytotoxic effect is not due merely to the acidity of the α-hydroxy acids. Regarding the degradation products, glycolic acid, and L(+) lactic acid, nonwovens of PLLA are more biocompatible with chondrocytes than nonwovens of polyglycolide. Collagen type II and poly-L-lysine generally improved cell seeding on resorbable polymers in tissue engineering; however, their efficiency varies depending on the type of fiber structure. © 1996 John Wiley & Sons, Inc.

Effect of titanium surface roughness on chondrocyte proliferation, matrix production, and differentiation depends on the state of cell maturation

Abstract: Although it is well accepted that implant success is dependent on various surface properties, little is known about the effect of surface roughness on cell metabolism or differentiation, or whether the effects vary with the maturational state of the cells interacting with the implant. In the current study, we examined the effect of titanium (Ti) surface roughness on chondrocyte proliferation, differentiation, and matrix synthesis using cells derived from known stages of endochondral development. Chondrocytes derived from the resting zone (RCs) and growth zone (GCs) of rat costochondral cartilage were cultured on Ti disks that were prepared as follows: HF-HNO3-treated and washed (PT); PT-treated and electropolished (EP); fine sand-blasted, HCl-H2SO4-etched, and washed (FA); coarse sand-blasted, HCl-H2SO4-etched, and washed (CA); or Ti plasma-sprayed (TPS). Based on surface analysis, the Ti surfaces were ranked from smoothest to roughest: EP, PT, FA, CA, and TPS. Cell proliferation was assessed by cell number and [3H]-thymidine incorporation, and RNA synthesis was assessed by [3H]-uridine incorporation. Differentiation was determined by alkaline phosphatase specific activity (AL-Pase). Matrix production was measured by [3H]-proline incorporation into collagenase-digestible (CDP) and noncollagenase-digestible (NCP) protein and by [35S]-sulfate incorporation into proteoglycan. GCs required two trypsinizations for complete removal from the culture disks; the number of cells released by the first trypsinization was generally decreased with increasing surface roughness while that released by the second trypsinization was increased. In RC cultures, cell number was similarly decreased on the rougher surfaces; only minimal numbers of RCs were released by a second trypsinization. [3H]-thymidine incorporation by RCs decreased with increasing surface roughness while that by GCs was increased. [3H]-Uridine incorporation by both GCs and RCs was greater on rough surfaces. Conversely, ALPase in the cell layer and isolated cells of both cell types was significantly decreased. GC CDP and NCP production was significantly decreased on rough surfaces while CDP production by RC cells was significantly decreased on smooth surfaces. [35S]-sulfate incorporation by RCs and GCs was decreased on all surfaces compared to tissue culture plastic. The results of this study indicate that surface roughness affects chondrocyte proliferation, differentiation, and matrix synthesis, and that this regulation is cell maturation dependent.

Effect of physical crosslinking methods on collagen-fiber durability in proteolytic solutions.

Abstract: We previously demonstrated that ultraviolet (UV) or dehydrothermal (DHT) crosslinking partially denatured fibers extruded from an insoluble type I collagen dispersion. In this study denaturation effects were evaluated by measuring collagen-fiber sensitivity to trypsin. Shrinkage-temperature measurements and sensitivity to collagenase served as indices of crosslinking. UV or DHT crosslinking increased the collagen-fiber shrinkage temperature, resistance to degradation in collagenase, and durability under load in collagenase. However, in trypsin solutions, solubility was significantly increased for UV (approximately 11%) or DHT (approximately 15%) crosslinked fibers compared with uncrosslinked fibers (approximately 4%). Size-exclusion chromatography indicated that no intact collagen alpha-chains were present in the soluble fraction of fibers exposed to trypsin (MW < 1 kD). Interestingly, UV-crosslinked collagen fibers remained intact an order of magnitude longer (4840 +/- 739 min) than DHT-crosslinked (473 +/- 39 min) or uncrosslinked (108 +/- 53 min) fibers when placed under load in trypsin solutions. These data indicate that mechanical loading during incubation in a trypsin solution measures denaturation effects not detected by the trypsin-solubility assay. Our results suggest that DHT-crosslinked collagen fibers should not be used as load-bearing implants. UV-crosslinked fibers may retain more native structure and should exhibit greater resistance to nonspecific proteases in vivo.

A highly porous 3‐dimensional polyphosphazene polymer matrix for skeletal tissue regeneration

Abstract: Current methods for the replacement of skeletal tissue in general involve the use of autografts or allografts. There are considerable drawbacks in the use of either of these tissues. In an effort to provide an alternative to traditional graft materials, a degradable 3-dimensional (3-D) osteoblast cell–polymer matrix was designed as a construct for skeletal tissue regeneration. A degradable amino acid containing polymer, poly[(methylphenoxy)(ethyl glycinato) phosphazene], was synthesized and a 3-D matrix system was prepared using a salt leaching technique. This 3-D polyphosphazene polymer matrix system, 3-D-PHOS, was then seeded with osteoblast cells for the creation of a cell–polymer matrix material. The 3-D-PHOS matrix possessed an average pore diameter of 165 μm. Environmental scanning electron microscopy revealed a reconnecting porous network throughout the polymer with an even distribution of pores over the surface of the matrix. Osteoblast cells were found attached and grew on the 3-D-PHOS at a steady rate throughout the 21-day period studied in vitro, in contrast to osteoblast growth kinetics on similar, but 2-D polyphosphazene matrices, that showed a decline in cell growth after 7 days. Characterization of 3-D-PHOS osteoblast-polymer matrices by light microscopy revealed cells growing within the pores as well as on surface of the polymer as early as day 1. This novel porous 3-D-PHOS matrix may be suitable for use as a bioerodible scaffold for regeneration of skeletal tissue. © 1996 John Wiley & Sons, Inc.

Poly(L-lactic acid) foams with cell seeding and controlled-release capacity.

Abstract: A synthetic porous three-dimensional structure that can mimic the architecture of actual tissues, provide sustained release of nutrients or growth factors, and serve as a template for cell seeding would be an ideal substrate for tissue engineering. Poly(l-lactic acid) (PLLA) foams were fabricated for this purpose, based on the principle of phase separation from homogeneous naphthalene solutions. Complex shapes could be readily fabricated, and resulting foams had relatively uniform, open cells throughout the matrix. Densities and total pore-surface areas were in the range of 0.05-0.1 g/cm3 and 0.8-1.3 m2/g, respectively. The loss tangent of these foams ranged from 0.07 to 0.128, as measured by thermomechanical analysis. Naphthalene residue in the resulting foams went below 0.2 wt% after extensive vacuum sublimation. Feasibility of incorporating drugs or nutrients into such a highly porous structure was demonstrated by the dispersion of two model compounds, bromothymol blue (BTB) and sulforhodamine B (SD), in the matrix. Sustained release of BTB from the foam with a porosity as high as 87% was observed for more than 2 months. Alkaline phosphatase, as a model protein to be incorporated, lost approximately 30% of its bioactivity during the fabrication. As a cell-culture substrate, the PLLA foams performed as well as the flat PLLA surface in supporting the growth of rat osteosarcoma cells (ROS 17/2.8) and in maintaining their functions such as alkaline phosphatase activity and osteocalcin synthesis. UMR-106 cells cultured in the foam also expressed a higher degree of mineralization than those cultured on the flat PLLA substrate.

Effect of size, concentration, surface area, and volume of polymethylmethacrylate particles on human macrophages in vitro.

Abstract: This study investigated effects of different sizes, concentrations, volumes, and surface areas of polymethylmethacrylate (PMMA) particles on human macrophages. Adherent peripheral blood monocytes isolated from five healthy individuals were exposed for 48 h to phagocytosable (0.325 micron and 5.5 microns) and nonphagocytosable (200 microns) spherical particles. Each particle size was tested over a range of concentrations (10(4)-10(11) particles per milliliter [0.325 micron], 10(2)-10(7) particles per milliliter [5.5 microns], 10(1)-10(4) particles per milliliter [200 microns]) to provide overlap in number, volume, and surface area. Primary human monocyte/macrophages were cultured in macrophage serum-free medium and 5% fetal calf serum. Macrophage viability was assessed by 3H-thymidine uptake and activation was quantified by release of interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, prostaglandin E2 (PGE2), and the lysosomal enzyme hexosaminidase. Medium alone served as a negative control; lipopolysaccharide (10 micrograms/mL) was also tested. PMMA particles were not toxic to human macrophages at any concentration tested. The smallest phagocytosable particles (0.325 micron) stimulated the release of interleukin-1 beta, interleukin-6, prostaglandin E2, and hexosaminidase at concentrations of 10(10)-10(11) particles/mL. The release of cytokines, PGE2, and hexosaminidase depended on the size, concentration, surface area, and volume of the phagocytosable particles. This study demonstrates that PMMA particle load Mi.e., the concentration of phagocytosable particles per tissue volume, characterized by size, surface area, and volume, rather than simply particle number-determines the degree of macrophage activation.

Macroporous biphasic calcium phosphate ceramics: Influence of five synthesis parameters on compressive strength

Abstract: Compressive strength measurements were conducted on 32 macroporous biphasic calcium phosphate (MBCP) samples to evaluate the influences and interactions of five synthesis factors: chemical composition, percentage of macropores, mean size of macropores, isostatic compaction pressure, and sintering temperature. These parameters were varied simultaneously between two limit levels. Experiments used a factorial design method (FDM) allowing optimization of the number of samples as well as statistical analysis of results. FDM showed that compressive strength, in a defined experimental area, can be described by a first-order polynomial equation in which the percentage of macroporosity and sintering temperature are the major influences. This study leads up to an isoresponse line diagram that will allow the manufacture of some classes of MBCP with fitted compressive strength. © 1996 John Wiley & Sons, Inc.

Characterization of silane-treated hydroxyapatite powders for use as filler in biodegradable composites.

Abstract: Hydroxyapatite (HA) powder was treated with different silane adhesion promoters, to optimize its performance as a filler in polymer composites. The silane coupling agents investigated possessed vinyl, methacryloxy, primary amine, secondary amine, and diamine functionality. The different coatings were evaluated with respect to their influence on ionic exchange. X-ray photoelectron spectroscopy revealed the presence of a few monolayers thin silane films on HA powder. Silane coupling agents were able to bond chemically on the HA surface because a thin coating remained after washing of the powder with water. The water stability of this bond was evaluated by successive extractions and was judged limited, especially in the case of the hydrophilic aminosilanes. Zeta-potential measurements indicated the "transparency" of the coatings for ionic transport, that was corroborated by two in vitro dissolution studies, in Gomori's Tris-maleate buffer, and in simulated body fluid. However, aminosilane coatings could delay the release of calcium and phosphate ions during the first 2 days of immersion of treated HA powder in Gomori's buffer.

Hydrogen peroxide toward enhanced oxide growth on titanium in PBS solution: blue coloration and clinical relevance.

Abstract: Oxide films formed on titanium exposed to a phosphate-buffered saline solution with and without hydrogen peroxide (H2O2) addition were investigated by means of electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS). The oxide growth at the titanium/electrolyte interface was monitored in situ by daily EIS measurements during periods of several weeks. The results suggest that the oxide film can be described by a two-layer model with a barrier inner layer and a porous outer layer. H2O2 addition results in an increased dissolution/oxidation rate that leads to an enhanced oxide growth of the porous outer layer. As a result, the total oxide film can reach a thickness corresponding to an interference blue color. Based on XPS results, H2O2 addition furthermore seems to facilitate the incorporation of phosphate ions into the thicker porous layer. This observation may be related to the so-called osseointegration properties of titanium. © 1996 John Wiley & Sons, Inc.

Enhanced cell attachment to anorganic bone mineral in the presence of a synthetic peptide related to collagen.

Abstract: We have examined the ability of a synthetic 15-residue peptide, P-15, related to a biologically active domain of type I collagen, to promote attachment of human dermal fibroblasts to anorganic bovine bone mineral (ABM) phase. The attachment of cells increased with increasing content of P-15 on the surface of ABM particles, as seen by the increased binding of radiolabeled cells, and by light microscopy and scanning electron microscopy. Incorporation of radioactive precursors of DNA and protein synthesis showed that cells on P-15-coated ABM synthesized over twofold the amount of DNA and protein than did cells on uncoated ABM. Fibroblasts attached to ABM in the presence of P-15 formed three-dimensional colonies. Cellular bridges formed between adjacent particles which aggregated in clusters with tissue-like structure. Cultures on ABM.P-15 stained for alkaline phosphatase. These observations suggest that P-15-coated ABM may be a useful matrix for bone repair.

Osteogenic differentiation of cultured marrow stromal stem cells on the surface of bioactive glass ceramics

Abstract: To investigate the significance of apatite-wollastonite-containing glass ceramic (AW ceramic) surfaces and the biological apatite layer formed on these surfaces, rat marrow cell culture, which shows osteogenic differentiation, was carried out on four different culture substrata (control culture dish, two AW ceramics, each having a different surface roughness, and a ceramic on which an apatite layer was formed. A culture period of 2 weeks in the presence of β-glycerophosphate, ascorbic acid, and dexamethasone resulted in abundant mineralized nodule formations that were positive for alkaline phosphatase (ALP) stain on all substrata. The stain on the apatite-formed AW ceramic was the most intense, the enzyme activity being about twice that of the control culture dish, which had the lowest stain and activity of the four substrata. Northern blot analysis of bone Gla protein (BGP) showed the same tendency, that is, the amount of BGP mRNA from cultured cells on the apatite-formed AW ceramics was the highest and the mRNA on the control dish was the lowest. These data indicate that the glass ceramic surface promotes osteoblastic differentiation and that the promotion can be further enhanced by the formation of a biological apatite layer on the ceramic surface. © 1996 John Wiley & Sons, Inc.

Characterization of electrolytically prepared brushite and hydroxyapatite coatings on orthopedic alloys

Abstract: Details of a procedure for electrolytically preparing highly pure brushite (CaHPO4.2H2O) coatings on high surface area metal substrates are provided. The influence of current density and deposition time on coating morphology is described. After a discussion of procedures used to convert the brushite coatings to hydroxyapatite, results from a preliminary animal study that demonstrate the propensity of these coatings to stimulate bony ingrowth into porous plugs are presented.

Comparison and quantitation of wear debris of failed total hip and total knee arthroplasty.

Abstract: To compare the physical properties of debris particles associated with failed total hip and total knee arthroplasty, we applied a recently developed assay to electronically characterize the size, number, and composition of debris particles isolated from tissues adjacent to failed implants. We identified 21 samples (from 20 patients) of hip synovia and 35 samples (from 32 patients) of knee tissues that had been obtained at the time of revision arthroplasty. There were 12 females and 9 males in the hip group, and 16 females and 19 males in the knee group. Primary arthroplasty was performed for osteoarthritis (OA, 15 cases) or rheumatoid arthritis (RA, 6 cases) in the hip, and for OA (23) or RA (12) in the knee. Patients ranged in age from 23 to 85 (mean 59 years) for total hip, and from 27 to 84 (mean 61 years) for total knee arthroplasty. Implantation duration was from 5 to 123 months (mean 37.8) for total hip, and from 11 to 123 months (mean 63.1) for total knee arthroplasty. All of the implants were composed of cobalt-chromium alloy articulating with ultrahigh-molecular-weight polyethylene. The number of particles smaller than 10 microns ranged from 1.04 x 10(8)/g to 1.91 x 10(10)/g in the hip, and from 6.69 x 10(8)/g to 2.13 x 10(10)/g in the knee. Energy-dispersive X-ray spectroscopy and polarized light analysis showed both polyethylene and metal particles in most cases. The mean diameter of particles smaller than 10 microns was 0.72 +/- 0.2 microns in the hip, and 0.74 +/- 0.1 microns in the knee. Evaluation of particles larger than 10 microns showed a larger range of particle size in knee tissues (maximum 6.1 mm, mean 283 microns), than in the hip tissues (maximum 826 microns, mean 81 microns) (p < 0.001). Very small particles are common in both groups, but it appears that a larger range of particle sizes is present adjacent to failed knee than to failed hip prostheses. The higher frequency of large particles in failed knee prostheses probably reflects the perceived higher rate of delamination and fragmentation of tibial and patellar compared to that of acetabular polyethylene.

Kinetic study of the setting reaction of a calcium phosphate bone cement

Abstract: The setting reaction of a calcium phosphate bone cement consisting of a mixture of 63.2 wt % alpha-tertiary calcium phosphate (TCP)[alpha-Ca3(PO4)2], 27.7 wt % dicalcium phosphate (DCP) (CaHPO4), and 9.1 wt % of precipitated hydroxyapatite [(PHA) used as seed material] was investigated. The cement samples were prepared at a liquid-to-powder ratio of: L/P = 0.30 ml/g. Bi-distilled water was used as liquid solution. After mixing the powder and liquid, some samples were molded and aged in Ringer's solution at 37 degrees C. At fixed time intervals they were unmolded and then immediately frozen in liquid nitrogen at a temperature of TN = -196 degrees C, lyofilized, and examined by X-ray diffraction as powder samples. The compressive strength versus time was also measured in setting samples of this calcium phosphate bone cement. The crystal entanglement morphology was examined by scanning electron microscopy. The results showed that: 1) alpha-TCP reacted to a calcium-deficient hydroxyapatite (CDHA), Ca9(HPO4)(PO4)5O H, whereas DCP did not react significantly; 2) the reaction was nearly finished within 32 h, during which both the reaction percentage and the compressive strength increased versus time, with a strong correlation between them; and 3) the calcium phosphate bone cement showed in general a structure of groups of interconnected large plates distributed among agglomerations of small crystal plates arranged in very dense packings.

Biocompatibility testing of NiTi screws using immunohistochemistry on sections containing metallic implants.

Abstract: NiTi is one of the most innovative concepts to have appeared in the field of metallic biomaterials in recent years but its biocompatibility remains controversial. We evaluated the biocompatibility of Nitinol screws using immunohistochemistry to observe the distribution of bone proteins during bone remodeling process around NiTi implant. Results were compared with screws made of Vitallium, c.p. titanium, Duplex austenitic-ferritic stainless steel (SAF), and Stainless Steel 316L. Screws were implanted in rabbit tibia for 3, 6, and 12 weeks. Embedding was performed in the hard resin Technovit, and for the immunohistochemical procedure undecalcified sections with bone-anchored implants could thus be used. The immunostaining method developed seemed to be a reliable technique to stain proteins in undecalcified sections. Biocompatibility results of the NiTi screws compared with the other screws showed a slower osteogenesis process characterized by no close contact between implant and bone, disorganized migration of osteoblasts around the implant, and a lower activity of osteonectin synthesis.

Bone-bonding behavior of plasma-sprayed coatings of BioglassR, AW-glass ceramic, and tricalcium phosphate on titanium alloy.

Abstract: The bone-bonding behavior of three kinds of bioactive ceramics coated on titanium alloy by the plasma-spray technique was investigated. Titanium alloy (Ti-6A1-4V) coated with BioglassR (45S5), apatite-wollastonite containing glass ceramic (AW), or β-tricalcium phosphate (TCP) was prepared, and rectangular specimens were implanted into the tibial bones of mature male rabbits, which were sacrificed 8 or 24 weeks after implantation. The tibiae containing the implants were dissected out and subjected to detachment tests to measure the failure load. The bone-implant interface was investigated by Giemsa surface staining, contact microradiography, and scanning electron microscopy-electron probe microanalysis (SEM-EPMA). Eight weeks after implantation, the failure loads for implants coated with BioglassR, AW, and TCP were 1.04 ± 0.94, 2.03 ± 1.17, and 3.91 ± 1.51 kg, respectively, and 24 weeks after implantation, the respective failure loads were 2.72 ± 1.33, 2.39 ± 1.30, and 4.23 ± 1.34 kg. Failure loads of AW- and TCP-coated implants did not increase significantly with time. After the detachment test, breakage of the coating layer was observed. Bioactive ceramics can act as stimulants that induce bonding between bone and metal implants. However, failure load of metal implants coated with the bioactive ceramics was lower than that of bulk AW or TCP. It appears impossible to obtain a higher failure load using a bioactive-ceramic coating on titanium alloy. Histologically, the coating layer was found to become detached from the metal implant and the bone tissue bonded to the coating layer. SEM-EPMA observation revealed breakage of the coating layer, although bonding between bone and the coating layer was evident. A Ca-P-rich layer was observed at the interface between bone and the AW coating, and a Ca-P-rich and a Si-rich layer were observed at the interface between bone and the BioglassR coating. For clinical application, it would seem better to use coated metal implants for short-term implantation. However, there is a possibility of breakage of the coating layer because of both dissolution of the bioactive ceramic and mechanical weakness at the interface between the coating layer and the metal implant. © 1996 John Wiley & Sons, Inc.

Hydrolytic degradation of films prepared from blends of high and low molecular weight poly(DL‐lactic acid)s

Abstract: Biodegradable films were prepared by casting acetone solutions of mixtures of a high molecular weight poly(DL-lactic acid) (HMW-PLA50) with 0, 10, and 30% w/w poly(DL-lactic acid) oligomers (LMW-PLA50), before drying. From size exclusion chromatography (SEC) it was shown that degradation occurred during film processing, in agreement with the acid-catalyzed degradation of polyesters. The higher the content of LMW-PLA50, the larger the decrease of the molar masses. The three selected film formulations were then allowed to age in isoosmolar 0.13M, pH 7.4 sodium phosphate buffer at 37 degrees C. The hydrolytic degradation was monitored by using various techniques, namely weighing to quantify water absorption and weight loss, SEC to evaluate molar mass changes, head space gas chromatography to assess the desorption of residual acetone, and enzymatic assay of the L-lactic acid released in the aging media. The presence of LMW-PLA50 clearly accelerated film degradation. Moreover, it was shown that the mechanism of degradation greatly depended on the content in the oligomers.