Decreased bacterial adhesion to surface-treated titanium

Osteointegrative dental implants are widely used in implantology for their well-known excellent performance once implanted in the host. Remarkable bacterial colonization along the transgingival region may result in a progressive loss of adhesion at gum-implant interface and an increase of the bone area exposed to pathogens. This phenomenon may negatively effect the osteointegration process and cause, in the most severe cases, implant failure. The presence of bacteria at implant site affect the growth of new bone tissue and consequently, the achievement of a mechanically stable bone-implant interface, key parameters for a suitable implant osteointegration. In the present work, a novel surface treatment has been developed and optimized in order to convert the amorphous titanium oxide in a crystalline layer enriched in anatase capable of providing not only antibacterial properties but also of stimulating the precipitation of apatite when placed in simulated body fluid. The collected data have shown that the tested treatment results in a crystalline anatase-type titanium oxide layer able to provide a remarkable decrease in bacterial attachment without negatively effecting cell metabolic activity. In conclusion, the surface modification treatment analyzed in the present study might be an elegant way to reduce the risk of bacterial adhesion and increase the lifetime of the transgingival component in the osteointegrated dental implant.     

Developmental platelet endothelial cell adhesion molecule expression suggests multiple roles for a vascular adhesion molecule

Platelet endothelial cell adhesion molecule (PECAM) is used extensively as a murine vascular marker. PECAM interactions have been implicated in both vasculogenesis and angiogenesis. To better understand the role of PECAM in mammalian development, PECAM expression was investigated during differentiation of murine embryonic stem (ES) cells and in early mouse embryos. Undifferentiated ES cells express PECAM, and as in vitro differentiation proceeds previously unidentified PECAM-positive cells that are distinct from vascular endothelial cells appear. PECAM expression is gradually restricted to endothelial cells and some hematopoietic cells of differentiated blood islands. In embryos, the preimplantation blastocyst contains PECAM-positive cells. PECAM expression is next documented in the postimplantation embryonic yolk sac, where clumps of mesodermal cells express PECAM before the development of mature blood islands. The patterns of PECAM expression suggest that undifferentiated cells, a prevascular cell type, and vascular endothelial cells express this marker during murine development. PECAM expression in blastocysts and by ES cells suggests that PECAM may function outside the vascular/hematopoietic lineage.     

Balancing osteoblast functions and bacterial adhesion on functionalized titanium surfaces

The demand for orthopedic and dental implants will continue to grow, and for these applications, titanium and its alloys have been used extensively. While these implants have achieved high success rates, two major complications may be encountered: the lack of bone tissue integration and implant-centered infection. The surface of the implant, through its interactions with proteins, bacteria and tissue cells, plays a determining role in the success or failure of the implant. Ideally, to enhance the success of implants, their surfaces should inhibit bacterial colonization and concomitantly promote osteoblast functions. In this article, we discuss strategies for tailoring implant surfaces by exploiting the differences in the response of bacteria and osteoblasts to proteins and surface structures. Nevertheless, limitations still exist in the quest for an ideal implant surface. Further advances in this field will require concurrent development in surface modification techniques and a better understanding of the complex and highly inter-related events occurring at the implant surface after implantation.     

The modulation of platelet and endothelial cell adhesion to vascular graft materials by perlecan

Controlled neo-endothelialisation is critical to the patency of small diameter vascular grafts. Endothelialisation and platelet adhesion to purified endothelial cell-derived perlecan, the major heparan sulfate (HS) proteoglycan in basement membranes, were investigated using in vivo and in vitro assays. Expanded polytetrafluoroethylene (ePTFE) vascular grafts were coated with perlecan and tested in an ovine carotid interposition model for a period of 6 weeks and assessed using light and scanning microscopy. Enhanced endothelial cell growth and reduced platelet adhesion were observed on the perlecan coated grafts when compared to uncoated controls implanted in the same sheep (n = 5). Perlecan was also found to stimulate endothelial cell proliferation in vitro over a period of 6 days in the presence of plasma proteins and fibroblastic growth factor 2 (FGF-2), however in the absence of FGF-2 endothelial cell growth could not be maintained during this period. Perlecan was found to be anti-adhesive for platelets, however after removal of the HS chains attached to perlecan, platelet adhesion and aggregation were supported. These results suggest a role for HS chains of perlecan in improving graft patency by selectively promoting endothelial cell proliferation while modulating platelet adhesion.     

Vascular endothelial growth factor, platelet endothelial cell adhesion molecule-1 and vascular cell adhesion molecule-1 in the follicular fluid of patients undergoing IVF.

BACKGROUND: The aim of our study was to measure concentrations of vascular endothelial growth factor (VEGF), platelet endothelial cell adhesion molecule-1 (PECAM-1) CD31 and vascular cell adhesion molecules (VCAM-1) in the follicular fluid of women treated with assisted reproduction technology to determine whether these proteins might be outcome markers. METHODS: Follicular fluid was collected from 75 patients < or =40 years undergoing oocyte retrieval procedures at our tertiary hospital during 1997 and 1998: 50 with tubal disease, 12 with endometriosis, and 13 whose partners had been diagnosed with severe oligoasthenoteratozoospermia. This retrospective analysis considered age and information about treatment and outcome for all these women who had undergone fewer than three assisted reproduction attempts. RESULTS: Nineteen women became pregnant (defined by human chorionic gonadotrophin concentrations and embryonic cardiac activity 1 month after follicular aspiration); 56 did not. Women did not differ significantly in their follicular fluid concentrations of VEGF, sCD31 and VCAM-1 according to cause of infertility, or assisted reproduction outcome, or age. Follicular fluid concentrations of VEGF were significantly correlated with the number of gonadotrophin ampoules administered (P < 0.012), and follicular fluid concentrations of sVCAM-1 with the fertilization rate (P < 0.01). Follicular fluid concentrations of VEGF and sVCAM-1 were also correlated (P < 0.007). CONCLUSIONS: Our results do not suggest that VEGF, CD31, or sVCAM-1 in follicular fluid predict assisted reproduction outcome, especially among patients < or =40 years old. The correlation of a high fertilization rate and sVCAM-1 in follicular fluid suggests that sVCAM-1 might be a marker of fertilization.     

Improved endothelial cell adhesion and proliferation on patterned titanium surfaces with rationally designed, micrometer to nanometer features

Previous in vitro studies have demonstrated increased vascular endothelial cell adhesion on random nanostructured titanium (Ti) surfaces compared with conventional (or nanometer smooth) Ti surfaces. These results indicated for the first time the potential nanophase metals have for improving vascular stent efficacy. However, considering the structural properties of the endothelium, which is composed of elongated vascular endothelial cells aligned with the direction of blood flow, it has been speculated that rationally designed, patterned nano-Ti surface features could further enhance endothelial cell functions by promoting a more native cellular morphology. To this end, patterned Ti surfaces consisting of periodic arrays of grooves with spacings ranging from 750 nm to 100 microm have been successfully fabricated in the present study by utilizing a novel plasma-based dry etching technique that enables machining of Ti with unprecedented resolution. In vitro rat aortic endothelial cell adhesion and growth assays performed on these substrates demonstrated enhanced endothelial cell coverage on nanometer-scale Ti patterns compared with larger micrometer-scale Ti patterns, as well as controls consisting of random nanostructured surface features. Furthermore, nanometer-patterned Ti surfaces induced endothelial cell alignment similar to the natural endothelium. Since the re-establishment of the endothelium on vascular stent surfaces is critical for stent success, the present study suggests that nanometer to submicrometer patterned Ti surface features should be further investigated for improving vascular stent efficacy.     

Blood compatibility of surfaces with immobilized albumin-heparin conjugate and effect of endothelial cell seeding on platelet adhesion.

Endothelial cell (EC) seeding significantly improves the blood compatibility of artificial surfaces. Although a coating consisting of albumin and heparin (alb-hep) is a suitable substrate for seeded ECs, binding of ECs to the substrate further improves when small amounts of fibronectin are present in the alb-hep coating. Alb-hep conjugate was immobilized on carbon dioxide gas plasma-treated polystyrene (PS-CO(2)), thereby significantly increasing the recalcification time of blood plasma exposed to this surface. Furthermore, surface-immobilized alb-hep conjugate inhibited exogenous thrombin. Heparin activity was reduced by adding fibronectin on top of a monolayer of alb-hep conjugate, but not by simultaneous coating of fibronectin and alb-hep conjugate. Coating of PS-CO(2) with alb-hep conjugate significantly decreased contact activation (FXII activation). The number of platelets deposited from blood plasma on PS-CO(2) coated with alb-hep conjugate was twice as high as on PS-CO(2) coated with albumin. Addition of fibronectin to alb-hep conjugate-coated PS-CO(2) had no significant effect on the number of adhered platelets. Seeding of the substrates with ECs significantly reduced the number of adhered platelets under stationary conditions. Platelets deposited onto endothelialized surfaces were primarily found on endothelial cell edges, and sparingly on areas between ECs. In conclusion, alb-hep conjugate-coated surfaces display anticoagulant activity. ECs adhering to and proliferating on this coating significantly decrease the number of platelets which adhere to the surface. Therefore, alb-hep conjugate-coated surfaces form a suitable substrate for seeding of ECs in low density. Although application of fibronectin on top of the coating decreases the anticoagulant activity to some extent, it might be useful in view of the improved adherence of ECs to the coating.     

Human alveolar bone cell adhesion and growth on ion-implanted titanium

Surface characteristics play a vital role in determining the biocompatibility of materials used as bone implants. Calcium ion implantation of titanium was previously reported to enhance osseointegration and bone formation in vivo, although the lack of consistent and reproducible effects highlight the need to understand the basic mechanisms involved in the response of target cells to such surfaces. The aim of this study was therefore to measure the precise effects of ion implantation of titanium on bone cells in vitro. Alveolar bone cells were seeded on the surface of polished titanium disks implanted with calcium, potassium, and argon ions. Using radioisotopically tagged bone cells, the results showed that although the calcium ion implanted surface reduced cell adhesion, it nevertheless significantly enhanced cell spreading and subsequent cell growth. In contrast, few differences in bone cell behavior were observed between the potassium- and argon-implanted titanium and the control nonimplanted titanium disks. These findings suggest the possibility that the calcium-implanted surface may significantly affect the biocompatibility of titanium implants by enhancing bone cell growth. Surface modification by ion implantation could thus prove to be a valuable tool for improving the clinical efficacy of titanium for bone repair and regeneration in vivo.     

Silk-functionalized titanium surfaces for enhancing osteoblast functions and reducing bacterial adhesion

It would be ideal to have implants which can simultaneously inhibit bacterial adhesion and promote osteoblast functions. In this work, titanium surfaces were modified with poly(methacrylic acid) (P(MAA)) followed by immobilization of silk sericin. Firstly a trichlorosilane coupling agent, which is an atom transfer radical polymerization (ATRP) initiator, was immobilized on the oxidized titanium surface to facilitate the surface-initiated ATRP of methacrylic acid sodium salt (MAAS). The pendant carboxyl end groups of the grafted and partially protonated MAA chains were subsequently coupled with silk sericin via carbodiimide chemistry. The functionalized Ti surfaces were characterized by X-ray photoelectron spectroscopy, and assayed for osteoblast cell functions and bacterial adhesion. The covalently immobilized MAA brushes significantly reduce the adhesion of the two bacterial strains (Staphylococcus aureus and Staphylococcus epidermidis) tested. The silk sericin-immobilized surfaces, at the same time, promote osteoblast cells' adhesion, proliferation, and alkaline phosphatase activity. Thus, the P(MAA) and silk sericin functionalized Ti surfaces have potential applications combating biomaterial-centered infection and promoting osseointegration.     

Mesenchymal stem cell adhesion and spreading on microwave plasma-nitrided titanium alloy

Improved methods to increase surface hardness of metallic biomedical implants are being developed in an effort to minimize the formation of wear debris particles that cause local pain and inflammation. However, for many implant surface treatments, there is a risk of film delamination due to the mismatch of mechanical properties between the hard surface and the softer underlying metal. In this article, we describe the surface modification of titanium alloy (Ti-6Al-4V), using microwave plasma chemical vapor deposition to induce titanium nitride formation by nitrogen diffusion. The result is a gradual transition from a titanium nitride surface to the bulk titanium alloy, without a sharp interface that could otherwise lead to delamination. We demonstrate that vitronectin adsorption, as well as the adhesion and spreading of human mesenchymal stem cells to plasma-nitrided titanium is equivalent to that of Ti-6Al-4V, while hardness is improved 3- to 4-fold. These in vitro results suggest that the plasma nitriding technique has the potential to reduce wear, and the resulting debris particle release, of biomedical implants without compromising osseointegration; thus, minimizing the possibility of implant loosening over time.     

内毒素致新生大鼠肺组织PECAM-1表达的研究

目的探讨内毒素作用于新生大鼠后,肺组织血小板内皮细胞粘附分子-1(PECAM-1)表达的变化及可能作用.方法用脂多糖(LPS)诱发7日龄新生大鼠肺损伤以至于肺出血模型,用免疫组化和RT-PCR方法观察肺组织PECAM-1蛋白水平和mRNA水平表达的变化.结果正常新生大鼠肺组织表达较高水平的PECAM-1蛋白水平和mRNA水平,LPS作用后,肺组织PECAM-1蛋白水平和mRNA水平表达均存在逐渐下降趋势,mRNA水平的表达在给药后1h即明显下降,蛋白水平在给药后4h明显下降,至LPS作用后8h降至最低水平,24h存活大鼠PECAM-1蛋白水平和mRNA水平有所恢复,二者的变化趋势基本一致.结论正常新生大鼠肺组织表达较高水平的PECAM-1蛋白及mRNA,PECAM-1可能在新生大鼠肺出血的发病机制中有一定的作用,并且PECAM-1表达变化的调节是在转录水平.     

脉冲电刺激对血管内皮细胞与其祖细胞黏附的影响

为探索脉冲电刺激下血管内皮细胞与内皮祖细胞(EPC)之间黏附强度的改变,诱导培养外周血EPC,荧光标记后与单层血管内皮细胞共培养,固定电压和频率为5 V和5 Hz,选择1、3、6、9 ms的脉宽对其进行干预,持续刺激24 h后检测贴壁EPC的荧光强度,以荧光比率衡量。结果显示,与对照组相比,3 ms刺激组荧光比率即显著增高,随着脉宽延长,6 ms组达到最大值,但9 ms刺激组却显著下降,提示适宜脉冲电刺激有利于血管内皮细胞与EPC之间的黏附,为电刺激促进血管新生的研究提供新的理论依据。     

Lack of platelet endothelial cell adhesion molecule-1 attenuates foreign body inflammation because of decreased angiogenesis

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kd member of the immunoglobulin superfamily of proteins, expressed on endothelial cells, leukocytes, and platelets. Antibody-blocking studies have implicated it in modulating leukocyte transmigration and angiogenesis. However, the generation of the PECAM-1 knockout mouse has shown that its function can be compensated for by similarly acting proteins because most acute inflammatory models proceed in a comparable manner in wild-type and knockout animals. We decided to examine the function of PECAM-1 in the chronic process of foreign body inflammation. We show that PECAM-1-deficient mice exhibit attenuated neutrophil infiltration in and around a subcutaneous polyvinyl acetyl implant. Bone marrow engraftment studies indicate that the lack of CD31 expression on the endothelium determines the diminished leukocyte accumulation in the knockout implants. Specifically, we find that decreased angiogenesis (as manifested by lower vessel density, decreased hemoglobin content, and less laminin deposition) correlates with lower neutrophil accumulation in the knockout animals. This study indicates that the absence of endothelial PECAM-1 results in decreased angiogenesis and therefore in diminished delivery of leukocytes to the foreign body implants.     

Effect of shear stress on platelet adhesion to expanded polytetrafluoroethylene, a silicone sheet, and an endothelial cell monolayer

We visualized in real-time platelets adhering to the surface of three representative biomaterials, by using an apparatus consisting of a modified cone and plate rheometer combined with an upright epifluorescence microscope under two shear flows (0.1 and 5.0 dyne/cm2). The materials were expanded polytetrafluoroethylene (ePTFE), silicone sheet, and a monolayer of bovine endothelial cells (ECs) formed on glass, all of which are opaque materials used for artificial blood vessels and medical devices. According to quantitative analysis, the monolayer of ECs formed on glass had better blood compatibility than did either the ePTFE or the silicone sheet under shear flow conditions. Under a shear flow condition of 0.1 dyne/cm2, platelet adhesion was silicone sheet > ePTFE. In contrast, under a shear flow condition of 5.0 dyne/cm2, ePTFE > silicone sheet. These results indicate that the intensity of shear stress could modify the order of hemocompatibility of the materials. Therefore, direct observation of platelet adhesion under shear flow conditions is indispensable for testing and screening biomaterials and for providing a precise quantitative evaluation of platelet adhesion.     

Cholesterol-derivatized polyurethane: characterization and endothelial cell adhesion

Endothelialization of synthetic surfaces has been challenging with limited success thus far. We investigated the hypothesis that covalent attachment of cholesterol to polyurethane via the urethane nitrogen groups would create a high-affinity surface for attachment and adhesion of endothelial cells. Cholesterol was covalently bound to the polyether polyurethane, Tecothane, by first derivatizing the polyurethane nitrogen groups with bromoalkyl side chains, followed by reacting mercapto-cholesterol to the bromoalkyl sites. Cholesterol-modified polyurethane demonstrated a qualitatively smoother surface per atomic force microscopy than nonmodified and increased surface energy (contact angle measurements) compared with unmodified polyurethane. Cell attachment assays showed a significantly greater number of attached bovine arterial endothelial cells (p = 0.0003) after 45 min of seeding on cholesterol-modified polyurethane versus unmodified polyurethane. Bovine arterial endothelial cells cultivated on cholesterol-modified Tecothane showed significantly greater levels of cell retention compared with unmodified Tecothane when exposed to arterial level shear stress for 2 h (25 dynes/cm2) with 90.0 +/- 6.23% cells remaining adherent compared with unmodified polyurethane, 41.4 +/- 11.7%, p = 0.0070. Furthermore, ovine endothelial precursors, obtained as blood outgrowth endothelial cells, were seeded on cholesterol-modified polyurethane and exposed to 25 dynes/cm2 shear conditions for 2 h, with the retention of 90.30 +/- 3.25% of seeded cells versus unmodified polyurethane, which retained only 4.56 +/- 0.85% (p < 0.001). It is concluded that covalently linking cholesterol to polyurethane results in improved material properties that permit increased endothelial cell retention compared with unmodified polyurethane.     

A new flow cell for platelet adhesion studies

A flow cell has been designed and tested for the purpose of exposing platelets to various substrates. The design adopts the principle of flow relaminarization by acceleration to dissipate secondary fluid motions and turbulence so that platelet diffusion will be controlled by Brownian diffusion coefficients. To assess the effectiveness of this flow mechanism two critical tests were undertaken. First, hot film anemometry signals were obtained to observe visually the local fluid velocity in the flow cell. Second, in vitro platelet adhesion results were obtained by exposing glass and silane coated glass to platelet suspensions. Surface platelet concentrations for varying exposure times were compared to a theoretical model which is based on a laminar diffusion model. Both tests confirm that hydrodynamically the flow cell behaves in a manner which is consistent with that of a fully developed, laminar flow with a constant diffusion coefficient.     

Dynamic heterodimer-functionalized surfaces for endothelial cell adhesion

The functionalization of hydrogels for receptor-mediated cell adhesion is one approach for targeted cell and tissue engineering applications. In this study, polyacrylamide gel surfaces were functionalized with specific cell adhesion ligands via the self-assembly of a peptide-based heterodimer. The system was comprised of a cysteine-terminated monomer, A (MW approximately 5400), grafted to the polyacrylamide gels and a complementary ligand presenting monomer, B(X) (MW approximately 5800) that was designed to heterodimerize with A. Two ligand presenting monomers were synthesized: one presenting the RGDS ligand, B(D), for receptor-mediated cell adhesion, and the other, a control monomer presenting the nonadhesive RGES ligand, B(E). Assembly of the peptide pair A-B(X) by association of the monomers into a coiled coil was verified by circular dichroism in solution. Binding studies were conducted to determine the dissociation constant of the pair A-B(X), which was found to be K(D) approximately 10(-8) m. Polyacrylamide gels functionalized with A-B(X) heterodimers were evaluated for cell adhesion using bovine aortic endothelial cells (BAECs). Endothelial cells cultured on the A-B(D) functionalized surfaces demonstrated typical cell morphologies and expected spreading behavior as a function of the density of RGDS ligand, calculated as the amount of B(D) associated with grafted A on the surface of the gels. In contrast, A-B(E) linked surfaces supported no cell adhesion. The adhesion of the substrate was dynamically altered through the reassembly of A-B(X) dimers as B(D) molecules in the solution replaced B(E) molecules at the substrate. The molecular constructs described here demonstrate the potential to design a broad family of switchable peptides that impart the dynamic control of biofunctionality at an interface, which would be useful for precise manipulation of cell physiology.     

Decreased lung carcinoma cell functions on select polymer nanometer surface features

Biomaterial nanotopographies have been proposed as a means to significantly influence cell functions (including osteoblasts, fibroblasts, endothelial cells, chondrocytes, immune cells, and bacteria). In this study, lung epithelial carcinoma cell functions including adhesion (up to 4 h), proliferation (up to 3 days), apoptosis (up to 5 days), and vascular endothelial growth factor (VEGF) synthesis (up to 5 days) on poly-lactic-co-glycolic (PLGA) films with various nanotopographies were systematically investigated. Importantly, this study created PLGA films with various nanotopographies (specifically, nano-smooth, 23, 300, and 400 nm hemispherical surface features) but similar surface chemistry in order to focus only on the effect of PLGA topography on cancer cell functions. Simple and effective cast-molding and solvent evaporation methods were used to accomplish this. Atomic force microscopy, electron spectroscopy for chemical analysis, and water contact angle measurements verified similar surface chemistry and energy but varied topographies for all of the PLGA films prepared in this study. Lung epithelial carcinoma cell adhesion, proliferation, and morphology results indicated less cell growth and spreading on nano-smooth and 400 nm surface-featured PLGA compared to any other samples. However, results also demonstrated decreased lung epithelial carcinoma cell VEGF (a key growth factor secreted for the vascularization of tumors) synthesis on the 23 nm surface-featured PLGA compared to the nano-smooth substrates for up to 5 days. In summary, these results provided the first insights into understanding the role that PLGA nanotography may play in mediating lung carcinoma cell functions for a wide range of applications in regenerative medicine.     

Peptides derived from fibronectin type III connecting segments promote endothelial cell adhesion but not platelet adhesion: implications in tissue-engineered vascular grafts

The development of a completely tissue-engineered small-caliber prosthesis suitable for incorporation into an in vivo vascular network is fraught with many challenges, including overcoming resistance to endothelialization and susceptibility to thrombogenesis. In this work, recombinant human fibronectin-derived low-molecular-weight peptide fragments were studied for their ability to promote cell type-specific alpha(4) integrin-mediated adhesion. Two populations of primary human endothelial cells were examined and found to express alpha(4) integrin receptors on their surfaces; on the contrary, human platelets were not found to be expressers of alpha(4) integrins. A peptide fragment isolated from the variably spliced human fibronectin type III connecting segment-1 (CS-1) domain was determined to mediate statistically significant endothelial cell alpha(4) integrin-mediated adhesion. In contrast, the fibronectin type III CS-1 fragment did not support human platelet adhesion under physiological fluid shear conditions, although fully intact human fibronectin molecules supported shear-induced platelet adhesion. This suggests that platelets bind to fibronectin in regions not encompassing the CS-1 domain. In conclusion, this work has demonstrated that the low-molecular-weight peptide CS-1 could serve as a cell-selective adhesion mediator in the engineering of a more-compatible small-caliber vascular graft lumen interface.