Effect of diamond-like carbon doped with chromium on cell differentiation, immune activation and apoptosis

Abstract

Diamond-like carbon (DLC) is a biocompatible material that has many potential biomedical applications, including in orthopaedics. DLC layers doped with Cr at atomic percent (at.%) of 0, 0.9, 1.8, 7.3, and 7.7 at.% were evaluated with reference to their osteoinductivity with human bone marrow mesenchymal stromal cells (hMSCs), immune activation potential with RAW 264.7 macrophage-like cells, and their effect on apoptosis in Saos-2 human osteoblast-like cells and neonatal human dermal fibroblasts (NHDFs). At mRNA level, hMSCs on DLC doped with 0.9 and 7.7 at.% of Cr reached higher maximum values of both RUNX2 and alkaline phosphatase. An earlier onset of mRNA production of type I collagen and osteocalcin was also observed on these samples; they also supported the production of both type I collagen and osteocalcin. RAW 264.7 macrophages were screened using a RayBio™ Human Cytokine Array for cytokine production. 10 cytokines were at a concentration more than 2 × as high as the concentration of a positive control, but the values for the DLC samples were only moderately higher than the values on glass. NHDF cells, but not Saos-2 cells, had a higher expression of pro-apoptotic markers Bax and Bim and a lower expression of anti-apoptotic factor BCL-XL in proportion to the Cr content. Increased apoptosis was also proven by annexin V staining. These results show that a Cr-doped DLC layer with a lower Cr content can act as an osteoinductive material with relatively low immunogenicity, but that a higher Cr content can induce cell apoptosis.

Titanium-Doped Diamond-like Carbon Layers as a Promising Coating for Joint Replacements Supporting Osteogenic Differentiation of Mesenchymal Stem Cells

“…The osteogenic differentiation of ADSCs and bmMSCs, which was supported by the appropriate composition of the medium, was successfully obtained during the experiments on all observed samples (i.e., pure DLC and Ti-doped DLC layers, control glass, and PS samples). The osteogenic differentiation of bmMSCs and ADSCs by a similar medium composition was proved in our previous publications [29,30]. In our current results, slight differences between bmMSCs and ADSCs were observed in terms of proliferation potential, the onset of osteogenic differentiation, and dependence on Ti content.…”

“…R7017, Sigma-Aldrich, St. Louis, MO, USA) conversion, which is brought about by the mitochondrial enzymes of the cells. A detailed description of the method was presented in our previous article [ 30 ]. In brief, the resazurin solution (40 μmol/L) in DMEM without phenol red was prepared and added to the cells that had previously been washed with PBS.…”

“…For a study of the early and/or the mid-term phase of the osteogenic differentiation of cells, the activity of ALP was measured on days 7, 14, and 21. The method was described in detail in our previous article [ 30 ]. In brief, 0.1 mg/mL p-nitrophenyl phosphate in the substrate buffer (50 mmol/L glycine, 1 mmol/L MgCl 2 , pH 10.5; Sigma-Aldrich) was added to the samples with cells.…”

“…To detect a late phase of osteogenic differentiation of cells and mainly the deposition of inorganic compounds by the cells, a calcium colorimetric assay was performed on days 14 and 21. The method was described in detail in our previous article [ 30 ]. In brief, the samples with cells were dried and treated with HCl (0.5 mol/L) overnight.…”

Overview on the Antimicrobial Activity and Biocompatibility of Sputtered Carbon-Based Coatings

“…Cr-doped DLC layers of different Cr concentrations have been used to evaluate osteoinductivity with human bone marrow mesenchymal stromal cells (hMSCs), along with the immune activation potential with RAW 264.7 macrophage-like cells and their effect on apoptosis in Saos-2 human osteoblast-like cells and neonatal human dermal fibroblasts (NHDFs). Cr-doped DLC layers with an optimized Cr content (0.9 at %) have improved osseointegration, long-term biocompatibility, and the functionality of metallic prostheses for use in humans [194].…”