Hydrogels of biocompatible calcium-crosslinkable polysaccharide gellan gum (GG) were enriched with bioglass particles to enhance (i) mineralization with calcium phosphate (CaP); (ii) antibacterial properties and (iii) growth of bone-forming cells for future bone regeneration applications. Three bioglasses were compared, namely one calcium-rich and one calcium-poor preparation both produced by a sol-gel technique (hereafter referred to as A2 and S2, respectively) and one preparation of composition close to that of the commonly used 45S5 type (hereafter referred to as NBG). Incubation in SBF for 7 d, 14 d and 21 d caused apatite formation in bioglass-containing but not in bioglass-free samples, as confirmed by FTIR, XRD, SEM, ICP-OES, and measurements of dry mass, i.e. mass attributable to polymer and mineral and not water. Mechanical testing revealed an increase in compressive modulus in samples containing S2 and NBG but not A2. Antibacterial testing using biofilm-forming meticillin-resistant staphylococcus aureus (MRSA) showed markedly higher antibacterial activity of samples containing A2 and S2 than samples containing NBG and bioglass-free samples. Cell biological characterization using rat mesenchymal stem cells (rMSCs) revealed a stimulatory effect of NBG on rMSC differentiation. The addition of bioglass thus promotes GG mineralizability and, depending on bioglass type, antibacterial properties and rMSC differentiation.
Idiopathic pes equinovarus (clubfoot) is a congenital deformity of the feet and lower legs. Clubfoot belongs to a group of fibro‐proliferative disorders but its origin remains unknown. Our study aimed to achieve the first complex proteomic comparison of clubfoot contracted tissue of the foot (medial side; n = 16), with non‐contracted tissue (lateral side; n = 13). We used label‐free mass spectrometry quantification and immunohistochemistry. Seven proteins were observed to be significantly upregulated in the medial side (asporin, collagen type III, V, and VI, versican, tenascin‐C, and transforming growth factor beta induced protein) and four in the lateral side (collagen types XII and XIV, fibromodulin, and cartilage intermediate layer protein 2) of the clubfoot. Comparison of control samples from cadavers brought only two different protein concentrations (collagen types I and VI). We also revealed pathological calcification and intracellular positivity of transforming growth factor beta only in the contracted tissue of clubfoot. Most of the 11 differently expressed proteins are strongly related to the extracellular matrix architecture and we assume that they may play specific roles in the pathogenesis of this deformity. These proteins seem to be promising targets for future investigations and treatment of this disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res
Nanocrystalline diamond (NCD) films are promising materials for bone implant coatings because of their biocompatibility, chemical resistance, and mechanical hardness. Moreover, NCD wettability can be tailored by grafting specific atoms. The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition and grafted by hydrogen atoms (H-termination) or oxygen atoms (O-termination). Human osteoblast-like Saos-2 cells were used for biological studies on H-terminated and O-terminated NCD films. The adhesion, growth, and subsequent differentiation of the osteoblasts on NCD films were examined, and the extracellular matrix production and composition were quantified. The osteoblasts that had been cultivated on the O-terminated NCD films exhibited a higher growth rate than those grown on the H-terminated NCD films. The mature collagen fibers were detected in Saos-2 cells on both the H-terminated and O-terminated NCD films; however, the quantity of total collagen in the extracellular matrix was higher on the O-terminated NCD films, as were the amounts of calcium deposition and alkaline phosphatase activity. Nevertheless, the expression of genes for osteogenic markers – type I collagen, alkaline phosphatase, and osteocalcin – was either comparable on the H-terminated and O-terminated films or even lower on the O-terminated films. In conclusion, the higher wettability of the O-terminated NCD films is promising for adhesion and growth of osteoblasts. In addition, the O-terminated surface also seems to support the deposition of extracellular matrix proteins and extracellular matrix mineralization, and this is promising for better osteoconductivity of potential bone implant coatings.
The following materials based on four allotrope types of nanocarbons were investigated: (1) fullerene C60 and hybrid C60/Ti films, (2) composites of synthetic polymers and carbon nanotubules (i.e., carbon nanohorns and carbon nanotubes), (3) graphene‐based materials (films and three‐dimensional scaffolds), and (4) nanocrystalline diamond‐based materials (films and nanofibrous scaffolds loaded with nanodiamond particles). In general, all these substrates provided a good support for colonization with human osteoblast‐like cells of the lines MG‐63, Saos‐2 and U‐2 OS, primary osteoblasts, and also human mesenchymal stem cells (hMSC). In the case of fullerenes C60, this was true for aged, i.e., 1‐year‐old, films. Fresh films, i.e., 1‐week‐old, had a decreased number of initially adhering cells, with less spreading, growth, metabolic activity and viability, though no DNA damage was detected. In the case of C60/Ti composite films, both fresh and aged films supported cell colonization well. The improved cell performance was attributed to structural changes in fullerene molecules, such as fragmentation, oxidation and polymerization, which occur during aging or co‐deposition of C60 and Ti. The addition of single‐wall carbon nanohorns or multi‐wall carbon nanotubes to a terpolymer of polytetrafluoroethylene, polyvinyldifluoride and polypropylene (PTFE/PVDF/PP) markedly improved the adhesion and growth of bone cells, while no significant changes in cell behavior were found on polysulfone after it had been enriched with the carbon nanotubules mentioned here. Graphene‐based films and scaffolds stimulated the adhesion and osteogenic differentiation of bone‐forming cells even in the absence of cell adhesion‐mediating molecules and differentiation factors in the cell culture medium. Nanocrystalline diamond films proved to be excellent substrates for cell adhesion, growth and osteogenic differentiation, and this cell behavior was further improved by boron doping (concentration of 133–6700 ppm) or by oxygen termination of these films. The addition of diamond nanoparticles to nanofibrous poly(lactide‐co‐glycolide) (PLGA) scaffolds increased the proliferation of hMSC and supported the adhesion and growth of MG‐63 cells in an extent similar to cell culture polystyrene. However, on nanofibrous poly(L‐lactide) scaffolds with diamond nanoparticles, the growth of MG‐63 cells decreased with increasing nanoparticle concentration.
Mineralization of hydrogel biomaterials is desirable to improve their suitability as materials for bone regeneration. In this study, gellan gum (GG) hydrogels were formed by simple mixing of GG solution with bioactive glass microparticles of 45S5 composition, leading to hydrogel formation by ion release from the amorphous bioactive glass microparticles. This resulted in novel injectable, self-gelling composites of GG hydrogels containing 20% bioactive glass. Gelation occurred within 20 min. Composites containing the standard 45S5 bioactive glass preparation were markedly less stiff. X-ray microcomputed tomography proved to be a highly sensitive technique capable of detecting microparticles of diameter approximately 8 μm, that is, individual microparticles, and accurately visualizing the size distribution of bioactive glass microparticles and their aggregates, and their distribution in GG hydrogels. The widely used melt-derived 45S5 preparation served as a standard and was compared with a calcium-rich, sol-gel derived preparation (A2), as well as A2 enriched with zinc (A2Zn5) and strontium (A2Sr5). A2, A2Zn, and A2Sr bioactive glass particles were more homogeneously dispersed in GG hydrogels than 45S5. Composites containing all four bioactive glass preparations exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus. Composites containing A2Zn5 and A2Sr5 bioactive glasses supported the adhesion and growth of osteoblast-like cells and were considerably more cytocompatible than 45S5. All composites underwent mineralization with calcium-deficient hydroxyapatite upon incubation in simulated body fluid. The extent of mineralization appeared to be greatest for composites containing A2Zn5 and 45S5. The results underline the importance of the choice of bioactive glass when preparing injectable, self-gelling composites.
The mechanical strength, durability, corrosion resistance, and biocompatibility of metal alloys based on zirconium (Zr) and titanium (Ti) make them desirable materials for orthopedic implants. However, as bioinert metals, they do not actively promote bone formation and integration. Here we report a plasma coating process for improving integration of such metal implants with local bone tissue. The coating is a stable carbonbased plasma polymer layer that increased surface wettability by 28%, improved surface elasticity to the range exhibited by natural bone, and additionally covalently bound the extracellular matrix protein, tropoelastin, in an active conformation. The thus biofunctionalized material was significantly more resistant to medical-grade sterilization by steam, autoclaving or gamma-ray irradiation, retaining >60% of the adhered tropoelastin molecules and preserving full bioactivity. The interface of the coating and metal was robust so as to resist delamination during surgical insertion and in vivo deployment, and the plasma process employed was utilized to also coat the complex 3D geometries typical of orthopedic implants. Osteoblast-like osteosarcoma cells cultured on the biofunctionalized Zr surface exhibited a significant 30% increase in adhesion and up to 70% improvement in proliferation. Cells on these materials also showed significant early stage up-regulation of bone marker expression (alkaline phosphatase, 1.8 fold; osteocalcin, 1.4 fold), and sustained up-regulation of these genes (alkaline phosphatase, 1.3 fold; osteocalcin, 1.2 fold) in osteogenic conditions. In addition, alkaline phosphatase production significantly increased (2-fold) on the functionalized surfaces, whereas bone mineral deposition increased by 30% above background levels compared to bare Zr. These findings have the potential to be readily translated to the development of improved Zr and Ti-based implants for accelerated bone repair.
Diamond in the allotrope form consists of carbon atoms arranged in a cubic crystal structure covalently bonded in sp 3 hybridization. Diamond has emerged as a very promising material for various biomedical applications due to its excellent mechanical properties (hardness, low friction coefficient, good adhesiveness to the underlying substrate, good interlayer cohesion), optical properties (the ability to emit intrinsic luminescence), electrical properties (good insulator in the pristine state and semiconductor after doping), chemical resistance (low chemical reactivity and resistance to wet etching) and biocompatibility (little if any toxicity and immunogenicity). For advanced biomedical applications, diamond is promising particularly in its nanostructured forms, namely nanoparticles, nanostructured diamond films and composite scaffolds in which diamond nanoparticles are dispersed in a matrix (mainly nanodiamond-loaded nanofibrous scaffolds). This chapter summarizes both our long-term experience and that of other research groups in studies focusing on the interaction of cells (particularly bone-derived cells) with nanodiamonds as nanoparticles, thin films and composites with synthetic polymers. Their potential applications in bioimaging, biosensing, drug delivery, biomaterial coating and tissue engineering are also reviewed.
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