RESUMO
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.
RESUMO
AIMS: Patients with type 1 diabetes and end-stage renal disease with simultaneous pancreas and kidney (SPK) or kidney transplants alone (KA) were recruited 9-12 years post transplantation. We investigated differences between these groups with regard to inflammatory parameters and long-term structural changes in kidneys. METHODS: Blood samples were analyzed by ELISA and multiplex for chemokines, cytokines, growth factors, cell adhesion molecules and matrix metalloproteinases. Kidney graft biopsies were analyzed by electron microscopy for glomerular basement membrane thickness. Heparan- and chondroitin sulfate disaccharide structures were determined by size exclusion chromatography mass-spectrometry. RESULTS: The SPK and the KA group had average glycated hemoglobin A1c (HbA1c) of 5.8% (40 mmol/mol) and 8.6% (70 mmol/mol) respectively. SPK recipients also had 16.2% lower body mass index (BMI) and 46.4% lower triglyceride levels compared with KA recipients, compatible with an improved metabolic profile in the SPK group. Plasminogen activator inhibitor (PAI-1), C-reactive protein (CRP) and vascular endothelial growth factor (VEGF) were lower in the SPK group. In kidney graft biopsies of the KA-patients an 81.2% increase in average glomerular basement membrane thickness was observed, accompanied by alterations in heparan sulfate proteoglycan structure. In addition to a decrease in 6-O-sulfated disaccharides, an increase in non-N-sulfated disaccharides with a corresponding slight decrease in N-sulfation was found in kidney biopsies from hyperglycemic patients. CONCLUSIONS: Patients with end stage renal disease subjected to KA transplantation showed impaired inflammatory profile, increased thickness of basement membranes and distinct changes in heparan sulfate structures compared with SPK recipients.
