Osteocyte-Like Cells Differentiated From Primary Osteoblasts in an Artificial Human Bone Tissue Model.

In vitro models of primary human osteocytes embedded in natural mineralized matrix without artificial scaffolds are lacking. We have established cell culture conditions that favored the natural 3D orientation of the bone cells and stimulated the cascade of signaling needed for primary human osteoblasts to differentiate into osteocytes with the characteristically phenotypical dendritic network between cells. Primary human osteoblasts cultured in a 3D rotating bioreactor and incubated with a combination of vitamins A, C, and D for up to 21 days produced osteospheres resembling native bone. Osteocyte-like cells were identified as entrapped, stellate-shaped cells interconnected through canaliculi embedded in a structured, mineralized, collagen matrix. These cells expressed late osteoblast and osteocyte markers such as osteocalcin (OCN), podoplanin (E11), dentin matrix acidic phosphoprotein 1 (DMP1), and sclerostin (SOST). Organized collagen fibrils, observed associated with the cell hydroxyapatite (HAp) crystals, were found throughout the spheroid and in between the collagen fibrils. In addition to osteocyte-like cells, the spheroids consisted of osteoblasts at various differentiation stages surrounded by a rim of cells resembling lining cells. This resemblance to native bone indicates a model system with potential for studying osteocyte-like cell differentiation, cross-talk between bone cells, and the mineralization process in a bonelike structure in vitro without artificial scaffolds. In addition, natural extracellular matrix may allow for the study of tissue-specific biochemical, biophysical, and mechanical properties. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Trypsin Inhibitor Isolated From Glycine max (Soya Bean) Extraction, Purification, and Characterization.

The current study aims to isolate, purify, and characterize the trypsin inhibitor protein from seeds of soya beans, scientifically known as Glycine max. Its seeds were ground, and the powder was soaked several times using n-hexane. It was added to phosphate buffer saline (PBS) followed by filtration and centrifugation of the PBS dissolved extract. The supernatant was subjected to ammonium sulfate precipitation and about six fractions, 30% to 80% were prepared. The centrifuged pellets obtained from each fraction were dialyzed and run on SDS-PAGE. The trypsin inhibitor protein was precipitated and characterized in 30% pellet and molecular weight was 21.5 kDa compared to protein ladder (ThermoFisher 10-170 kDa). GC-MS analysis revealed the steroid derivatives such as stigmasterol, campesterol, beta-sitosterol, and gamma-tocopherol. Glycine max trypsin inhibitor could be used as a plant-derived drug to overcome the over-activation of trypsin without its real substrate (proteins) becoming activated and start auto digestion leading to pancreatitis.

Using Copper-Doped Mesoporous Bioactive Glass Nanospheres to Impart Anti-Bacterial Properties to Dental Composites.

Experimental dental resin composites containing copper-doped mesoporous bioactive glass nanospheres (Cu-MBGN) were developed to impart anti-bacterial properties. Increasing amounts of Cu-MBGN (0, 1, 5 and 10 wt%) were added to the BisGMA/TEGDMA resin matrix containing micro- and nano-fillers of inert glass, keeping the resin/filler ratio constant. Surface micromorphology and elemental analysis were performed to evaluate the homogeneous distribution of filler particles. The study investigated the effects of Cu-MBGN on the degree of conversion, polymerization shrinkage, porosity, ion release and anti-bacterial activity on S. mutans and A. naeslundii. Experimental materials containing Cu-MBGN showed a dose-dependent Cu release with an initial burst and a further increase after 28 days. The composite containing 10% Cu-MBGN had the best anti-bacterial effect on S. mutans, as evidenced by the lowest adherence of free-floating bacteria and biofilm formation. In contrast, the 45S5-containing materials had the highest S. mutans adherence. Ca release was highest in the bioactive control containing 15% 45S5, which correlated with the highest number of open porosities on the surface. Polymerization shrinkage was similar for all tested materials, ranging from 3.8 to 4.2%, while the degree of conversion was lower for Cu-MBGN materials. Cu-MBGN composites showed better anti-bacterial properties than composites with 45S5 BG.

Efficacy of copolymer scaffolds delivering human demineralised dentine matrix for bone regeneration.

Poly(L-lactide-co-ε-caprolactone) scaffolds were functionalised by 10 or 20 µg/mL of human demineralised dentine matrix. Release kinetics up to 21 days and their osteogenic potential on human bone marrow stromal cells after 7 and 21 days were studied. A total of 390 proteins were identified by mass spectrometry. Bone regeneration proteins showed initial burst of release. Human bone marrow stromal cells were cultured on scaffolds physisorbed with 20 µg/mL and cultured in basal medium (DDM group) or physisorbed and cultured in osteogenic medium or cultured on non-functionalised scaffolds in osteogenic medium. The human bone marrow stromal cells proliferated less in demineralised dentine matrix group and activated ERK/1/2 after both time points. Cells on DDM group showed highest expression of IL-6 and IL-8 at 7 days and expressed higher collagen type 1 alpha 2, SPP1 and bone morphogenetic protein-2 until 21 days. Extracellular protein revealed higher collagen type 1 and bone morphogenetic protein-2 at 21 days in demineralised dentine matrix group. Cells on DDM group showed signs of mineralisation. The functionalised scaffolds were able to stimulate osteogenic differentiation of human bone marrow stromal cells.