|Articles about Biomaterials|
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| Properties of osteoconductive biomaterials: calcium phosphates
LeGeros, R. Z. (2002), Clin Orthop Relat Res(395): 81-98.
Abstract: Bone is formed by a series of complex events involving the mineralization of extracellular matrix proteins rigidly orchestrated by cells with specific functions of maintaining the integrity of the bone. Bone, similar to other calcified tissues, is an intimate composite of the organic (collagen and noncollagenous proteins) and inorganic or mineral phases. The bone mineral idealized as calcium hydroxyapatite, Ca10 (PO4)(6)(OH)2, is a carbonatehydroxyapatite, approximated by the formula: (Ca,X)(10)(PO4,HPO4,CO3)(6)(OH,Y)2, where X are cations (magnesium, sodium, strontium ions) that can substitute for the calcium ions, and Y are anions (chloride or fluoride ions) that can substitute for the hydroxyl group. The current author presents a brief review of CaP biomaterials that now are used as grafts for bone repair, augmentation, or substitution. Commercially-available CaP biomaterials differ in origin (natural or synthetic), composition (hydroxyapatite, beta-tricalcium phosphate, and biphasic CaP), or physical forms (particulates, blocks, cements, coatings on metal implants, composites with polymers), and in physicochemical properties. CaP biomaterials have outstanding properties: similarity in composition to bone mineral; bioactivity (ability to form bone apatitelike material or carbonate hydroxyapatite on their surfaces), ability to promote cellular function and expression leading to formation of a uniquely strong bone-CaP biomaterial interface; and osteoconductivity (ability to provide the appropriate scaffold or template for bone formation). In addition, CaP biomaterials with appropriate three-dimensional geometry are able to bind and concentrate endogenous bone morphogenetic proteins in circulation, and may become osteoinductive (capable of osteogenesis), and can be effective carriers of bone cell seeds. Therefore, CaP biomaterials potentially are useful in tissue engineering for regeneration of hard tissues.
| Properties of titanium-silver alloys for dental application
Oh, K. T., H. M. Shim, et al. (2005), J Biomed Mater Res B Appl Biomater 74(1): 649-58.
Abstract: The purpose of this study was to develop titanium-silver alloys with biocompatibility, high corrosion resistance, and low ion-release rate, and to evaluate the electrochemical properties of titanium-silver alloys in artificial saliva. Titanium-silver alloys with silver contents ranging from 0 to 4.5 at % in steps of 0.5 at % were designed. The alloys were arc melted, homogenized at 950 degrees C for 72 h, hot rolled to 2 mm in thickness, and finally solution heat treated at 950 degrees C for 1 h and quenched in water. Chemical compositions, phases, hardnesses, electrochemical properties, and the cytotoxicity of the alloys were investigated. The purity of titanium-silver alloys was maintained above 99.9%, because few impurities were introduced through their manufacture. In the case of alloys containing silver in the range 2.0-4.0 at %, the formation of an acicular alpha phase was observed inside the beta phase. The acicular phase got thinner with increasing amounts of silver. This means that silver is a beta-phase stabilizing element in titanium-silver alloys. The hardness value tended to rise with increasing silver content and increased largely over 3.5 at %, and the increase of the hardness value versus pure titanium was about 33%. It is believed that the substantial increases in hardness was due to the effects of solid solution strengthening and of alpha-beta phase transition. Moreover, titanium-silver alloys had higher corrosion resistances than pure titanium. These results mean that silver additions to titanium can improve alloy corrosion resistance. Passive current densities in the potentiodynamic polarization curves were dependent on the chemical compositions of the titanium-silver alloys. However, they did not show a linear relationship with respect to silver contents. Titanium-silver alloys did not show pitting corrosion in artificial saliva. It is believed that silver addition to titanium strengthened the passive film due to titanium dissolution induced by the different electromotive forces of titanium and silver. In the agar overlay test, the cytotoxicity of the titanium-silver alloys and of titanium were none or mild. In summary, titanium-silver alloys had higher mechanical properties and corrosion resistance than titanium, and toxicities that were similar to titanium. Therefore, it is recommended that titanium-silver alloys be adopted cautiously by the biomedical and dental fields.
| Property influence of polyanilines on photovoltaic behaviors of dye-sensitized solar cells
Tan, S., J. Zhai, et al. (2004), Langmuir 20(7): 2934-7.
Abstract: The influence of polyanilines (PANIs) as hole conductors on the photovoltaic behaviors of dye-sensitized solar cells is studied. The current-voltage (I-V) characteristics and the incident photon to current conversion efficiency (IPCE) curves of the devices are determined as the function of different conductivities and morphologies of PANIs. The results show that the conductivity of PANIs affects the performance of the devices greatly, and PANI with the intermediate conductivity value (3.5 S/cm) is optimum. In addition, the effects of both the film formation property and the cluster size of polyanilines on the photovoltaic behaviors of the devices are also discussed.
| Prospective assessment of the use of enamel matrix proteins with minimally invasive surgery
Harrel, S. K., T. G. Wilson, et al. (2005), J Periodontol 76(3): 380-4.
Abstract: BACKGROUND: Minimally invasive surgery (MIS) is a surgical technique using very small incisions, indicated for performing regenerative therapy in periodontal defects. Enamel matrix proteins (EMP) have been shown to enhance periodontal regeneration. This study was undertaken to determine the effect of using EMP in combination with an MIS approach. METHODS: Patients from two private periodontal practices with chronic periodontitis who, following non-surgical therapy, had one or more sites with probing depths (PD) of > or =6 mm were included in the study. An MIS approach was utilized for surgical access. Following surgical debridement, EMP was placed into the bony defect. The surgical sites were reevaluated after at least 11 months. RESULTS: Surgical treatment was performed at 160 sites in 16 patients. No significant differences were noted in the results between the two offices and the data were combined. Mean PD reduction (P = 0.002) and attachment level improvements (P = 0.012) were significantly greater than 3 mm with mean post-surgical PD of 3.17 mm and attachment levels of 4.05 mm, based on subject means. Mean change in recession following surgery was negligible (0.01 mm). All sites were considered to be clinically successful. CONCLUSION: The combination of MIS and EMP yields significant reductions in probing depths and improvements in attachment levels while producing little or no increase in recession.
| Prospective native coronary artery stenosis treated with sirolimus-eluting stent (ONASSIS) registry--acute results and mid-term outcomes: a single-center experience
Voudris, V., E. Alexopoulos, et al. (2005), J Invasive Cardiol 17(8): 401-5.
Abstract: BACKGROUND: Encouraging results with the use of the sirolimus-eluting stent (SES) have been recently presented in several multi-center trials. In the present study, the short- and mid-term clinical outcomes of the SES in everyday clinical practice of interventional cardiology were compared with a strategy using conventional bare metal stents. METHODS: In a total of 530 consecutive patients (males 86%, mean age 61 +/- 10 years) who had been treated with a SES were compared with a control group of 398 patients (males 87%, mean age 59 +/- 11 years) treated with a bare metal stent before the use of SES. In-hospital results and clinical outcomes during follow-up (11.22 +/- 3.4 versus 11.41 +/- 3.1 months) were obtained. RESULTS: Patients treated with SES had more risk factors for coronary artery disease or multivessel disease compared to those with treated bare metal stent. The clinical success rate was 99.6% in the SES group and 98.5% in the bare metal stent group (p = ns), and non-Q-wave myocardial infarction (MI) occurred in 5.7% and 4.0% of patients, respectively. The incidence of death or MI during follow-up was not different (1.1% versus 1.3% and 0.8% versus 1.8%; p = ns). Percutaneous or surgical revascularization for target lesion restenosis was required in 2.1% of patients treated with SES and in 10.1% of those with bare metal stents (p < 0.001), and the event-free survival from death, cerebrovascular accident, MI or any percutaneous or surgical revascularization was 93.13% and 83.63%, respectively (p < 0.01). CONCLUSIONS: The implantation of the SES is associated with excellent in-hospital and mid-term results, mainly because of dramatic reductions in the need for repeat revascularization, despite a higher risk factor profile and more complex lesion characteristics.
| Prospective randomized comparison of early and late results of a carbonized stent versus a high-grade stainless steel stent of identical design: the PREVENT Trial [corrected]
Sick, P. B., O. Brosteanu, et al. (2005), Am Heart J 149(4): 681-8.
Abstract: BACKGROUND: Restenosis after coronary interventions with stent implantation is still the main obstacle of interventional cardiology. The aim of this study was to compare a carbonized and high-grade stainless steel stent of identical design with regard to early and late adverse events. METHODS: In this prospective randomized trial the carbonized MAC stent (amg GmbH, Raesfeld-Erle, Germany) was compared with the stainless steel MAC stent of identical design. Primary end point was diameter stenosis at follow-up; secondary end points were angiographic parameters, rate of restenosis, and major cardiac adverse events (MACE; myocardial infarction, reintervention, and death). RESULTS: Between August 1999 and June 2002, 396 patients were randomized in 2 centers of Germany. Diameter stenosis at follow-up (38.6% +/- 23.4% vs 39.1% +/- 22.2%, P =.49) as primary end point, relative late lumen loss (26.8% +/- 23.7% vs 27.7% +/- 22.3%, P =.26), absolute late lumen loss (0.92 +/- 0.71 vs 0.92 +/- 0.66 mm, P =.58), net gain (1.4 +/- 0.8 vs 1.4 +/- 0.8 mm, P =.96), as well as restenosis rates (18.0% vs 19.0%, P =.81) and MACE (13.5% vs 12.2%, P =.71) were not significantly different between the carbonized and the pure stainless steel study arm, respectively. CONCLUSION: The hypothesis of superiority of the carbonized stent over a stainless steel stent of identical design with regard to restenosis and MACE could not be proved. Inactive coating of stents seems to have no advantage over pure stainless steel stents, which was also demonstrated in other trials. The future probably lies in active coating of stents with drugs that reduce the neointimal proliferation process.
| Prospects for application of biotechnology-derived biomaterials
Hellman, K. B., G. L. Picciolo, et al. (1994), J Cell Biochem 56(2): 210-24.
| Prospects of plasma immersion ion implantation and deposition (PIlI & D) in the biomaterials field in Malaysia and south East Asia
Mohamed, M. H. (2004), Med J Malaysia 59 Suppl B: 19-20.
Abstract: One of the emerging technologies in the area of plasma processing is plasma immersion ion implantation (PSII). The paper addresses the merits offered by the PSII technique especially in the area of biomaterial processing. Worldwide development status as well as the drive towards commercial applications is elaborated in an attempt to draw the attention to the importance of the process for Malaysia as well as south East Asia.
| Prostaglandin E2 modulates monocyte MHC-II (Ia) suppression in biomaterial infection
Henke, P. K., T. M. Bergamini, et al. (1997), J Surg Res 69(2): 372-8.
Abstract: Staphylococcus epidermidis biomaterial infection is associated with local cellular immune suppression measured by a depressed monocyte (M phi) Ia expression. The purpose of this study was to define the effect of proinflammatory mediators on Ia expression and bacterial clearance in experimental biomaterial infection. A 1-cm-long Dacron tube graft, sterile or colonized with Staphylococcus epidermidis (1 x 10(7) cfu/ml2), was implanted in Swiss-Webster mice. Perigraft fluid was collected at 7, 10, 14, and 28 days and assayed by enzyme-linked immunoassays for tumor necrosis factor alpha (TNF alpha), interleukin (IL)-I alpha, IL-4, IL-10, and prostaglandin E2 (PGE2). Grafts were sonicated and plated for quantitative growth. In vivo effector inhibitions was accomplished with anti-TNF alpha, anti-IL-1 alpha antibodies (7 micrograms/24 hr), or indomethacin (50 micrograms/24 hr) via an Alzet 7-day microinfusion pump. M phi Ia expression was determined by flow cytometry. A significant elevation of TNF alpha, IL-1 alpha, and PGE2 was found during the first 10 days in the infected compared with sterile (P < or = 0.05) grafts and correlated with maximal Ia suppression. Neither IL-4 nor IL-10 was significantly different in the sterile or infected perigraft fluid. Indomethacin completely prevented M phi Ia suppression, while anti-IL-1 alpha only partially (94%) prevented M phi Ia suppression with a corresponding decrease in PGE2 production in infected grafts. Anti-TNF alpha increased PGE2 production by 189% and was associated with depressed M phi Ia expression. Indomethacin treatment improved mean graft-adherent bacterial clearance by 54% at 7 days and 75% at 28 days compared with control (not significant). Interleukin-1 alpha but not TNF alpha increases PGE2 production which modulates M phi Ia suppression. To improve treatment of biomaterial infections, local immunomodulation of PGE2 and IL-1 alpha is promising.
| Prostaglandin EP4 receptor agonist augments fixation of hydroxyapatite-coated implants in a rat model of osteoporosis
Hayashi, K., A. Fotovati, et al. (2005), J Bone Joint Surg Br 87(8): 1150-6.
Abstract: The reduced stability of hydroxyapatite (HA)-coated implants in osteopenic conditions is considered to be a major problem. We therefore developed a model of a boosted cementless implantation in osteopenic rats.Twelve-week-old rats were either ovariectomised (OVX) or sham-operated (SO), and after 24 weeks plain or HA-coated implants were inserted. They were treated with either a prostaglandin EP4 receptor agonist (ONO-4819) or saline for one month.The EP4 agonist considerably improved the osteoporosis in the OVX group. Ultrastructural analysis and mechanical testing showed an improvement in the implant-bone attachment in the HA-coated implants, which was further enhanced by the EP4 agonist. Although the stability of the HA-coated implants in the saline-treated OVX rats was less than in the SO normal rats, the administration of the EP4 agonist significantly compensated for this shortage. Our results showed that the osteogenic effect of the EP4 agonist augmented the osteoconductivity of HA and significantly improved the stability of the implant-bone attachment in the osteoporotic rat model.
| Prosthodontic biomaterials and adverse reactions: a critical review of the clinical and research literature
Lygre, H. (2002), Acta Odontol Scand 60(1): 1-9.
Abstract: Prosthodontic biomaterials include impression materials, luting cements, and restorative materials. They consist of metals and alloys ceramics, and polymer materials and are retained in patients for <60 min or for decades. Oral release of compounds from biomaterials occurs, and adverse reactions may follow dental treatment. Especially in allergically vulnerable patients contact allergy may occur. There are reports from many different countries on contact allergy from gold/palladium alloys, components from polymer-based materials, chromium/cobalt alloys, and nickel. Notifications on adverse reactions in Norway, Sweden, and England are handled by a registry in which patient reactions and occupational exposure are recorded. Data from The Adverse Reaction Unit in Bergen and Umea have been a most valuable basis in extending knowledge in a field of current interest in dentistry. A review of the clinical and research literature relating to prosthodontic biomaterials and adverse reactions shows that reliable methods seem necessary to expose the frequency of adverse reactions in general dentistry, including prosthodontic treatment.
| Protease-activated quantum dot probes
Chang, E., J. S. Miller, et al. (2005), Biochem Biophys Res Commun 334(4): 1317-21.
Abstract: We have developed a novel nanoparticulate luminescent probe with inherent signal amplification upon interaction with a targeted proteolytic enzyme. This construct may be useful for imaging in cancer detection and diagnosis. In this system, quantum dots (QDs) are bound to gold nanoparticles (AuNPs) via a proteolytically degradable peptide sequence to non-radiatively suppress luminescence. A 71% reduction in luminescence was achieved with conjugation of AuNPs to QDs. Release of AuNPs by peptide cleavage restores radiative QD photoluminescence. Initial studies observed a 52% rise in luminescence over 47 h of exposure to 0.2 mg/mL collagenase. These probes can be customized for targeted degradation simply by changing the sequence of the peptide linker.
| Protein absorption and ellipsometry in biomaterial research
Elwing, H. (1998), Biomaterials 19(4-5): 397-406.
Abstract: Ellipsometry is an optical surface-sensitive method for the investigation of various aspects of protein adsorption mainly at reflecting metal surfaces and ceramic surfaces. One interesting feature of the method is the possibility of detailed and accurate determination of real-time adsorption kinetics of proteins without labelling of the protein. It is also possible to detect protein adsorption with the use of antibodies that adsorb onto the antigen-coated surfaces and to detect antibodies by their adsorption behaviour with regard to antigen-coated surfaces. Compared to other solid phase methods such as enzyme linked immunosorbent assay (ELISA), immunofluorescence and radioimmunoassay, ellipsometry has the advantage of not involving any labelling of the reactant and it is a relatively inexpensive method to maintain. This review is a current report of 15 years of contributions in biomaterials and biochemical research. Special consideration has been given to biologically related surface phenomena such as protein conformation changes, protein displacement effects, early events in blood clotting and complement activation. Among the technical achievements given prominence are the wettability gradient method and the rational use of silicon as an experimental surface. It is clear that ellipsometry and related methods such as reflectometry and surface plasmon resonance (Biacore) are now being increasingly used in biomaterial research as well as in other areas of research.
| Protein adsorption and osteoblast precursor cell attachment to hydroxyapatite of different crystallinities
Yang, Y., D. Dennison, et al. (2005), Int J Oral Maxillofac Implants 20(2): 187-92.
Abstract: PURPOSE: The effect of hydroxyapatite (HA) crystallinity on protein adsorption and osteoblast precursor cell attachment to HA was investigated. MATERIALS AND METHODS: Different weight ratios of 100% crystalline HA and 100% amorphous calcium phosphate powders were mixed and pressed into disks (0.5 g) of different crystallinities--either 0% (HAO), 30% (HA30), 50% (HA50), 70% (HA70), or 100% (HA100). RESULTS: X-ray diffraction indicated differences in HA crystallinities. In addition, dissolution of the HA was dependent on its crystallinity, with an increase in phosphorus dissolution as the degree of crystallinity was decreased. No significant difference in albumin adsorption and initial osteoblast precursor cell attachment was observed in the range of HA0 to HA70 surfaces. However, a significantly lower albumin adsorption and initial osteoblast precursor cell attachment were observed on HA100. DISCUSSION: It was suggested that changes in ionic interactions as a result of a change in crystallinity affect the amount of calcium ion ligands readily available to electrostatically bind to proteins. CONCLUSION: It was thus concluded from this study that HA crystallinity affects the amount of albumin adsorbed and initial osteoblast attachment.
| Protein adsorption in vitro onto biomaterial surfaces covered with ULTI carbon
Borovetz, H. S., G. E. Molek, et al. (1982), Biomater Med Devices Artif Organs 10(3): 187-203.
Abstract: A study is presented which describes the adsorption in vitro of albumin and fibrinogen onto a mini-shunt oxygenation unit. The unit is characterized by flow through 10 etched microchannel conduits with a microporous membrane and conduits are pretreated with a uniform layer of ULTI carbon and evaluated for the steady-state and time varying kinetics of protein adsorption. Corresponding results for oxygenator components not pretreated with ULTI carbon are also included.
| Protein adsorption on 18-alkyl chains immobilized on hydroxyl-terminated self-assembled monolayers
Goncalves, I. C., M. C. Martins, et al. (2005), Biomaterials 26(18): 3891-99.
Abstract: Surfaces of devices that contact blood accumulate adsorbed and denatured proteins perhaps triggering activation of the coagulation system. A renewable layer of albumin would biologically "passivate" the surface and prevent thrombus formation. Based on the approach of selectively binding albumin to fatty acids, different percentages of a compound with 18 carbons (C18) were immobilized on OH-terminated self-assembled monolayers (SAMs). Fourier transform infrared reflection absorption spectroscopy (IRAS), ellipsometry, contact angle (and surface free energy) and X-ray photoelectron spectroscopy (XPS) measurements were used to characterize these surfaces and proved that there is an efficient immobilization of C18. There is an increase of the thickness and hydrophobicity of SAMs with an increasing percentage of C18. Adsorption of human serum albumin (HSA) was evaluated using radiolabelled (125)I-HSA and IRAS. This study showed a gradual increase of HSA adsorption with the increase of surface hydrophobicity. Regarding competitive binding and exchangeability of albumin towards fibrinogen, it was proved, by radiolabelling, that SAMs prepared from solutions with 2.5% C18 presented considerable adsorption in a selective and reversible way.
| Protein array consisting of sol-gel bioactive platform for detection of E. coli O157:H7
Lee, W., K. S. Park, et al. (2005), Biosens Bioelectron 20(11): 2292-9.
Abstract: Sol-gel-derived bioactive platform was fabricated for detection of pathogenic microbes, E. coli O157:H7. Design flexibility of sol-gel technique and ease of fabrication can fulfill to create the surfaces with structural and chemical features that are compatible with biomaterials such as antibody, enzymes, etc. In this study, the bioactive platform was prepared based on the silica gels, which were produced by hydrolyzing tetraethylorthosilane (TEOS) in ethanol. The mercaptopropyl triethoxysilane (MPTS) was mixed with the TEOS solution for the surface functionalization of bioactive platform. During TEOS hydrolysis, the modified thin film was prepared by sol-gel dip coating. Antibody against E. coli O157:H7 was immobilized with a configuration of protein array using piezo-type dispensing system. Surface morphology of the prepared bioactive platform was analyzed using atomic force microscopy (AFM). The antibody-antigen interaction was investigated with fluorescence microscopy and sandwich type immunoassay using fluorescein isothiocyanate (FITC)-labeled antibody. The results showed that antibody was sequestered within the sol-gel-derived bio-gel due to physical adsorption. The measurement of E. coli O157:H7 was done using the fabricated antibody surface. The fluorescence intensity was proportional to the concentration of E. coli O157:H7, of which the detection limit was 10(2)CFU/ml.
| Protein array method for assessing in vitro biomaterial-induced cytokine expression
Li, Y., R. J. Schutte, et al. (2005), Biomaterials 26(10): 1081-5.
Abstract: This study demonstrates the feasibility of a cytokine-based in vitro test for biomaterials. The combination of monocyte culture and protein array technology tested in this study permitted the detection of subtle changes in cytokine expression following an exposure to titanium (Ti) particles. However, a broader range of materials and sample configurations must be examined before these promising results can be translated into a reliable and predictive in vitro biomaterials testing protocol. Modified glass slides were robotically printed with eight identical arrays consisting of capture antibodies against four mouse cytokines [IL-6, TNF-alpha, MIP-2, TGF-beta1] and two positive and two negative detection controls. RAW 264.7 mouse monocytes seeded into six-well plates at 10(5)cells/well were treated with either sterilized Ti particles (test biomaterial), or lipopolysaccharide (LPS; positive control), or untreated (negative control). Aliquots (80 microl) of culture medium collected at 1, 6, 24, 48, and 72 h were applied to the protein arrays for simultaneous sandwich fluoroimmunoassay, followed by imaging the fluorescent intensities on a conventional microarray scanner. LPS induced the release of all four cytokines between 1 and 6h treatment periods, whereas Ti induction of cytokines showed a gradual and subtle increase in cytokine expression for >24 h. Among the four cytokines assayed, TNF-alpha and MIP-2 were most prominently expressed, while IL-6 was slightly elevated and TGF-beta1 was undetected above background.
| Protein binding properties of surface-modified porous polyethylene membranes
Greene, G., H. Radhakrishna, et al. (2005), Biomaterials 26(30): 5972-82.
Abstract: In this study, we quantified the adsorption of immunoglobulin G (IgG) protein onto several polyelectrolyte-modified sintered porous polyethylene (PPE) membranes. The polymer surfaces had both cationic and anionic charges obtained via the adsorption of polyethylenimine (PEI) and polyacrylic acid (PAA), respectively, onto plasma-activated PPE. The amount of IgG adsorption was determined by measuring the gamma radiation emitted by [125I]-IgG radio labeled protein. By studying the impact of pH and ionic strength on IgG adsorption, we attempted to characterize the role and nature of the electrostatic interactions involved in the adsorption process to better understand how these interactions were influenced by the charge and structure of immobilized polyelectrolyte complexes at modified membrane surfaces. We were able to show that surface modification of PPE membranes with adsorbed PEI monolayers and PEI-PAA bilayers can greatly improve the IgG binding ability of the membrane under optimized conditions. We also showed that the observed improvement in the IgG binding is derived from electrostatic interactions between IgG and the polyelectrolyte surface. In addition, we found that the greatest IgG adsorption occurred when the IgG and the surface possessed predominantly opposite charges, rather than when the surface possessed the greatest electrostatic charge. Finally, we have found that the molecular weight of the terminating polyelectrolyte has a noticeable effect upon the electrostatic interactions between IgG and the PEI-PAA bilayer-modified PPE surfaces.
| Protein crystallization in hydrogel beads
Willaert, R., I. Zegers, et al. (2005), Acta Crystallogr D Biol Crystallogr 61(Pt 9): 1280-8.
Abstract: The use of hydrogel beads for the crystallization of proteins is explored in this contribution. The dynamic behaviour of the internal precipitant, protein concentration and relative supersaturation in a gel bead upon submerging the bead in a precipitant solution is characterized theoretically using a transient diffusion model. Agarose and calcium alginate beads have been used for the crystallization of a low-molecular-weight (14.4 kDa, hen egg-white lysozyme) and a high-molecular-weight (636.0 kDa, alcohol oxidase) protein. Entrapment of the protein in the agarose-gel matrix was accomplished using two methods. In the first method, a protein solution is mixed with the agarose sol solution. Gel beads are produced by immersing drops of the protein-agarose sol mixture in a cold paraffin solution. In the second method (which was used to produce calcium alginate and agarose beads), empty gel beads are first produced and subsequently filled with protein by diffusion from a bulk solution into the bead. This latter method has the advantage that a supplementary purification step is introduced (for protein aggregates and large impurities) owing to the diffusion process in the gel matrix. Increasing the precipitant, gel concentration and protein loading resulted in a larger number of crystals of smaller size. Consequently, agarose as well as alginate gels act as nucleation promoters. The supersaturation in a gel bead can be dynamically controlled by changing the precipitant and/or the protein concentration in the bulk solution. Manipulation of the supersaturation allowed the nucleation rate to be varied and led to the production of large crystals which were homogeneously distributed in the gel bead.
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