Grygoryan1 A.S., Filonov2 M.R., Shtansky2 D.V., Selezneva3 I.V., Toporkova1 A.K.
(1 - Central Research Institute for Stomatology, Timur Frunze Street 16, Moscow, 119021 Russia; 2 - Moscow State Institute of Steel and Alloys, Leninsky Prospect 4, Moscow 119991, Russia; 3 - Institute of Theoretical and Experimental Biophysics RAS, Institutskaja Street 3, Pushchino, Moscow Region, 142290, Russia; Contact: Alexey Grigoryan, Central Research Institute for Stomatology, Timur Frunze Street 16, Moscow, 119021 Russia; e-mail: asgrian@gmail.com)
The work was made with the help of financial support of the Fund SRDF (RUE1-1506-MO-05) and Fund RFBR (06-04-49472-a).
Design of bioresorbable scaffolds in composite implants without disadvantages of metal carriers remains a significant problem in oral and maxillofacial surgery. The objective of the present study is to search for synthetic materials for scaffold in hybrid implants made of stem-cell carriers or cells-persecutors and individual cells.
We propose a new technology for preparating carriers for composite implants with scaffolds made of synthetic polymer polytetrafluoroethylene (PTFE) with different coatings.
Coating was deposited by SHS-compacting of composite targets TiC0.5, TiC0.5+CaO(10%), TiC0.5+CaO(10%)+KMnO4(2%). Titanium plates (size 1.0x1.0x0.5 cm) or PTFE plates (size 1.0x1.0x0.5 cm; 36% porosity) were used as substrates. Titanium plates were prepared by mechanical polishing and ultrasonic cleaning in ethanol. Titanium substrates were additionally cleaned under the action of a highly negative bias UBias = - 400 to -500 V and by Ar+ ions using a slit ion source (ion energy, 2.5 keV) in 10 min. The targets were magnetron sputtered for 60 min in a mixture of argon and nitrogen (at 14% nitrogen partial pressure). Sputtering was carried out at a pressure of 0.1-0.2 Pa in a vacuum chamber. The substrate temperature was in the range of 150-170°С.
The applicability of the composite material as a scaffold for hybrid implants was evaluated by testing adhesion and spreading of human embryonic fibroblasts on the surface of known compositions. Skin-muscle fibroblasts were obtained from human 6-month-old embryos. Cells were cultivated in 5% CO2 (v/v) atmosphere in DMEM/199 media (1:1) with 10% horse serum and 100 U/ml penicillin/streptomycin. After 10 passages, cell culture (CD133-, Cd117-, CD45-, CD90+, CD54-, CD62L-, CD62P-, CD9+, CD34-, CD31-, CD71-, CD20-, CD157-, CD106+, CD62E+) was used for testing the composite material. Cells were seeded on the surface of samples at density 35x103 per cm and incubated for 72 hr.
Four samples were examined by luminescent microscopy after incubation of fibroblasts on their surfaces: 1) Metal (Ti) deposited with Ti-Ca-C-O-N, 2) PTFE deposited with Ti-Ca-C-O-N, 3) Metal (Ti) deposited with Ti-Ca-Mn-C-O-N, and 4) PTFE deposited with Ti-Ca-Mn-C-O-N.
Morphology and cell viability was investigated using a LUMAM-I2 microscope equipped with FS-1-4 filter and 0.0002% (w/v in phosphate buffer) acridine orange as staining. This metachromatic stain selectively interacts with DNA and RNA by intercalation and electrostatic attraction, respectively. DNA-intercalated dye fluoresces green (525 nm); RNA-electrostatically bound acridine orange fluoresces red (>630 nm). Therefore, this approach may help determine an overall activity, proliferation and apoptosis of cells.
Luminescent microscopy showed many oblong cells often interlaced with one another, confusedly spread or formed clusters. These cells had large elongate nucleus, which gave pale-green color. Several areas of orange and red luminescence were observed in cell cytoplasm. Bright green inclusions were visible in nucleuses of single cells (Ti deposited with Ti-Ca-Mn-C-O-N).
Samples seeded with embryonic fibroblast cells were investigated by scanning electron microscopy (SEM): 1) Ti without coating; 2) Ti deposited with Ti-Ca-C-O-N; 3) Ti deposited with Ti-Ca-Mn-C-O-N; 4) PTFE without coating; 5) PTFE deposited with Ti; 6) PTFE deposited with Ti-Ca-C-O-N; 7) PTFE deposited with Ti-Ca-Mn-C-O-N.
SEM investigation showed that only a small number of human embryonic fibroblasts attached to the surface of Ti samples without coating, and typically cells did not cover them completely. We suggest that it is an indication of a week binding capacity of the tested material. (Fig. 1A). At the same time, embryonic fibroblasts bound to and covered the surfaces of samples Ti coated with Ti-Ca-C-O-N or Ti-Ca-Mn-C-O-N.
No bound cells were observed on PTFE samples without coating. (Fig. 1B). However, good attachment and cell coverage were found on the samples of PTFE coated with Ti, Ti-Ca-C-O-N and Ti-Ca-Mn-C-O-N. The most effective distribution was found on the samples of PTFE with Ca-contained coating.
Thus, this investigation showed that PTFE coated with the above types of ceramic provides a promising methodology for making scaffolds for stem cells or cells-precusores in composite implants.
Figure 1. Scanning electron microscopy images. A. An isolated group of cells displayed on the surface of Ti sample; B. PTFE without coating. No bound cells are observed on the surface. C. PTFE coated with Ti-Ca-C-O-N. Multiple bound cells covering the surface are observed. D. PTFE coated with Ti-Ca-Mn-C-O-N. Attached cells are connected with appendixes and formed syncytium.
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