Ultrananocrystalline Diamond-Coated Microporous Silicon Nitride Membranes for Medical Implant Applications
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- Skoog, S.A., Sumant, A.V., Monteiro-Riviere, N.A. et al. JOM (2012) 64: 520. doi:10.1007/s11837-012-0300-x
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Ultrananocrystalline diamond (UNCD) exhibits excellent biological and mechanical properties, which make it an appropriate choice for promoting epidermal cell migration on the surfaces of percutaneous implants. We deposited a ~150 nm thick UNCD film on a microporous silicon nitride membrane using microwave plasma chemical vapor deposition. Scanning electron microscopy and Raman spectroscopy were used to examine the pore structure and chemical bonding of this material, respectively. Growth of human epidermal keratinocytes on UNCD-coated microporous silicon nitride membranes and uncoated microporous silicon nitride membranes was compared using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. The results show that the UNCD coating did not significantly alter the viability of human epidermal keratinocytes, indicating potential use of this material for improving skin sealing around percutaneous implants.
Percutaneous implants are commonly used for treatment of medical and dental conditions; according to Jacobsson and Tjellstrom,1 over one million percutaneous medical devices are implanted in the USA each year. In these implants, the foreign object crosses the skin, resulting in a permanent defect at the skin–implant interface.2 Early percutaneous implants included devices for charging cardiac pacemakers and hemodialysis.3,4 More recent applications of percutaneous implants have involved attachment of prostheses to bony tissue, such as prosthetic limb attachments.5–7 Although many cochlear implants use wireless technology, percutaneous implants known as bone-anchored hearing aids remain in use.8 In addition, percutaneous active medical devices, such as glucose sensors, are being developed.9
Suboptimal skin sealing around the implant is observed with many conventional percutaneous implant materials;9 for example, work by Snyder et al.10 showed that full-thickness skin grafts that were placed around bone-anchored hearing aids were ineffective in 46.7% of the patients; diabetes mellitus, smoking, and use of inhaled steroids may impair wound healing at the implant site. Infection of percutaneous implants may lead to loss of implant functionality or even patient mortality.9 Recent research efforts have sought to optimize the skin–material interface of percutaneous implants.5
von Recum and Park11 noted that implant materials with porous surfaces can facilitate migration of epidermal cells, enabling the development of a seal that resists movement of fluid and microorganisms. Pioneering work by Winter12 indicated that pore size larger than 40 μm is necessary for migration of connective tissue. Migration of epithelial basal cells, known as permigration, follows that of connective tissue.13
Several researchers have investigated use of smooth carbon materials in percutaneous implants due to the fact that these materials are associated with resistance to fatigue, resistance to degradation, chemical inertness, and an absence of tissue irritation;14–17 for example, Krouskop et al.18 developed a porous vitreous carbon material with 200–500 μm average pore diameter for growth of tissue into percutaneous devices They implanted this material in canine, leporine, and porcine models and showed that the implant sites functioned appropriately and resisted infiltration of normal skin bacteria for periods of time up to 48 months.19 In subsequent work, Nowicki et al.20 evaluated porous vitreous carbon in porcine and leporine models. They showed that Escherichia coli and Staphylococcus aureus were able to bind to porous carbon; however, the skin–porous carbon interface resisted infection by bacteria in the same manner as control skin. Tagusari et al.17 created a fine trabecular carbon material with a maximal pore size greater than 200 μm by infiltrating pyrolytic carbon on the surface of carbon fiber yarn-wrapped carbon rod They performed percutaneous implantation in a bovine model and found that the pores filled with mature connective tissue and blood vessels.
Recent research efforts have evaluated the use of another form of carbon, ultrananocrystalline diamond (UNCD), in medical devices.21 Microwave plasma-enhanced chemical vapor deposition is used to prepare UNCD films. This material is deposited in a hydrogen-deficient, argon-rich atmosphere at temperatures as low as 400°C. Due to the limited amount of hydrogen, minimal regasification of small grains and high renucleation are observed. UNCD contains grains below 10 nm, commonly 2–5 nm.22,23 Material in these grains (95–98% of the entire material) is sp3-hybridized carbon, and material at the grain boundaries is sp2-hybridized carbon. Hamilton et al.24 noted that UNCD is an exceptionally smooth material, exhibiting a coefficient of friction below 0.007 in an environment that contains sufficient humidity. Bajaj et al.25 compared MC3T3 (osteoblastic), PC12 (neuronal), and HeLa (epithelial) cell behavior on UNCD, platinum, and silicon surfaces. Cell spreading was noted to be greater on UNCD than on silicon or platinum; in addition, cells grown on UNCD showed an increase in cell attachment. Furthermore, cell rounding occurred less on UNCD surfaces than on silicon surfaces or platinum surfaces. These results indicate that UNCD is biocompatible and is not cytotoxic. Xiao et al.26 considered use of UNCD thin films in retinal microchips. They implanted UNCD-coated silicon in the eyes of a leporine model for up to 6 months and showed that the completely coated materials were biologically inert. On the other hand, acute tissue reactions were associated with incompletely coated materials.
In the present study, we deposited a ~150 nm thick UNCD film on a commercially available microporous silicon nitride membrane and evaluated the growth of human epidermal keratinocytes on this material. Silicon nitride is an appropriate substrate material since there is a low thermal expansion coefficient mismatch between silicon nitride and diamond, enhancing film–substrate adhesive strength and minimizing interfacial residual stresses at the film–substrate interface.31,32 In addition, silicon nitride exhibits excellent wear resistance, high hardness, high fracture toughness, and low coefficient of friction.31,32 Several researchers have demonstrated that silicon nitride is a biocompatible material. Roy et al., Cappi et al., and Neumann et al. used in vitro assays involving human lung (WI-38) cells, mouse fibroblast cells (L929) cells, and human mesenchymal stem cells (hMSC) to demonstrate biocompatibility of silicon nitride.33–35 The structural, chemical, and biological properties of the UNCD-coated microporous silicon nitride membranes were examined using SEM, Raman spectroscopy, and an in vitro cell viability assay.
Microporous silicon nitride membranes were obtained from a commercial source (Ted Pella Inc., Redding, CA). In this material, a 500 μm × 500 μm membrane is supported by a 200-μm-thick silicon frame that facilitates handling. The 200-nm-thick membrane contains an array of 100 × 100 2.5-μm-diameter pores with a high-density hexagonal layout and 4.5 μm pitch. The root-mean-square (Rq) and mean roughness (Ra) of the membrane are 0.65 ± 0.06 nm and 0.45 ± 0.02 nm, respectively. As noted by the manufacturer, silicon nitride membranes have potential use in biological applications, including use in cell attachment and cell growth studies.36,37
Microwave plasma chemical vapor deposition was used to deposit UNCD thin films on silicon nitride membranes. Nucleation pretreatment was carried out in an ultrasonic bath. Pretreatment involved placement in a nanodiamond suspension in methanol for 3 min; sequential rinsing in methanol, acetone, and isopropanol was subsequently performed. It is important to note that the nucleation pretreatment is a crucial step for controlling lateral UNCD growth. Since the silicon nitride membranes are only 200 nm thick, they need to be carefully handled during the ultrasonication process. A Lambda Technologies microwave plasma chemical vapor deposition system (Lambda Technologies, Raleigh, NC) in the clean room at Center for Nanoscale Materials (Argonne National Laboratory, Argonne, IL) was used for UNCD deposition. The deposition was conducted using a gas ratio of Ar/CH4/H2 of 400 sccm/1.2 sccm/8 sccm. The deposition was performed at substrate temperature of 800°C, working pressure of 120 mbar, and input power of 2100 W. The duration of the deposition process was 2 h.
The pore structure and surface morphology of the UNCD-coated silicon nitride membranes were examined using a Nova 600 NanoLab dual-beam scanning electron microscope/focused ion beam instrument (FEI, Hillsboro, OR). SEM was performed in field-free mode at voltage of 5 kV and current of 0.4 mA. Confocal Raman microscopy was conducted on the UNCD-coated silicon nitride membrane with a Renishaw inVia Raman spectrometer (Renishaw, Gloucestershire, UK). A He-Ne laser with wavelength of 633 nm was used in this study. The laser was introduced to the membrane sample through a ×50 objective lens.
The 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay is a colorimetric assay that evaluates the activity of mitochondrial succinic dehydrogenase, specifically reduction of a yellow tetrazolium salt (MTT) to a purple formazan dye, in living cells.38 Glass coverslips, silicon nitride membranes, and UNCD-coated silicon nitride membranes were cleaned and sterilized for cell culture studies. The substrates were initially sterilized in sterile Petri plates using ultraviolet radiation. Each substrate was then dipped in 70% ethanol and placed in sterile Hank’s balanced salt solution within a 24-well plate. The substrates were rinsed in medium and then equilibrated in medium in the incubator until seeding with human epidermal keratinocytes.
Cryopreserved first pass neonatal human epidermal keratinocytes (Lonza, Walkersville, MD) were seeded in 75 cm2 flasks, grown to 75% confluency, harvested, and seeded on each substrate (n = 4) in a 24-well plate at 90,000 cells per well. Human epidermal keratinocytes were also seeded in the wells alone (n = 4) in order to monitor cell growth (well control). Once the human epidermal keratinocytes in the well controls reached approximately 70% confluency, the medium was changed and the cells were grown for an additional 24 h. To assess cell viability, the medium was replaced with MTT medium and then incubated for 3 h. The medium was aspirated, the cells were rinsed in Hank’s balanced salt solution, and 1 ml of isopropyl alcohol added to cells in each well in order to solubilize the formazan crystals within the cells. Then 100 μl of isopropyl alcohol was transferred to a new 96-well plate and the absorbance was quantitated at 550 nm in a Multiskan RC plate reader (Labsystems Oy, Helsinki, Finland). The well and coverslip controls were normalized to the membrane area. The data presented as percent viability relative to the well controls.
The mean viability data were calculated, and significant differences (p < 0.05) were determined using the PROC GLM procedure (SAS 9.1 for Windows, Cary, NC). When significant differences were found, comparisons were performed using Dunnett’s t test at p < 0.05 level of significance.
Results and Discussion
The results of this work show that microwave plasma chemical vapor deposition may be used to deposit UNCD thin films on microporous silicon nitride membranes. SEM indicated that the pores of the UNCD-coated silicon nitride membrane exhibited uniform geometry and size. The MTT assay depicted that the UNCD coating did not significantly alter the viability of human epithelial keratinocytes. We anticipate that optimization of the deposition process will enable the development of UNCD-coated membranes with appropriate microscale and nanoscale pore dimensions for numerous medical implant applications, including use in percutaneous implants. Studies are necessary to determine the relationship between epithelial cell growth, pore size, and porosity. Natural structures that mimic percutaneous implants (e.g., deer antlers) may also be used to guide percutaneous implant development.44 In addition, seeding pores with cells and functionalizing pores with biologically relevant materials are being considered.45 Biocompatible microporous membranes that facilitate in vitro growth of keratinocytes may also find use as scaffolds for tissue regeneration and for three-dimensional skin models; for example, Pu et al. and Ohsawa et al. demonstrated growth of keratinocytes on microporous membranes at the air–liquid interface.46,47 Bernstram et al.48 initially grew primary human keratinocytes on submerged microporous nylon membranes for 2 weeks and subsequently raised these structures to the air–liquid interface for 3 weeks. The cells on these membranes exhibited characteristics comparable to those of in vivo epidermal cells, including the presence of cornified cell layers, lamellar granules, keratohyaline-like granules, and desmosomes. Such microporous systems may be used for screening potential skin irritants and other types of toxicological studies.49–52
The authors would like to acknowledge use of the Center for Nanoscale Materials, which is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.