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Imparting mineral affinity to proteins with thiol-labile disulfide linkages
Bansal, G., J. E. Wright, et al. (2005), J Biomed Mater Res A 74(4): 618-28.
Abstract: Chemical conjugation of bisphosphonates (BPs) to proteins is an effective means to enhance binding of proteins to mineral-containing biomaterials. BPs linked to proteins with reversible (i.e., cleavable) linkages were considered desirable over the conjugates linked with stable linkages because cleavable linkages allow protein release in free form from the mineral-containing biomaterials. To explore the feasibility of creating cleavable BP-protein conjugates, an amine- and a thiol-containing BP were conjugated to the model protein Bovine Serum Albumin (BSA) with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), which resulted in disulfide-linked BP-BSA conjugates. Although disulfide-linked conjugates were stable under aqueous conditions, the conjugates in solution were readily cleaved in the presence of physiological concentrations (approximately 0.3 mM) of the thiol compound, cysteine. The imparted mineral affinity as a result of BP conjugation, as assessed by hydroxyapatite (HA) binding in vitro, was lost upon cleavage of the disulfide-linked BP. The conjugates bound to HA were also cleavable with cysteine, but their cleavage rate was significantly reduced as compared to the conjugates in solution. In conclusion, disulfide-linked BP conjugates were shown to be readily cleavable by the amino acid cysteine and this resulted in the loss of imparted mineral affinity of the proteins. The proposed approach will be useful for modulating in vivo delivery of proteins implanted with mineral-containing biomaterials.

Impedance characterization and modeling of electrodes for biomedical applications
Franks, W., I. Schenker, et al. (2005), IEEE Trans Biomed Eng 52(7): 1295-302.
Abstract: A low electrode-electrolyte impedance interface is critical in the design of electrodes for biomedical applications. To design low-impedance interfaces a complete understanding of the physical processes contributing to the impedance is required. In this work a model describing these physical processes is validated and extended to quantify the effect of organic coatings and incubation time. Electrochemical impedance spectroscopy has been used to electrically characterize the interface for various electrode materials: platinum, platinum black, and titanium nitride; and varying electrode sizes: 1 cm2, and 900 microm2. An equivalent circuit model comprising an interface capacitance, shunted by a charge transfer resistance, in series with the solution resistance has been fitted to the experimental results. Theoretical equations have been used to calculate the interface capacitance impedance and the solution resistance, yielding results that correspond well with the fitted parameter values, thereby confirming the validity of the equations. The effect of incubation time, and two organic cell-adhesion promoting coatings, poly-L-lysine and laminin, on the interface impedance has been quantified using the model. This demonstrates the benefits of using this model in developing better understanding of the physical processes occurring at the interface in more complex, biomedically relevant situations.

Implant infection and infection resistant materials: A mini review
Arciola, C. R., F. I. Alvi, et al. (2005), Int J Artif Organs 28(11): 1119-25.
Abstract: Implant infection is an aggressive, often irreducible post-surgical infection. It remains the primary cause of implant failure. Bacterial contamination during surgery and subsequent adhesion onto biomaterial surface of opportunistic microorganisms, such as staphylococcal species, exopolysaccharidic slimes or specific adhesins, initiates the implant infection. Pathogenesis of periprosthestic infection is the focus of studies aimed at developing infection resistant materials.

Implant migration after early weightbearing in cementless hip replacement
Bottner, F., M. Zawadsky, et al. (2005), Clin Orthop Relat Res(436): 132-7.
Abstract: Twenty-nine patients (five women and 24 men) with an average age of 47 years (range, 24-59 years) had 37 total hip arthroplasties using a hydroxyapatite-coated double-wedge press-fit femoral component. All patients had a Type A bone quality. Patients were either mobilized with weightbearing as tolerated or toe-touch weightbearing for 6 weeks postoperatively. After 6 weeks all patients were advanced to weightbearing as tolerated. Radiostereometric analysis radiographs were taken at 3 days, 6 weeks, and 6 months postoperatively to measure migration of the femoral component. Radiostereometric analysis revealed no difference in stem migration between the two groups as defined by maximal total point migration. There was a difference in the vertical (proximal-distal) migration within the first 6 weeks between groups (0.81 mm versus 0.13 mm), but not afterwards (0.17 mm versus 0.18 mm). Continuous migration after 6 weeks was observed in three patients from each group. There was no loosening in either group within a 2-year followup. Weightbearing as tolerated is recommended for young patients with excellent bone quality after cementless total hip arthroplasty with a double-wedge press-fit femoral component. LEVEL OF EVIDENCE: Diagnostic study, Level I (testing of previously developed diagnostic criteria in series of consecutive patients--with previously applied reference gold standard). See the Guidelines for Authors for a complete description of levels of evidence.

Implant surface roughness influences osteoclast proliferation and differentiation
Marchisio, M., M. Di Carmine, et al. (2005), J Biomed Mater Res B Appl Biomater 75(2): 251-6.
Abstract: The osteoclast is a hematopoietic cell derived from CFU-GM and branches from the monocyte-macrophage lineage during the differentiation process. Biological environment appears to be crucial for osteoclast formation and activity. It has been reported that bone remodeling following implant placement requires a coordinated activity by osteoclasts and osteoblasts. The response of such cells at the bone-implant interface has been suggested to be affected by the structural and morphological features of the biomaterial surface. To shed more light on this topic we performed a multiparametric analysis of murine monocytes response to different titanium surfaces. These cells, RAW 264.7 type TIB-71, represent a very useful system because they differentiate into osteoclasts following treatment of definite doses of the osteoclast-differentiation factor RANKL and macrophage colony-stimulating factor (M-CSF). Cells, cultured on glass (control), on grade 3 machined and on titanium pull-spray superficial-TPSS surfaces disclosed profound different responses in terms of morphological rearrangements, adhesion, and differentiation abilities. Indeed, after 14 days, cells cultured on glass and machined surfaces were uniformly distributed, while, on the TPSS surface cells strictly aggregated into small isolated clusters were observed. In addition, cells cultured on the machined surface displayed a higher adhesion ability, while cells cultured on the rougher surface disclosed a more evident capability to differentiate. These results could explain the higher bone-implant contact percentage found around implants with rougher surfaces and suggest that osteoclasts may play an important role in the initial period after implant placement to prime or prepare the implant surface for the osteoblast activity.

Implantation of preadipocyte-loaded hyaluronic acid-based scaffolds into nude mice to evaluate potential for soft tissue engineering
Hemmrich, K., D. von Heimburg, et al. (2005), Biomaterials 26(34): 7025-37.
Abstract: The reconstruction of soft tissue defects following extensive deep burns or tumor resections remains an unresolved problem in plastic and reconstructive surgery since adequate implant materials are still not available. Preadipocytes, immature precursor cells found between mature adipocytes in adipose tissue, are a potential material for soft tissue engineering since they can proliferate and differentiate into adipose tissue after transplantation. In previous studies, we identified hyaluronan benzyl ester (HYAFF 11) sponges to be promising carrier matrices. This study now evaluates, in vitro and in vivo, a new sponge architecture with pores of 400 microm either made of plain HYAFF 11 or HYAFF 11 coated with the extracellular matrix glycosaminoglycan hyaluronic acid. Human preadipocytes were isolated, seeded onto carriers and implanted into nude athymic mice. Explants harvested after 3, 8, and 12 weeks were examined for macroscopical appearance, thickness, weight, pore structure, histology, and immunohistochemistry. Compared to previous studies, we found better penetration of cells into both types of scaffolds, with more extensive formation of new vessels throughout the construct but with only minor adipose tissue. Our encouraging results contribute towards a better seeded and vascularised scaffold but also show that the enhancement of adipogenic conversion of preadipocytes remains a major task for further in vivo experiments.

Implantation of recombinant human bone morphogenetic proteins with biomaterial carriers: A correlation between protein pharmacokinetics and osteoinduction in the rat ectopic model
Uludag, H., D. D'Augusta, et al. (2000), J Biomed Mater Res 50(2): 227-38.
Abstract: This study was carried out to determine the effect of recombinant human bone morphogenetic protein (rhBMP) pharmacokinetics (PK) on rhBMP-induced osteoinductive activity. It was our working hypothesis that the PK of a rhBMP significantly affects its osteoinductive activity. The PK of various rhBMPs (rhBMP-2, rhBMP-4, rhBMP-6, and chemically modified rhBMP-2) implanted with four biomaterial carries (Helistat, hDBM, Osteograf/N, and Dexon) was determined using (125)I-labeled proteins in the rat ectopic assay. A select combination of rhBMP and carriers then was evaluated in the rat ectopic assay for osteoinductive activity using a semi-quantitative histologic scoring system. The results indicate that initial protein retention is dependent on protein isoelectric point (pI); proteins with a higher pI yielded a higher implant retention. Subsequent PK was not strongly dependent on the pI or on the carrier. Because of the difference in early retention, the rhBMP-carrier combinations exhibited a >100-fold difference in implant-retained protein dose. When rhBMP-2 and rhBMP-4 were implanted with the carriers, more rhBMP-2 was retained in an implant, and the osteoinductive potency of rhBMP-2 typically was higher than rhBMP-4 at low implantation doses. We conclude that protein pI plays a significant role in the local retention of implanted rhBMP and that higher retention yields a higher osteoinductive activity.

Implantation of stimulation electrodes in the subretinal space to demonstrate cortical responses in Yucatan minipig in the course of visual prosthesis development
Sachs, H. G., F. Gekeler, et al. (2005), Eur J Ophthalmol 15(4): 493-9.
Abstract: PURPOSE: During the course of the development of visual prostheses, subretinal stimulation films were implanted in micropigs in order to prove the feasibility of subretinal electrical stimulation with subsequent cortical response. One aim was to demonstrate that epidural recording of visual evoked potentials is possible in the micropig. METHODS: Film-bound stimulation electrode arrays were placed in the subretinal space of micropigs. This enabled the retina to be stimulated subretinally. Since conventional visual evoked potential (VEP) measuring is virtually impossible in the pig from the neurosurgical point of view, epidural recording electrode arrays were positioned over the visual cortex as permanent electrodes. RESULTS: The feasibility of temporary implantation of film-bound stimulation electrode arrays was successfully demonstrated in the micropig model. On stimulation with monopolar voltage pulses (1000 to 3000 mV), reproducible epidural VEP measurements (5 to 10 micronV) were detected. CONCLUSIONS: The feasibility of subretinal stimulation of the retina was demonstrated in a retinal model that is similar to the human retina. This animal model therefore offers a suitable means of studying the tolerability of stimulation situations in the course of visual prosthesis development.

Implants in the posterior maxilla: a comparative clinical and radiologic study
Rodoni, L. R., R. Glauser, et al. (2005), Int J Oral Maxillofac Implants 20(2): 231-7.
Abstract: PURPOSE: The aim of this study was to evaluate implants placed according to several methods of sinus floor augmentation. MATERIALS AND METHODS: Forty-eight patients (median age of 62 years, range 23 to 89) had been treated at least 3 years prior to examination with screw-type implants in the posterior maxilla. Depending on the vertical dimension of the residual bone, 1 of 3 surgical procedures had been performed: sinus lift by lateral antrostomy (SL) in 13 patients; osteotome technique (OT) in 18 patients; standard implantation in 17 patients (control). In each patient 1 implant was randomly chosen for analysis (48 implants with a mean observation time of 4.6 +/- 1.4 years). Examination included probing pocket depth (PPD) measurement and radiographic examination. Radiographs were digitized to assess the marginal bone level. Differences between the groups were tested using analysis of variance, the Student t test and the Kruskal-Wallis test. RESULTS: Mean PPD was 3.0 mm for the SL, 3.1 mm for OT, and 3.1 mm for control. The mean radiographic bone level was 1.53 mm for SL, 2.40 mm for OT, and 1.96 mm for control. No statistically significant differences were found between the groups for either of these parameters. DISCUSSION AND CONCLUSION: Clinical examinations as well as radiographically stable bone levels indicated similar biomechanical conditions for prosthetic restorations when applying the 3 surgical procedures tested.

Implants. Biomaterials and biomechanics
Brunski, J. B. (1988), Cda J 16(1): 66-77.

Importance of adsorption-desorption processes of plasma proteins in biomaterials hemocompatibility
Sevastianov, V. I., E. A. Tseytlina, et al. (1984), Trans Am Soc Artif Intern Organs 30: 137-42.

Importance of microanalysis in understanding mechanism of transformation in active glassy biomaterials
Gatti, A. M., G. Valdre, et al. (1996), J Biomed Mater Res 31(4): 475-80.
Abstract: This work deals with a method to study bioactive glassy materials used for repair of bone defects in order to understand the mechanism of the bioactivity and thus have its in vivo behavior optimized. The study of these materials takes into account their changes in morphology, by means of electron microscopy, after implantation in different animal models and evaluates the mechanism of the bioactivity through semi- and quantitative microanalytical evaluations in order to quantify the phenomenon of glass corrosion and the formation of a chemical bond with bone. Special preparations of glass granules before implantation were prepared to microanalytically study surfaces with different permeabilities.

Importance of phosphorylation for osteopontin regulation of biomineralization
Gericke, A., C. Qin, et al. (2005), Calcif Tissue Int 77(1): 45-54.
Abstract: Previous in vitro and in vivo studies demonstrated that osteopontin (OPN) is an inhibitor of the formation and growth of hydroxyapatite (HA) and other biominerals. The present study tests the hypotheses that the interaction of OPN with HA is determined by the extent of protein phosphorylation and that this interaction regulates the mineralization process. Bone OPN as previously reported inhibited HA formation and HA-seeded growth in a gelatin-gel system. A transglutaminase-linked OPN polymer had similar effects. Recombinant, nonphosphorylated OPN and chemically dephosphorylated OPN, had no effect on HA formation or growth in this system. In contrast, highly phosphorylated milk OPN (mOPN) promoted HA formation. The mOPN stabilized the conversion of amorphous calcium phosphate (a non-crystalline constituent of milk) to HA, whereas bone OPN had a lesser effect on this conversion. Mixtures of OPN and osteocalcin known to form a complex in vitro, unexpectedly promoted HA formation. To test the hypothesis that small alterations in protein conformation caused by phosphorylation account for the differences in the observed ability of OPN to interact with HA, the conformation of bone OPN and mOPN in the presence and absence of crystalline HA was determined by attenuated total reflection (ATR) infrared (IR) spectroscopy. Both proteins exhibited a predominantly random coil structure, which was unaffected by the addition of Ca(2+). Binding to HA did not alter the secondary structure of bone OPN, but induced a small increase of beta-sheet (few percent) in mOPN. These data taken together suggest that the phosphorylation of OPN is an important factor in regulating the OPN-mediated mineralization process.

Impregnation of plasmid DNA into three-dimensional scaffolds and medium perfusion enhance in vitro DNA expression of mesenchymal stem cells
Hosseinkhani, H., Y. Inatsugu, et al. (2005), Tissue Eng 11(9-10): 1459-75.
Abstract: This article describes the development of an in vitro culture system to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSCs) by a combination of plasmid DNA impregnation into three-dimensional cell scaffolds and culture methods. Gelatin was cationized by introducing spermine to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, poly(glycolic acid) (PGA) fiber fabrics, collagen sponges, and collagen sponges reinforced by incorporation of PGA fibers were used. A complex of cationized gelatin and plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) was impregnated into the scaffolds. Plasmid DNA was released from PGA-reinforced collagen sponge for longer than from the other scaffolds. MCS were seeded into each type of scaffold and cultured by static, stirring, and perfusion methods. When MSCs were cultured in PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by perfusion culture compared with the other culture methods, and the time of expression was prolonged. Irrespective of the culture method, the expression level was significantly higher from plasmid DNA impregnated in scaffold than by plasmid DNA in medium. The alkaline phosphatase activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method were significantly higher compared with those of other methods, and a significantly higher amount of plasmid DNA internalized into MSCs was observed. We conclude that a combination of plasmid DNA-impregnated PGA-reinforced sponge and the perfusion method was promising to promote in vitro gene expression for MSCs.

Improved chemical analysis of cellulose ethers using dialkylamine derivatization and mass spectrometry
Momcilovic, D., H. Schagerlof, et al. (2005), Biomacromolecules 6(5): 2793-9.
Abstract: Oligosaccharides of hydroxypropylmethyl cellulose, hydroxypropyl cellulose, and methyl cellulose were investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The cellulose ether oligosaccharides were produced either by enzymatic depolymerization utilizing the purified family 5 endoglucanase from Bacillus agaradhaerens or by partial acidic depolymerization. To lower the limit of detection in MALDI-MS three dilakylamines, dimethyl-, diethyl-, and dipropylamine were studied as reagents for reductive amination of the oligosaccharides. All three amines contributed to a significant increase in sensitivity in MALDI-MS, especially for oligosaccharides with a degree of polymerization (DP) < 3. These reagents were also attractive due to their high volatility, which facilitated the purification of the reaction mixtures. It was established that low-mass discrimination in MALDI-MS in the DP range 1-7 was substantially reduced with dialkylamine derivatization. Hence, dialkylamine derivatization of cellulose ether oligosaccharides obtained by endoglucanase depolymerization increased the number of detected analyte components. Dimethylamine was concluded to be the preferred reagent of those evaluated.

Improved CZE capabilities with new dynamic coatings
Chang, W. W., D. C. Bomberger, et al. (2005), J Capillary Electrophor 9(3-4): 53-6.
Abstract: Electroosmotic flow (EOF) is one of the fundamental processes affecting both resolution and separation times in capillary zone electrophoresis (CZE). EOF is a function of pH and buffer composition (zeta potential) in bare-silica capillaries at any pH above 3 and runs in the cathodal direction, decreasing the effective separation length for cationic species. On the other hand, the absence of EOF at low pH can significantly increase separation times, particularly for low pl zwitterionic species. This interdependence of pH and EOF limits the ability to design effective separations by capillary zone electrophoresis. The EOTrol family of dynamic coatings (Target Discovery, Inc., Palo Alto, CA, U.S.A.) allows both the magnitude and direction of EOF to be adjusted independently of the buffer composition and pH. Here we report the use of EOTrol in two challenging CZE separations: (1) inorganic cations with small mobility differences, and (2) the rapid separation of organic zwitterions that are only resolvable at low pH.

Improved electrochemical analysis of neuropathy target esterase activity by a tyrosinase carbon paste electrode modified by 1-methoxyphenazine methosulfate
Sokolovskaya, L. G., L. V. Sigolaeva, et al. (2005), Biotechnol Lett 27(16): 1211-8.
Abstract: A graphite-paste tyrosinase biosensor was improved by adding 1-methoxyphenazine methosulfate as a mediator. Mediator modification enhanced sensitivity to phenol 4-fold and long-term stability 3-fold. Phenol could be detected at 25 nM (S/N = 2) using an Ag/AgCl reference electrode. The biosensor was used to measure the activity of a toxicologically significant enzyme, neuropathy target esterase (NTE), which yields phenol by hydrolysis of the substrate, phenyl valerate. Using the new biosensor, blood and brain NTE inhibition by organophosphorus (OP) compounds with different neuropathic potencies were well correlated (r = 0.990, n = 7), supporting the use of blood NTE as a biochemical marker of exposure to neuropathic OP compounds.

Improved Epon embedding for biomaterials
Blaauw, E. H., J. A. Oosterbaan, et al. (1989), Biomaterials 10(5): 356-8.
Abstract: To obtain improved transmission electron microscopy sections for cell biological and interface evaluation of implanted biomaterials we present an improved embedding procedure. Standard problems in preparation and sectioning, like dissolution of the biomaterial, or holes and chatter in the sections can be prevented by introducing butyl-2,3-epoxypropylether as an intermedium between the dehydration series and the Epon resin. Most biomaterials were not affected by this chemical agent. The introduction of butyl-2,3-epoxypropylether resulted in completely homogeneous Epon blocks which enabled us to cut 50 nm sections, free of holes and chatter. The biomaterials did not dislodge during the process of sectioning and the cell-polymer interface remained intact for electron microscopical evaluation.

Improved performance of primary rat hepatocytes on blended natural polymers
Li, K., X. Qu, et al. (2005), J Biomed Mater Res A 75(2): 268-74.
Abstract: Alginate (Alg), chitosan (Chi), collagen (Col), gelatin (Gel), and the mixtures of every two of them were screened to determine their suitability for hepatocyte culture. The test materials were fabricated as films and then evaluated on the basis of their abilities to promote the attachment and functions of rat hepatocytes cultured on them. Cellular attachment on Col and Gel was favorable. However, cellular viability, cytoskeleton organization and function, as evaluated by albumin production, ureagenesis, and enzyme activity of cytochrome P450 as well as expression levels of hepatocyte nuclear factor 4 alpha deteriorated. Reverse cellular behavior was observed on Alg and Chi. Two blends, composed of Chi and Col or Gel, were found to be superior to other materials and sustained viability and differentiated functions of hepatocyte.

Improved sample preparation of biomaterials for in vitro genotoxicity testing using reference materials
Muller, B. P., S. Ensslen, et al. (2002), J Biomed Mater Res 61(1): 83-90.
Abstract: New biomaterial related reference materials with known genotoxic properties were produced in order to study the sample preparation and in vitro genotoxicity testing of biomaterials. We incorporated genotoxic substances like benzo[a]pyrene into the biomaterial Tecoflex, a polyurethane frequently used for catheters and other applications. We demonstrated that the model compound benzo[a]pyrene is sufficiently extracted by organic solvents, whereas cell culture medium only extracts very limited quantities. By changing the medium several times during extraction the extracted amount was augmented. Using higher amounts of organic solvent in relation to the reference material's surface led to a higher recovery of extracted benzo[a]pyrene. For the in vitro genotoxicity testing using the Mammalian Cell Gene Mutation Test (HPRT test), Mammalian Chromosome Aberration Test, and bacterial umu- and SOS-tests, concentration of extracts is a prerequisite because of the low sensitivity of the test systems. Often cytotoxicity interferes with the evaluation of genotoxic effects. We demonstrated that some recommendations of the ISO 10993-Part 3 and 12,(1),(2) dealing with the biological evaluation of medical devices, seem to be insufficient, and new rules for the in vitro genotoxicity testing of biomaterials have to be established.

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Last Modified: 8 February 2006