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.
- MeSH
- aktiny metabolismus MeSH
- alkalická fosfatasa genetika metabolismus MeSH
- apoptóza účinky léků imunologie MeSH
- buněčná adheze účinky léků MeSH
- buněčná diferenciace účinky léků imunologie MeSH
- buněčné linie MeSH
- chrom farmakologie MeSH
- cytokiny metabolismus MeSH
- diamant farmakologie MeSH
- fibroblasty cytologie účinky léků MeSH
- kolagen typu I genetika metabolismus MeSH
- lidé MeSH
- makrofágy účinky léků metabolismus MeSH
- mezenchymální kmenové buňky cytologie účinky léků imunologie metabolismus MeSH
- myši MeSH
- osteogeneze účinky léků MeSH
- osteokalcin genetika metabolismus MeSH
- počet buněk MeSH
- proliferace buněk účinky léků MeSH
- protein PEBP2alfaA genetika metabolismus MeSH
- regulace genové exprese účinky léků MeSH
- RNA metabolismus MeSH
- tvar buňky účinky léků MeSH
- vápník metabolismus MeSH
- vinkulin metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aktiny MeSH
- alkalická fosfatasa MeSH
- chrom MeSH
- cytokiny MeSH
- diamant MeSH
- kolagen typu I MeSH
- osteokalcin MeSH
- protein PEBP2alfaA MeSH
- RNA MeSH
- vápník MeSH
- vinkulin MeSH
Mandibular/alveolar (m/a) bone, as a component of the periodontal apparatus, allows for the proper tooth anchorage and function of dentition. Bone formation around the tooth germs starts prenatally and, in the mouse model, the mesenchymal condensation turns into a complex vascularized bone (containing osteo-blasts, -cytes, -clasts) within only two days. This very short but critical period is characterized by synchronized cellular and molecular events. The m/a bone, as others, is subjected to endocrine regulations. This not only requires vasculature to allow the circulation of active molecules (ligands), but also the expression of corresponding cell receptors to define target tissues. This contribution aimed at following the dynamics of calciotropic receptors´ expression during morphological transformation of a mesenchymal condensation into the initial m/a bone structure. Receptors for all three calciotropic systemic regulators: parathormone, calcitonin and activated vitamin D (calcitriol), were localized on serial histological sections using immunochemistry and their relative expression was quantified by q-PCR. The onset of calciotropic receptors was followed along with bone cell differentiation (as checked using osteocalcin, sclerostin, RANK and TRAP) and vascularization (CD31) during mouse prenatal/embryonic (E) days 13-15 and 18. Additionally, the timing of calciotropic receptor appearance was compared with that of estrogen receptors (ESR1, ESR2). PTH receptor (PTH1r) appeared in the bone already at E13, when the first osteocalcin-positive cells were detected within the mesenchymal condensation forming the bone anlage. At this stage, blood vessels were only lining the condensation. At E14, the osteoblasts started to express the receptor for activated vitamin D (VDR). At this stage, the vasculature just penetrated the forming bone. On the same day, the first TRAP-positive (but not yet multinucleated) osteoclastic cells were identified. However, calcitonin receptor was detected only one day later. The first Sost-positive osteocytes, present at E15, were PTH1r and VDR positive. ESR1 almost copied the expression pattern of PTH1r, and ESR2 appearance was similar with VDR with a significant increase between E15 and E18. This report focuses on the in vivo situation and links morphological transformation of the mesenchymal cell condensation into a bone structure with dynamics of cell differentiation/maturation, vascularization and onset of receptors for calciotropic endocrine signalling in developing m/a bone.
- Klíčová slova
- Activated vitamin D, Calcitonin, Osteogenesis, Parathormone, Receptors,
- MeSH
- adaptorové proteiny signální transdukční analýza genetika MeSH
- buněčná diferenciace MeSH
- imunohistochemie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- mandibula růst a vývoj MeSH
- messenger RNA analýza MeSH
- myši MeSH
- osteoblasty fyziologie MeSH
- osteocyty fyziologie MeSH
- osteogeneze fyziologie MeSH
- osteokalcin analýza genetika MeSH
- osteoklasty fyziologie MeSH
- receptory kalcitoninu metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- adaptorové proteiny signální transdukční MeSH
- messenger RNA MeSH
- osteokalcin MeSH
- receptory kalcitoninu MeSH
- Sost protein, mouse MeSH Prohlížeč
OBJECTIVES: Bioactive peptides derived from receptor binding motifs of native proteins are a potent source of bioactive molecules that can induce signalling pathways. These peptides could substitute for osteogenesis promoting supplements. The work presented here compares three kinds of bioactive peptides derived from collagen III, bone morphogenetic protein 7 (BMP-7) and BMP-2 with their potential osteogenic activity on the model of porcine mesenchymal stem cells (pMSCs). MATERIALS AND METHODS: pMSCs were cultured on electrospun polycaprolactone nanofibrous scaffolds with different concentrations of the bioactive peptides without addition of any osteogenic supplement. Analysis of pMSCs cultures included measurement of the metabolic activity and proliferation, immunofluorescence staining and also qPCR. RESULTS: Results showed no detrimental effect of the bioactive peptides to cultured pMSCs. Based on qPCR analysis, the bioactive peptides are specific for osteogenic differentiation with no detectable expression of collagen II. Our results further indicate that peptide derived from BMP-2 protein promoted the expression of mRNA for osteocalcin (OCN) and collagen I significantly compared to control groups and also supported deposition of OCN as observed by immunostaining method. CONCLUSION: The data suggest that bioactive peptide with an amino acid sequence of KIPKASSVPTELSAISTLYL derived from BMP-2 protein was the most potent for triggering osteogenic differentiation of pMSCs.
- Klíčová slova
- bioactive peptides, electrospun scaffold, mesenchymal stem cells, osteogenic differentiation,
- MeSH
- buněčná diferenciace účinky léků MeSH
- buněčné kultury MeSH
- kolagen typ II genetika metabolismus MeSH
- kolagen typu I genetika metabolismus MeSH
- konfokální mikroskopie MeSH
- kultivované buňky MeSH
- mezenchymální kmenové buňky cytologie účinky léků metabolismus MeSH
- mikroskopie elektronová rastrovací MeSH
- miniaturní prasata MeSH
- osteogeneze účinky léků MeSH
- osteokalcin genetika metabolismus MeSH
- peptidy chemie farmakologie MeSH
- prasata MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- kolagen typ II MeSH
- kolagen typu I MeSH
- osteokalcin MeSH
- peptidy MeSH
