Glial cell and fibroblast cytotoxicity study on 4026-cyclotene photosensitive benzocyclobutene (BCB) polymer films

Photosensitive benzocyclobutene (photo-BCB) is a class of polymers with the trade name Cyclotene. The photoimagable property of Cyclotene makes it suitable for the manufacture of microelectronic devices. The motivation behind this study is that we see an exciting application of photo-BCB as substrates in implantable microelectronic biomedical devices due to several desirable properties distinctive from other polymer materials. To our knowledge, however, photo-BCB has never been tested for biomedical implant applications, as evidenced by the lack reported data on its biocompatibility. This study takes the first step towards assessing photo-BCB biocompatibility by evaluating the cytotoxicity and cell adhesion behavior of Cyclotene 4026 coatings exposed to monolayers of glial and fibroblast cells in vitro. It can be concluded from these studies that photo-BCB films deposited on silicon wafers using microfabrication processes did not adversely affect 3T3 fibroblast and T98-G glial cell function in vitro. We also successfully rendered photo-BCB films non-adhesive (no significant fibroblast or glial cell adhesion) with surface immobilized dextran using methods developed for other biomaterials and applications. Future work will further develop prototype photo-BCB microelectrode devices for chronic neural implant applications.     

In vitro assessment of bioactive coatings for neural implant applications

Recent efforts in our laboratory have focused on developing methods for immobilizing bioactive peptides to low cell-adhesive dextran monolayer coatings and promoting biospecific cell adhesion for biomaterial implant applications. In the current study, this dextran-based bioactive coating technology was developed for silicon, polyimide, and gold, the base materials utilized to fabricate our prototype neural implants. Chemical composition of all modified surfaces was verified by X-ray photoelectron spectroscopy (XPS). We observed that surface-immobilized dextran supported very little cell adhesion in vitro (24-h incubation with serum-supplemented medium) on all base materials. Inactive nonadhesion-promoting Gly-Arg-Ala-Asp-Ser-Pro peptides immobilized on dextran-coated materials promoted adhesion and spreading at low levels not significantly different from dextran-coated substrates. Arg-Gly-Asp (RGD) peptide-grafted surfaces were observed to promote substantial fibroblast and glial cell adhesion with minimal PC12 (neuronal cell) adhesion. In contrast, dextran-coated materials with surface-grafted laminin-based, neurite-promoting Ile-Lys-Val-Ala-Val (IKVAV) peptide promoted substantial neuron cell adhesion and minimal fibroblast and glial cell adhesion. It was concluded that neuron-selective substrates are feasible using dextran-based surface chemistry strategies. The chemical surface modifications could be utilized to establish a stable neural tissue-implant interface for long-term performance of neural prosthetic devices.     

Biomedical applications of diamond-like carbon coatings: a review

Owing to its superior tribological and mechanical properties with corrosion resistance, biocompatibility, and hemocompatibility, diamond-like carbon (DLC) has emerged as a promising material for biomedical applications. DLC films with various atomic bond structures and compositions are finding places in orthopedic, cardiovascular, and dental applications. Cells grew on to DLC coating without any cytotoxity and inflammation. DLC coatings in orthopedic applications reduced wear, corrosion, and debris formation. DLC coating also reduced thrombogenicity by minimizing the platelet adhesion and activation. However, some contradictory results (Airoldi et al., Am J Cardiol 2004;93:474-477, Taeger et al., Mat-wiss u Werkstofftech 2003;34:1094-1100) were also reported that no significant improvement was observed in the performance of DLC-coated stainless stent or DLC-coated femoral head. This controversy should be discussed based on the detailed information of the coating such as atomic bond structure, composition, and/or electronic structure. In addition, instability of the DLC coating caused by its high level of residual stress and poor adhesion in aqueous environment should be carefully considered. Further in vitro and in vivo studies are thus required to confirm its use for medical devices.     

Interactions of human bone cells with diamond-like carbon polymer hybrid coatings

Diamond-like carbon (DLC) coatings produced using the plasma-accelerating filtered pulsed arc discharge (FPAD) method display excellent adherence to the substrate and improve its corrosion resistance. This article reports the interactions of human osteoblastic cells with DLC and two DLC polymer hybrid (DLC-p-h) coatings deposited on smooth, matt and rough silicon wafers by the FPAD method. The DLC-p-h materials were DLC–polytetrafluoroethylene hybrid (DLC-PTFE-h) and DLC–polydimethylsiloxane hybrid (DLC-PDMS-h) coatings. The biocompatibility of the coatings was assayed by using mesenchymal stem cells, primary osteoblasts and Saos-2 cells. Human mesenchymal stem cells proliferated when cultured on DLC and DLC-PTFE-h, but their numbers diminished on DLC-PDMS-h. In all three cell types studied, phalloidin–TRITC staining disclosed cell-type organization typical of an actin cytoskeleton on DLC and DLC-PTFE-h, but minimal and disorganized stress fibers on cells cultured on DLC-PDMS-h. The microtubular cytoskeleton was similarly disorganized on DLC-PDMS-h. Cells on DLC-PDMS-h developed a peculiar form of membrane damage, with nuclear staining by propidium iodide associated with granular calcein staining of the cytoplasm. Active caspase-3 labeling was only seen in cells cultured on DLC-PDMS-h, indicating that these cells undergo apoptosis induced by defective cell adhesion. Results suggest that DLC-PDMS-h coatings might be useful in orthopedic applications where an implant or implant-facet should be protected against bone overgrowth while DLC and DLC-PTFE-h coatings might improve osseointegration.     

Surface-immobilized dextran limits cell adhesion and spreading

Dextran has recently been investigated as an alternative to polyethylene glycol (PEG) for low protein-binding, cell-resistant coatings on biomaterial surfaces. Although anti-fouling properties of surface-grafted dextran and PEG are quite similar, the multivalent properties of dextran are advantageous when high-density surface immobilization of biologically active molecules to low protein-binding surface coatings is desired. The preferred methods of dextran immobilization for biomaterial applications should be simple with minimal toxicity. In this report, a method is described for covalent immobilization of dextran to material surfaces which involves low residual toxicity reagents in mild aqueous reaction conditions. 70 kDa MW dextran was immobilized on glass and polyethylene terephthalate (PET) surfaces. 3T3 fibroblast cell adhesion was compared on untreated, aminated, and dextran-coated materials. Dextran coatings effectively limited cell adhesion and spreading on glass and PET surfaces in the presence of serum-borne cell adhesion proteins. With dextran-based surface coatings, it will be possible to develop well-defined surface modifications that promote specific cell interactions and perhaps better performance in long-term biomaterial implants.     

Fluorinated diamond-like carbon as antithrombogenic coating for blood-contacting devices

Diamond-like carbon (DLC) is being considered for widespread clinical use as a surface coating for cardiovascular devices. We synthesized fluorinated DLC (F-DLC) coatings in order to create a more hydrophobic surface with improved antithrombogenicity and flexibility when compared with conventional DLC coatings by combining the inertness of DLC films with the advantage of fluorination. The purpose of this study was to evaluate the in vitro hemocompatibility and in vivo biocompatibility of the F-DLC coating for medical devices. The in vitro whole blood model confirmed that platelet loss was lower in the F-DLC group than in the noncoated group (SUS316L), which suggests the adhesion of a smaller number of platelets to F-DLC-coated materials. Furthermore, the biomarkers of mechanically induced platelet activation (beta-thromboglobulin) and activated coagulation (thrombin-antithrombin-three complex) were markedly reduced in the F-DLC-coated group. In vivo rat implant model studies revealed no excessive local and systemic inflammatory responses in the F-DLC group. The thickness of the fibrous tissue capsule surrounding the F-DLC-coated disk was almost equal to that of the noncoated SUS316L disk, which has the favorable biocompatibility for metallic implant materials. F-DLC coating thus appears to be a promising candidate for use as a coating material in blood-contacting devices.     

Polyethylene glycol-containing polyurethane hydrogel coatings for improving the biocompatibility of neural electrodes

The instability of the interface between chronically implanted neuroprosthetic devices and neural tissue is a major obstacle to the long-term use of such devices in clinical practice. In this study, we investigate the feasibility of polyethylene glycol (PEG)-containing polyurethane (PU) hydrogel as coatings for polydimethylsiloxane (PDMS)-based neural electrodes in order to achieve a stable neural interface. The influence of PU hydrogel coatings on electrode electrochemical behaviour was investigated. Importantly, the biocompatibility of PU hydrogel coatings was evaluated in vitro and in vivo. Changes in the electrochemical impedance of microelectrodes with PU coatings were negligible. The amount of protein adsorption on the PDMS substrate was reduced by 93% after coating. Rat pheochromocytoma (PC12) cells exhibited more and longer neurites on PU films than on PDMS substrates. Furthermore, PDMS implants with (n=10) and without (n=8) PU coatings were implanted into the cortex of rats and the tissue response to the implants was evaluated 6 weeks post-implantation. GFAP staining for astrocytes and NeuN staining for neurons revealed that PU coatings attenuated glial scarring and reduced the neuronal cell loss around the implants. All of these findings suggest that PU hydrogel coating is feasible and favourable for neural electrode applications.     

In vitro and in vivo investigations into the biocompatibility of diamond-like carbon (DLC) coatings for orthopedic applications

Diamond-like carbon (DLC) shows great promise as a durable, wear- and corrosion-resistant coating for biomedical implants. The effects of DLC coatings on the musculoskeletal system have not been investigated in detail. In this study, DLC coatings were deposited on polystyrene 24-well tissue culture plates by fast-atom bombardment from a hexane precursor. Two osteoblast-like cell lines were cultured on uncoated and DLC-coated plates for periods of up to 72 h. The effects of DLC coatings on cellular metabolism were investigated by measuring the production of three osteoblast-specific marker proteins: alkaline phosphatase, osteocalcin, and type I collagen. There was no evidence that the presence of the DLC coating had any adverse effect on any of the parameters measured in this study. In a second series of experiments, DLC-coated cobalt-chromium cylinders were implanted in intramuscular locations in rats and in transcortical sites in sheep. Histologic analysis of specimens retrieved 90 days after surgery showed that the DLC-coated specimens were well tolerated in both sites. These data indicate that DLC coatings are biocompatible in vitro and in vivo, and further investigations into their long-term biological and tribological performance are now warranted.     

Immobilized RGD peptides on surface-grafted dextran promote biospecific cell attachment

Dextran has recently been investigated as an alternative to poly(ethylene glycol) (PEG) for low protein-binding, cell-resistant coatings on biomaterial surfaces. Although antifouling properties of surface-grafted dextran and PEG are quite similar, surface-bound dextran has multiple reactive sites for high-density surface immobilization of biologically active molecules. We recently reported nontoxic aqueous methods to covalently immobilize dextran on material surfaces. These dextran coatings effectively limited cell adhesion and spreading in the presence of serum-borne cell adhesion proteins. In this study we utilized the same nontoxic aqueous methods to graft cell adhesion peptides on low protein-binding dextran monolayer surfaces. Chemical composition of all modified surfaces was verified by X-ray photoelectron spectroscopy (XPS). Surface-grafted cell adhesion peptides stimulated endothelial cell, fibroblast, and smooth muscle cell attachment and spreading in vitro. In contrast, surface-grafted inactive peptide sequences did not promote high levels of cell interaction. Surface-grafted high affinity cyclic RGD peptides promoted cell type-dependent interactions. With dextran-based surface coatings, it will be possible to develop well-defined surface modifications that promote specific cell interactions and perhaps better performance in long-term biomaterial implants.     

类金刚石膜的生物医学性能与应用

类金刚石膜具有高硬度、高光洁度、低摩擦系数、高耐磨性及耐腐蚀性等很多优异性能,并且易于掺杂而改变性能以适应不同需要.良好的生物医学性能也使其在体内植入物及医疗器械的表面改性方面有广阔的应用前景.主要就类金刚石膜在生物医学方面的性能与应用进行综述.     

Alleviation of capsular formations on silicone implants in rats using biomembrane-mimicking coatings

Despite their popular use in breast augmentation and reconstruction surgeries, the limited biocompatibility of silicone implants can induce severe side effects, including capsular contracture – an excessive foreign body reaction that forms a tight and hard fibrous capsule around the implant. This study examines the effects of using biomembrane-mimicking surface coatings to prevent capsular formations on silicone implants. The covalently attached biomembrane-mimicking polymer, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), prevented nonspecific protein adsorption and fibroblast adhesion on the silicone surface. More importantly, in vivo capsule formations around PMPC-grafted silicone implants in rats were significantly thinner and exhibited lower collagen densities and more regular collagen alignments than bare silicone implants. The observed decrease in α-smooth muscle actin also supported the alleviation of capsular formations by the biomembrane-mimicking coating. Decreases in inflammation-related cells, myeloperoxidase and transforming growth factor-β resulted in reduced inflammation in the capsular tissue. The biomembrane-mimicking coatings used on these silicone implants demonstrate great potential for preventing capsular contracture and developing biocompatible materials for various biomedical applications.     

Adhesion, cytoskeletal architecture and activation status of primary human macrophages on a diamond-like carbon coated surface.

Diamond-like carbon is a promising surface coating for biomedicinal implants like coronary stents or hip joints. Before widespread clinical use of this material, its biocompatibility has to be thoroughly assessed. Cells likely to encounter a diamond-like coated implant in the human body are cells of the monocytic lineage. Their interaction with the diamond-like carbon coated surface will probably critically influence the fate of the implant, as monocytes orchestrate inflammatory reactions and also affect osseointegration of implants. We therefore investigated adhesion, cytoarchitecture and activation status of primary human monocytes and their differentiated derivatives, macrophages, on diamond-like coated glass coverslips using immunofluorescence technique. We show that adhesion of primary monocytes to a diamond-like-coated coverslip is slightly, but not significantly, enhanced in comparison to uncoated coverslips, while the actin and microtubule cytoskeletons of mature macrophages show a normal development. The activation status of macrophages, as judged by polarization of the cell body, was not affected by growth on a diamond-like carbon surface. We conclude that diamond-like carbon shows good indications for biocompatibility to blood monocytes in vitro. It is therefore unlikely that contact with a diamond-like carbon coated surface in the human body will elicit inflammatory signals by these cells.     

热化学法制备CaTiO3涂层及涂层的生物相容性

背景：钛酸钙(CaTiO₃)作为一种有前途的涂层,可应用于钛基医用植入体表面。 目的：通过一种简单的热化学处理技术,在Ti6Al4V基体上制备均匀的CaTiO₃涂层,同时通过培养成骨细胞观察涂层生物相容性。 方法：通过将Ti6Al4V基体埋于无水硝酸钙(Ca(NO₃)₂)粉末中,并且升高温度;当温度处在Ca(NO₃)2熔点以上时,在Ti6Al4V基体上能够生成一层均匀的CaTiO₃层。同时在热化学处理温度为570 ℃的Ti6Al4V基体材料上进行成骨细胞培养,观察所制备涂层的生物相容性。 结果与结论：该涂层转化只有在(Ca(NO₃)₂)熔点(561 ℃)以上时才会发生,同时随着处理温度的升高,CaTiO₃晶体尺寸随之增大;经过热化学处理后的样品具有良好的生物相容性,对成骨细胞黏附、增殖具有更好的促进作用。该方法简单、有效,所制备的涂层具有良好的生物相容性,有望在钛植入体表面处理中获得应用,以提高金属表面与细胞、组织的相容性。     

Osteoblast interaction with DLC-coated Si substrates

Diamond-like carbon (DLC) coating is a convenient means of modifying material surfaces that are sensitive to wear, such as titanium and silica substrates. This work aims to evaluate the osteoblast-like cells' response to DLC-coated Si (Si-DLC), which was treated under different conditions. DLC and deuterated DLC films were deposited by plasma-enhanced chemical vapor deposition to obtain a 200-nm-thick layer on all the samples. Three types of precursor gas were applied for deposition: pure methane (CH(4)), pure deuterated methane (CD(4)) and their half/half mixture. All surface treatments were performed under two different self-bias voltages (V(sb)): -400 and -600V. The modified surfaces were characterized by X-ray photoelectron spectroscopy, Raman spectroscopy, Rutherford backscattering spectroscopy, elastic recoil detection analysis, X-ray reflectometry and the sessile-drop method. MC3T3-E1 osteoblasts were cultured on the Si-DLC wafers for 3 and 6 days. Biological tests to measure cell proliferation, cell vitality, cell morphology and cell adhesion were performed. All DLC coatings produced a slightly more hydrophobic state than non-treated Si. Certain types of amorphous DLC coating, such as the surface treated under the V(sb) of -600V in pure methane (600CH(4)) or in pure deuterated methane (600CD(4)), offered a significantly higher cell proliferation rate to Si substrate. Scanning electron microscopy observations confirmed that the optimal cell adhesion behavior, among all the treated surfaces, occurred on the surface of the 600CH(4) and 600CD(4) groups, which showed increased amounts of filopodia and microvilli to enhance cell-environment exchange. In conclusion, DLC coating on Si could produce better surface stability and improved cellular responses.     

聚酰亚胺薄膜电极与大鼠视神经胶质细胞的生物相容性研究

为了研究视神经胶质细胞与聚酰亚胺薄膜电极的黏附性及生物相容性、最佳体外电脉冲刺激参数,为人工视觉假体材料的植入提供实验依据,本实验从大鼠神经胶质细胞的体外培养入手,采用细胞毒性试验、黏附试验等多种生物学方法对材料的生物相容性进行系统的评价,并建立电刺激大鼠神经胶质细胞体外模型,观察脉冲电流对神经胶质细胞生物学特性的影响。结果显示:聚酰亚胺薄膜材料的细胞毒性为0～1级,无明显细胞毒性。聚酰亚胺薄膜电极表面的细胞黏附生长良好,该材料对大鼠体外培养的细胞形态无损害,对细胞的生长和增殖均无明显的抑制作用;扫描电镜下观察到神经胶质细胞紧密贴附在材料表面,铺展良好并连接成片。在固定刺激脉冲的宽度和幅度以及刺激时间的情况下,频率越小,对细胞影响越小。以上研究结果提示聚酰亚胺薄膜电极与大鼠视神经胶质细胞有较好的黏附性和生物相容性,并能形成有效的电脉冲刺激,是一种优良的视神经植入材料。     

Tribological behavior of DLC-coated articulating joint implants

Coatings from diamond-like carbon (DLC) have been proven to be an excellent choice for wear reduction in many technical applications. However, for successful adaption to the total joint replacement field, layer performance, stability and adhesion in realistic physiological setups are quintessential and these aspects have not been consistently researched. In our team’s efforts to develop long-term stable DLC implant coatings, test results gained from a simplified linear spinal simulator setup are presented. It is shown that metal-on-metal (MoM) pairs perform well up to 7 million loading cycles, after which they start to generate wear volumes in excess of 20 times those of DLC-coated implants. This is attributed to the roughening observed on unprotected metal surfaces. Furthermore, we illustrate that in contrast to DLC-on-DLC, MoM tribopairs require protein-containing media to establish low-friction conditions. Finally, results of defect monitoring during testing are presented, showing catastrophic failure of layers whose interfaces are too weak with respect to the stress-corrosion-cracking mechanism encountered in vivo.     

Biocompatibility and antibacterial activity of plasma sprayed titania coating grafting collagen and gentamicin

In this article, the plasma sprayed titania coatings were treated by grafting pure and gentamicin loaded collagen to improve the biocompatibility and antibacterial activity. The biocompatibility of the titania coating grafting collagen was evaluated by in vitro cell culturing test. The release rate of gentamicin from collagen was measured in tris-HCl buffer using UV spectrophotometer, and the antibacterial activity of the titania coating with gentamicin against Staphylococcus aureus was examined using the zone of inhibition test. The results showed that collagen was successfully grafted on the surface of titania coatings treated by sulfuric acid. The in vitro cell culturing test revealed that collagen significantly improved the cell adhesion and proliferation on the surface of titania coatings. The gentamicin loaded in collagen matrix could retain a sustained release in tris-HCl for 30 days, which was efficient to protect against the postoperative infection caused by S. aureus. The results indicated that the plasma sprayed titania coating grafting collagen and gentamicin would have the antibacterial activity together with the biocompatibility, which might be beneficial for the long term stability and surgical success rate of implants.     

Hemocompatibility of nitrogen-doped, hydrogen-free diamond-like carbon prepared by nitrogen plasma immersion ion implantation-deposition

Amorphous hydrogenated carbon (a-C:H) has been shown to be a potential material in biomedical devices such as artificial heart valves, bone implants, and so on because of its chemical inertness, low coefficient of friction, high wear resistance, and good biocompatibility. However, the biomedical characteristics such as blood compatibility of doped hydrogen-free diamond-like carbon (DLC) have not been investigated in details. We recently began to investigate the potential use of nitrogen-doped, hydrogen-free DLC in artificial heart valves. In our experiments, a series of hydrogen-free DLC films doped with nitrogen were synthesized by plasma immersion ion implantation-deposition (PIII-D) utilizing a pulsed vacuum arc plasma source and different N to Ar (FN/FAr) gas mixtures in the plasma chamber. The structures and properties of the film were evaluated by Raman spectroscopy, Rutherford backscattering spectrometry (RBS), and X-ray photoelectron spectroscopy (XPS). To assess the blood compatibility of the films and the impact on the blood compatibility by the presence of nitrogen, platelet adhesion tests were conducted. Our results indicate that the blood compatibility of both hydrogen-free carbon films (a-C) and amorphous carbon nitride films are better than that of low-temperature isotropic pyrolytic carbon (LTIC). The experimental results are consistent with the relative theory of interfacial energy and surface tension including both dispersion and polar components. Our results also indicate that an optimal fraction of sp2 bonding is desirable, but an excessively high nitrogen concentration degrades the properties to an extent that the biocompatibility can be worse than that of LTIC.     

Nanometer surface roughness increases select osteoblast adhesion on carbon nanofiber compacts

Carbon nanofibers have exceptional theoretical mechanical properties (such as low weight-to-strength ratios) that, along with possessing nanoscale fiber dimensions similar to crystalline hydroxyapatite found in bone, suggest strong possibilities for use as an orthopedic/dental implant material. To determine, for the first time, cytocompatibility properties pertinent for bone prosthetic applications, osteoblast (bone-forming cells), fibroblast (cells contributing to callus formation and fibrous encapsulation events that result in implant loosening), chondrocyte (cartilage-forming cells), and smooth muscle cell (for comparison purposes) adhesion were determined on carbon nanofibers in the present in vitro study. Results provided evidence that, compared to conventional carbon fibers, nanometer dimension carbon fibers promoted select osteoblast adhesion. Moreover, adhesion of other cells was not influenced by carbon fiber dimensions. In fact, smooth muscle cell, fibroblast, and chondrocyte adhesion decreased with an increase in either carbon nanofiber surface energy or simultaneous change in carbon nanofiber chemistry. To determine properties that selectively enhanced osteoblast adhesion, similar cell adhesion assays were performed on polymer (specifically, poly-lactic-co-glycolic; PLGA) casts of carbon fiber compacts previously tested. Compared to PLGA casts of conventional carbon fibers, results provided the first evidence of enhanced select osteoblast adhesion on PLGA casts of nanophase carbon fibers. The summation of these results demonstrate that due to a high degree of nanometer surface roughness, carbon fibers with nanometer dimensions may be optimal materials to selectively increase osteoblast adhesion necessary for successful orthopedic/dental implant applications.     

Titanium containing amorphous hydrogenated carbon films (a-C: H/Ti): surface analysis and evaluation of cellular reactions using bone marrow cell cultures in vitro

Amorphous hydrogenated carbon (a-C : H) coatings, also called diamond-like carbon (DLC), have many properties required for a protective coating material in biomedical applications. The purpose of this study is to evaluate a new surface coating for bone-related implants by combining the hardness and inertness of a-C : H films with the biological acceptance of titanium. For this purpose, different amounts of titanium were incorporated into a-C : H films by a combined radio frequency (rf) and magnetron sputtering set-up. The X-ray photoelectron spectroscopy (XPS) of air-exposed a-C : H/titanium (a-C : H/Ti) films revealed that the films were composed of TiO2 and TiC embedded in and connected to an a-C : H matrix. Cell culture tests using primary adult rat bone marrow cell cultures (BMC) were performed to determine effects on cell number and on osteoblast and osteoclast differentiation. By adding titanium to the carbon matrix, cellular reactions such as increased proliferation and reduced osteoclast-like cell activity could be obtained, while these reactions were not seen on pure a-C : H films and on glass control samples. In summary, a-C : H/Ti could be a valuable coating for bone implants, by supporting bone cell proliferation while reducing osteoclast-like cell activation.