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Cell-based therapies and tissue engineering
Rice, M. A., B. T. Dodson, et al. (2005), Otolaryngol Clin North Am 38(2): 199-214, v.
Abstract: Tissue engineering is a rapidly evolving discipline that may some-day afford surgeons a limitless supply of autologous tissue for transplantation or allow in situ tissue regeneration. A number of biologic, engineering, and clinical challenges continue to face tissue engineers and surgeons alike. One important example is the choice of an appropriate cell scaffold that promotes growth and is eventually resorbed by the body. Although the application of bioengineered tissue is specific to the anatomic areas of interest,continued advances bring tissue engineering closer to reality in all areas of otolaryngology.

Cell-cycle control in cell-biomaterial interactions: expression of p53 and Ki67 in human umbilical vein endothelial cells in direct contact and extract testing of biomaterials
van Kooten, T. G., C. L. Klein, et al. (2000), J Biomed Mater Res 52(1): 199-209.
Abstract: Current biocompatibility testing involves the demonstration of cell proliferation, which is usually interpreted as a sign of positive biocompatibility when the materials sustain cell proliferation. As the field of biomaterials research is rapidly moving toward tissue-engineered devices and hybrid organs, control of cell function has become a main topic. Cell function, which involves specific differentiation pathways, cannot be separated from cell-cycle control. The study of cell-cycle control is an important extension of routine proliferation assays and has extensive roots in developmental and tumor biology. We studied the expression of the tumour suppressor gene p53 and the proliferation-associated antigen Ki67 of endothelial cells in response to biomaterial contact. Cells were seeded in six- or 24-well plates, in which one or three 12-mm-diameter biomaterial disks were laid down. After 48- and 72-h incubation periods, cells were processed for flow cytometry, immunofluorescence, or Western blotting. The following materials were used: titanium, NiCr alloy, and CoCr alloy. Cells were also exposed to 24-h (ISO-norm) extracts in 25-cm(2) culture flasks (600, 000 cells) for 24 and 48 h. For extract testing, serially diluted Ni-ion suspensions were also used. Human umbilical vein endothelial cells adhered to metal surfaces and started forming a monolayer within 3 days. Ki67 expression was positive in more than 60% after 2 days and decreased markedly after 3 days of adhesion. During this time cells developed focal contacts and produced a fibronectin matrix. p53 expression could be demonstrated with Western blotting and flow cytometry, but not with immunofluorescence. Differences due to both culturing time and material were found in expression patterns with both methods. Inverse correlations between Ki67 and p53 expression were detected, which are probably based on culture kinetics. The results indicate that expression of p53 and also Ki67 is clearly influenced by biomaterials in direct contact testing, despite the absence of obvious morphological differences. The p53 marker can be used for defining cell function in more detail, although the correlation with specific physiological function has still to be clarified.

Cell-ingrowth in a silicone plombe. Interactions between biomaterial and scleral tissue after 8 years in situ: a SEM and TEM investigation
Kalicharan, D., W. L. Jongebloed, et al. (1991), Doc Ophthalmol 78(3-4): 307-15.
Abstract: A male patients (42 years) who had been treated for retinal detachment by the implantation of a silicone plombe into the sclera, returned to the clinic 8 years after implantation because of inflammation of the tissue and partial protrusion from the sclera. After removal of the plombe this was processed for TEM and SEM and examination of the plombe material after 8 years in situ could be carried out in order to get information about the cell-biomaterial interface. A large proportion of the silicone pores was filled with cellular material, including macrophages, giant cells and erythrocytes thus indicating a foreign body granuloma. The external surface of the pores showed a granular osmiophilic dense amorphous layer including extracellular debris. Engulfing of silicone particles by macrophages and the evidence of long-term tissue response suggest partial biodegradation of the silicone and certainly not complete inertness as was formerly claimed.

Cellular and molecular events during chondrogenesis of human mesenchymal stromal cells grown in a three-dimensional hyaluronan based scaffold
Lisignoli, G., S. Cristino, et al. (2005), Biomaterials 26(28): 5677-86.
Abstract: Mesenchymal stromal cells (MSCs) seem to be a good alternative to chondrocytes for cartilage regeneration. To obtain new information on the sequence of cellular and molecular events during in vitro chondrogenic differentiation we analysed MSCs on a widely used hyaluronic acid biomaterial (Hyaff-11). Cellular differentiation was induced using two different concentrations of TGFbeta1 (10 and 20 ng/ml) and the process was analysed at different time points (24 h, and 7, 14, 21 and 28 days) using techniques of light and electron microscopy, real-time PCR and immunohistochemistry. We found that without TGFbeta MSCs did not survive while in the presence of TGFbeta the cells significantly proliferated from day 7 until day 28. Light and electron microscopy showed that TGFbeta at 20 ng/ml better induced the formation of cartilage-like tissue. Real-time PCR showed an increased expression of collagen type II, IX and aggrecan associated to a down-regulation of collagen type I. Immunohistochemical analysis confirmed that collagen type I was down-modulated while collagen type II increased from day 14 to day 28. These data clearly showed that higher concentrations of TGFbeta (20 ng/ml) induce chondrogenesis of MSCs on Hyaff-11 scaffold better than 10 ng/ml of TGFbeta. This process is characterized by a sequence of cellular and molecular events that deal with the in vitro formation of a cartilage-like tissue.

Cellular cross-linking of peptide modified hydrogels
Drury, J. L., T. Boontheekul, et al. (2005), J Biomech Eng 127(2): 220-8.
Abstract: Peptide modification of hydrogel-forming materials is being widely explored as a means to regulate the phenotype of cells immobilized within the gels. Alternatively, we hypothesized that the adhesive interactions between cells and peptides coupled to the gel-forming materials would also enhance the overall mechanical properties of the gels. To test this hypothesis, alginate polymers were modified with RGDSP-containing peptides and the resultant polymer was used to encapsulate C2C12 myoblasts. The mechanical properties of these gels were then assessed as a function of both peptide and cell density using compression and tensile tests. Overall, it was found that above a critical peptide and cell density, encapsulated myoblasts were able to provide additional mechanical integrity to hydrogels composed of peptide-modified alginate. This occurred presumably by means of cell-peptide cross-linking of the alginate polymers, in addition to the usual Ca++ cross-linking. These results are potentially applicable to other polymer systems and important for a range of tissue engineering applications.

Cellular interactions with biomaterials: in vivo cracking of pre-stressed Pellethane 2363-80A
Zhao, Q., M. P. Agger, et al. (1990), J Biomed Mater Res 24(5): 621-37.
Abstract: The phenomenon of stress cracking of Pellethane 2363-80A (PEU) was investigated using the cage implant system. A cytotoxic polyvinylchloride (PVC) and a silicone rubber containing an anti-inflammatory steroid were used to create inflammatory environments in which the biostability of the pre-stressed PEU was tested. These coimplants provided alternative in vivo environments to study in vivo polymer interactions. The inflammatory responses to the implanted cages were monitored by analyzing the exudates aspirated from the cages at different implantation times over 21 days. The pre-stressed PEU specimens were retrieved after 5, 10, and 15 weeks postimplantation and examined by optical microscopy (OM) and scanning electron microscopy (SEM). The results support the conclusion that in vivo cracking of stressed (strained) Pellethane 80A is related to cell-polymer interactions. Severe cracking or rupture of the implanted PEU specimens was observed as early as 5 weeks postimplantation. Molecular chain degradation of the implanted specimens was evident from molecular weight measurements. Neither surface cracking nor degradation of macromolecules was found on the pre-stressed PEU specimens with the added cytotoxic PVC implanted over 15 weeks. No cracking was observed on the pre-stressed specimens in the presence of steroid silicone rubber, even after 10 weeks implantation.

Cellular response to gelatin- and fibronectin-coated multilayer polyelectrolyte nanofilms
Li, M., D. K. Mills, et al. (2005), IEEE Trans Nanobioscience 4(2): 170-9.
Abstract: Surface engineering is a critical effort in defining substrates for cell culture and tissue engineering. In this context, multilayer self-assembly is an attractive method for creating novel composites with specialized chemical and physical properties that is currently drawing attention for potential application in this area. In this work, effects of thickness, surface roughness, and surface material of multilayer polymer nanofilms on the growth of rat aortic smooth muscle cells were studied. Polyelectrolyte multilayers (PEMs) electrostatically constructed from poly(allylamine hydrochloride) and poly(sodium 4-styrenesulfonate) (PSS) with gelatin, fibronectin, and PSS surface coatings were evaluated for interactions with smooth muscle cells (SMCs) in an in vitro environment. The results prove that PEMs terminated with cell-adhesive proteins promote the attachment and further growth of SMCs, and that this property is dependent upon the number of layers in the underlying multilayer film architecture. Cell roundness and number of pseudopodia were also influenced by the number of layers in the nanofilms. These findings are significant in that they demonstrate that both surface coatings and underlying architecture of nanofilms affect the morphology and growth of SMCs, which means additional degrees of freedom are available for design of biomaterials. This work supports the excellent potential of nanoassembled ultrathin films for biosurface engineering, and points to a novel perspective for controlling cell-material interaction that can lead to an elegant system for defining the extracellular in vitro environment.

Cellular response to zinc-containing organoapatite: an in vitro study of proliferation, alkaline phosphatase activity and biomineralization
Storrie, H. and S. I. Stupp (2005), Biomaterials 26(27): 5492-9.
Abstract: We present a series of experiments investigating the in vitro biological activity of zinc-containing organoapatite (ZnOA)-coated titanium meshes. ZnOA is a hydroxyapatite-based material that contains poly(l-lysine) and zinc ions and can be coated onto titanium by treating the metal surface with poly(amino acids) that allow for electrostatic bonding of the mineral to the titanium surface. Preosteoblastic mouse calyaria cells were cultured on ZnOA-coated titanium meshes in a three-dimensional (3D) bioreactor, which provides an in vitro culture environment that better simulates what cells experience in vivo, compared to traditional 2D cultures. Results of these studies show a time-dependent cascade of events leading to an earlier onset of alkaline phosphatase (ALP) expression and biomineralization of ZnOA-coated samples as compared to controls. After the observation of peak ALP levels in ZnOA-coated titanium samples, mineralized bone nodules were observed by scanning electron microscopy. Tetracycline staining confirmed that the observed mineral nodules were newly synthesized biomineral, and not due to the inorganic coating. ZnOA-coated titanium substrates represent a new class of materials for human repair that provide, mechanical stability, as well as chemical and biochemical signals to promote new bone growth.

Cellular responses to chemical and morphologic aspects of biomaterial surfaces. I. A novel in vitro model system
Chesmel, K. D. and J. Black (1995), J Biomed Mater Res 29(9): 1089-99.
Abstract: The clinical success of any implant is directly dependent upon the cellular behavior in the immediate vicinity of the interface established between the host tissue and the biomaterial(s) used to fabricate the device. All biomaterials have morphologic, chemical, and electrical surface characteristics that influence the cellular response to the implant. Quantitative measurement of specific aspects of this local host response to different but well-characterized biomaterial surfaces provides a crucial link in the understanding of the overall phenomenon of implant biocompatibility. A system has been devised for in vitro examination of responses of cells to controlled but independent changes in both the chemistry and morphology of polystyrene (PS) tissue culture surfaces. Micromachined silicon wafers were used as templates to solvent-cast PS replicas [using 0, 1, or 2 wt % styrene (S) monomer additions] with either none, 0.5- or 5.0-microns-deep surface grooves arranged in a radial array. When all possible morphologies were combined with all possible polymers, nine model biomaterial surfaces (MBSs) were produced. The chemical characteristics of the MBSs were determined using electron spectroscopy for chemical analysis, secondary ion mass spectroscopy, and contact angle techniques and were found to be distinct. The types and amount of proteins that adsorb onto these surfaces from serum containing media were examined and found to consist of multiple molecular layers of relatively uniform composition. Self-contained tissue culture vessels formed from the MBSs were capable of supporting the growth of confluent cultures of rat calvarial cells. The model biomaterial system described here can be used to examine how simultaneous stimuli resulting from the chemical and morphological characteristics of a test material may influence biologic responses. Such multifactorial biocompatibility research is needed to properly document material-host interactions.

Cellular responses to chemical and morphologic aspects of biomaterial surfaces. II. The biosynthetic and migratory response of bone cell populations
Chesmel, K. D., C. C. Clark, et al. (1995), J Biomed Mater Res 29(9): 1101-10.
Abstract: The biosynthetic and migratory response of bone cells to changes in both surface composition and morphology of polystyrene (PS) substrates was examined. A system was devised wherein micromachined silicon wafers were used as templates to solvent-cast PS replicas [using 0, 1, or 2 wt % styrene (S) monomer additions] with either 0.5- or 5.0- microns-deep surface grooves. Smooth replicas (0% S) served as the control surfaces. The chemical and morphologic characteristics of the nine unique model biomaterial surfaces (MBSs) produced using this system were documented and were found to be distinct. For the biosynthetic studies, bone cells isolated from neonatal rat calvaria were plated onto the MBSs and labeled at postconfluence with [14C]proline for 24 h. Total DNA per surface, total newly synthesized collagenous (CP), and noncollagenous protein (NCP) (cell associated and secreted) were determined. Cell-associated CP was found to increase significantly for the bone cells cultured on the substrates with 0.5-micron grooves and 2% S (P <.05). Cell-associated NCP was found to be elevated for all 2% S substrates and for the 0.5-micron grooves substrates with 1% S. For the migration studies, bone cells were plated first onto 5-mm nitrocellulose disks that were attached to standard Petri dishes using a plasma clot. At confluence, the disks were removed aseptically and placed on the replicas. The cellular area occupied as a result of the outward migration of the bone cells was measured after 4 days of culture using an image analysis system. An average velocity for the leading edge of bone cell populations on each of the nine MBSs was calculated: Cells on surfaces with either 1% S or 5.0-microns grooves displayed significantly higher velocities than did the control cultures. A significant interaction effect between chemistry and morphology was observed. The biosynthetic and migratory responses of in vitro cultures of bone cells were not predictable from the observations of the cellular responses to the individual features, but appeared to depend on cellular responses to more than one substrate factor.

Cellular, histomorphologic, and clinical characteristics of a new octyl-2-cyanoacrylate skin adhesive
Nitsch, A., A. Pabyk, et al. (2005), Aesthetic Plast Surg 29(1): 53-8.
Abstract: Short-chained cyanoacrylates have been used for many years for topical skin closure. Toxic effects in cell culture of a new long-chained octyl-2-cyanoacrylate tissue adhesive are compared with those of short-chained ethyl-2- and butyl-2-cyanoacrylates. Two cellular tests were used: the agar overlay test and the MTT test. An in vitro test using copper plates coated with the three types of cyanoacrylates serves for evaluating the stability of polymerized skin adhesives. Bilateral neck skin incisions in Goettingen miniature pigs were glued on one side with Dermabond. On the other side, conventional sutures were applied. After the pigs were killed, the resulting skin samples were tested for the tensile strength of their wound stability. Samples of pig dermis were exemplarily and histomorphologically characterized. A clinical examination after submandibular lymph node dissection should examine the application in humans. Cell culture tests were used to show the toxic effects of the three cyanoacrylates. In a copper test, octyl-2-cyanoacrylate was more stabile than ethyl- and butyl-cyanoacrylates. Breaking strength was 30% lower 28 days after operation with the new product than with sutures. In electron microscopy, octyl-2-cyanoacrylate showed no disadvantages with regard to tissue regeneration and no histotoxicity. For plastic surgery, this new topical skin adhesive is a real alternative with attractive results, as compared with conventional suture.

Cellulose phosphates as biomaterials. In vivo biocompatibility studies
Fricain, J. C., P. L. Granja, et al. (2002), Biomaterials 23(4): 971-80.
Abstract: Femoral implantation of regenerated cellulose hydrogels revealed their biocompatibility, but a complete osseointegration could not be observed. Phosphorylation was therefore envisaged as the means to enhance cellulose bioactivity. In vitro studies showed that regenerated cellulose hydrogels promote bone cells attachment and proliferation but do not mineralize in acellular simulated physiological conditions. On the contrary, phosphorylated cellulose has shown an opposite behavior, by inducing the formation of a calcium phosphate layer in simulated physiological conditions, but behaving as a poor substrate for bone cells attachment and proliferation. In order to investigate the in vivo behavior of these materials, and assess the influence of mineralization induction ability vs. bone cells compatibility, unmodified and phosphorylated cellulose hydrogels were implanted in rabbits for a maximum period of 6 months and bone regeneration was investigated. Despite the difficulties arising from the retraction of cellulose hydrogels upon dehydration during the preparation of retrieved implants, histological observations showed no inflammatory response after implantation, with bone intra-spongious regeneration of cells and the integration of the unmodified as well as the phosphorylated cellulose implants. After a maximum implantation period of 6 months, histological observations, histomorphometry and the measurement of the amount of 45Ca incorporated in the surrounding tissue indicated a slightly better osseointegration of phosphorylated cellulose, although no significant differences between the two materials were found.

Cellulose: fascinating biopolymer and sustainable raw material
Klemm, D., B. Heublein, et al. (2005), Angew Chem Int Ed Engl 44(22): 3358-93.
Abstract: As the most important skeletal component in plants, the polysaccharide cellulose is an almost inexhaustible polymeric raw material with fascinating structure and properties. Formed by the repeated connection of D-glucose building blocks, the highly functionalized, linear stiff-chain homopolymer is characterized by its hydrophilicity, chirality, biodegradability, broad chemical modifying capacity, and its formation of versatile semicrystalline fiber morphologies. In view of the considerable increase in interdisciplinary cellulose research and product development over the past decade worldwide, this paper assembles the current knowledge in the structure and chemistry of cellulose, and in the development of innovative cellulose esters and ethers for coatings, films, membranes, building materials, drilling techniques, pharmaceuticals, and foodstuffs. New frontiers, including environmentally friendly cellulose fiber technologies, bacterial cellulose biomaterials, and in-vitro syntheses of cellulose are highlighted together with future aims, strategies, and perspectives of cellulose research and its applications.

Cement from magnesium substituted hydroxyapatite
Lilley, K. J., U. Gbureck, et al. (2005), J Mater Sci Mater Med 16(5): 455-60.
Abstract: Brushite cement may be used as a bone graft material and is more soluble than apatite in physiological conditions. Consequently it is considerably more resorbable in vivo than apatite forming cements. Brushite cement formation has previously been reported by our group following the mixture of nanocrystalline hydroxyapatite and phosphoric acid. In this study, brushite cement was formed from the reaction of nanocrystalline magnesium-substituted hydroxyapatite with phosphoric acid in an attempt to produce a magnesium substituted brushite cement. The presence of magnesium was shown to have a strong effect on cement composition and strength. Additionally the presence of magnesium in brushite cement was found to reduce the extent of brushite hydrolysis resulting in the formation of HA. By incorporating magnesium ions in the apatite reactant structure the concentration of magnesium ions in the liquid phase of the cement was controlled by the dissolution rate of the apatite. This approach may be used to supply other ions to cement systems during setting as a means to manipulate the clinical performance and characteristics of brushite cements.

Cemented cup stability during lever-out testing after acetabular bone impaction grafting with bone graft substitutes mixes containing morselized cancellous bone and tricalcium phosphate--hydroxyapatite granules
Arts, J. J., B. W. Schreurs, et al. (2005), Proc Inst Mech Eng [H] 219(4): 257-63.
Abstract: Bone defects after failed total hip arthroplasty can be reconstructed with impacted morselized bone grafts and a cemented cup. In the near future the amount of bone grafts available for surgical purposes will be insufficient. Ceramic calcium phosphates [tricalcium phosphate (TCP) and hydroxyapatite (HA)] have been widely considered as potential bone graft substitutes or bone graft extenders. In the past, mechanical experiments have been performed to determine implant stability of bone grafts and ceramic TCP-HA granules mixes under a compressive load. However, in-vivo migration studies suggest that shear loading may be equally important. This in-vitro study investigated the initial stability of cups reconstructed with various mixes of bone grafts and ceramic TCP-HA granules in a lever-out situation, where shearing is the predominant loading mode. It was found that the cups reconstructed with mixes of bone graft and TCP-HA granules exhibited greater mechanical stability than the cups reconstructed with bone grafts only. It is concluded that from a mechanical standpoint, when considering shear force resistance, 50-50 per cent volume mix and 25-75 per cent volume mix of morselized cancellous bone graft and TCP-HA granules both provide adequate initial cup stability and can be used for acetabular reconstructions with the bone impaction grafting technique.

Cemented tibial component fixation performs better than cementless fixation: a randomized radiostereometric study comparing porous-coated, hydroxyapatite-coated and cemented tibial components over 5 years
Carlsson, A., A. Bjorkman, et al. (2005), Acta Orthop 76(3): 362-9.
Abstract: BACKGROUND: The question whether the tibial component of a total knee arthroplasty should be fixed to bone with or without bone cement has not yet been definitely answered. We studied movements between the tibial component and bone by radiostereometry (RSA) in total knee replacement (TKR) for 3 different types of fixation: cemented fixation (C-F), uncemented porous fixation (UC-F) and uncemented porous hydroxyapatite fixation (UCHA-F). PATIENTS: 116 patients with osteoarthrosis, who had 146 TKRs, were included in 2 randomized series. The first series included 86 unilateral TKRs stratified into 1 of the 3 types of fixation. The second series included 30 patients who had simultaneous bilateral TKR surgery, and who were stratified into 3 subgroups of pairwise comparisons of the 3 types of fixation. RESULTS: After 5 years 2 knees had been revised, neither of which were due to loosening. 1 UCHA-F knee in the unilateral series showed a large and continuous migration and a poor clinical result, and is a pending failure. The C-F knees rotated and migrated less than UC-F and UCHA-F knees over 5 years. UCHA-F migrated less than UC-F after 1 year. INTERPRETATION: Cementing of the tibial component offers more stable bone-implant contact for 5 years compared to uncemented fixation. When using uncemented components, however, there is evidence that augmenting a porous surface with hydroxyapatite may mean less motion between implant and bone after the initial postoperative year.

Ceramic bonding strength of Au-1.6 wt% Ti alloy
Ito, M., M. Kikuchi, et al. (2005), Dent Mater J 24(2): 268-74.
Abstract: The aim of this investigation was to evaluate the metal-ceramic bonding of Au-1.6 wt% Ti alloy. Therefore, the coefficient of thermal expansion, modulus of elasticity, ceramic bonding strength, and hardness of Au-1.6 wt% Ti alloy were measured. The metal-ceramic interface was observed by EPMA to investigate its bonding mechanism. The Au-1.6 wt% Ti alloy showed a coefficient of thermal expansion close to that of conventional precious metal alloys for metal-ceramics. Bonding strength was 25 MPa or higher for all specimens--whether ceramic-fired directly after casting, or following oxidation treatment or age hardening. Specimens subjected to oxidation treatment showed slightly lower post-firing hardness than those age-hardened. EPMA observation showed an accumulation of Ti and O contents on the alloy side at the metal-ceramic interface.

Ceramic corundum materials with modified surface as implantation materials
Lewandowski, R., R. Rutowski, et al. (2005), Polim Med 35(2): 3-14.
Abstract: Investigations on implants have proved that not only the kind of material structure, but also the surface character of grafts influences the local reaction of biomaterials. In recent years electrochemical reactions on implants surface leading to the so called zeta potential arouse interests. Applications of these properties has made us carry on experiments on use of biomaterials with active potential on their bio-compatibility. The aim of this work is evaluation of influence of electrical charge with zeta potentials character on the local reaction of bone tissue after implantation. Two kinds of ceramics were used in experiments: solid and solid-silane with active surface. Implantation experiments were made on 20 rabbits. Selections were carried of 12, 26, 36 and 54 weeks after implantation of the tested materials. After implantation of solid ceramics, not only bone tissue but also fibrous tissue was observed around the graft. But in case of solid silane ceramics, in all terms of tests, bone tissue tightly sticking to the implant was observed around the graft. Investigations of electrokinetic zeta potential carried out 36 and 54 weeks after implantation showed that the values of that potential originally introduced on the grafts surface were maintained on stable level. The carried out investigations confirmed that activation of implants surface through silaning with sol-gel method allows to introduce the planned zeta potential depending on the characteristic for a given kind of tissue.

Changes in binding affinity of a monoclonal antibody to a platelet binding domain of fibrinogen adsorbed to biomaterials
Grunkemeier, J., C. Wan, et al. (1996), J Biomater Sci Polym Ed 8(3): 189-209.
Abstract: Previously, we found that when fibrinogen-coated polyurethanes resided in a buffer for a period of time (the 'residence time') platelet adhesion to these materials decreased. Other changes in adsorbed fibrinogen such as decreases in polyclonal antibody binding and SDS elutability supported the conclusion that fibrinogen undergoes postadsorptive conformational changes. Subsequently we measured the binding of monoclonal antibodies to the three putative platelet binding sites on fibrinogen, using a single mid-range concentration of antibody. We found that binding of a monoclonal antibody to the platelet binding site at the C-terminus of the gamma chain of fibrinogen changed little with residence time, while binding of monoclonal antibodies to the other two putative binding sites on fibrinogen either increased with residence time (RGDF at A alpha 95-98), or first increased and then decreased with residence time (RGDS at A alpha 572-575). In the current study, we measured antibody binding affinity, Ka, by measuring antibody binding at a series of antibody concentrations. This is a more sensitive method for detecting changes in adsorbed fibrinogen than measuring antibody binding from a single antibody concentration. The Ka was determined for two antibodies, M1 (4A5), which binds to a platelet binding domain of fibrinogen (gamma 402-411) and R1 (155 B 1616), which binds to residues 87-100 of the A alpha chain (containing an RGDF site). A summary of the results for the M1 antibody are as follows. The Ka was higher for M1 binding to fibrinogen adsorbed to Immulon I than to Biomer, Biospan or poly(ethylene terephthalate), suggesting that fibrinogen adsorbed to Immulon I is more platelet adhesive than fibrinogen adsorbed to the other polymers. On Biospan, the Ka decreased from 2.8 x 10(9) to 1.0 x 10(9) M-1 after a 24 h 37 degrees C residence time, which correlated with the decrease in platelet adhesiveness of adsorbed fibrinogen observed previously under these conditions. The change in Ka was greater when adsorbed fibrinogen was kept under denaturing conditions. For example, the Ka decreased from 2.8 x 10(9) to 0.8 x 10(9) M-1 after a 1 h 70 degrees C residence time whereas it remained approximately the same, 2.9 x 10(9) M-1, after a 24 h 0 degree C residence time.

Changes in integrin expression during adipocyte differentiation
Liu, J., S. M. DeYoung, et al. (2005), Cell Metab 2(3): 165-77.
Abstract: 3T3-L1 preadipocytes require cAMP for maximal differentiation. Microarray analysis reveals that the integrins alpha5 and alpha6 are coordinately regulated by cAMP. alpha5 expression is gradually diminished during adipogenesis, whereas alpha6 is increased. Overexpression of alpha5 in preadipocytes results in enhanced proliferation and attenuated differentiation. Conversely, alpha6 overexpression is without effect. The GTPase Rac is normally inhibited during differentiation. However, overexpression of integrin alpha5 increases Rac activity. Constitutively active but not dominant-negative Rac inhibits differentiation when overexpressed in preadipocytes, implying its role downstream of alpha5 integrin in maintaining preadipocytes in an undifferentiated state. Moreover, alpha6 integrin is critically involved in clustering growth-arrested preadipocytes on basement membrane Matrigel. Perturbation of such clustering enhances Rho activity and promotes growth-arrested preadipocytes to reenter the cell cycle. These findings demonstrate a role for integrin alpha6 in connecting morphogenesis with signaling processes leading to terminal differentiation.

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Last Modified: 8 February 2006