Modeling In Vitro Osteoarthritis Phenotypes in a Vascularized Bone Model Based on a Bone-Marrow Derived Mesenchymal Cell Line and Endothelial Cells

Abstract

The subchondral bone and its associated vasculature play an important role in the onset of osteoarthritis (OA). Integration of different aspects of the OA environment into multi-cellular and complex human, in vitro models is therefore needed to properly represent the pathology. In this study, we exploited a mesenchymal stromal cell line/endothelial cell co-culture to produce an in vitro human model of vascularized osteogenic tissue. A cocktail of inflammatory cytokines, or conditioned medium from mechanically-induced OA engineered microcartilage, was administered to this vascularized bone model to mimic the inflamed OA environment, hypothesizing that these treatments could induce the onset of specific pathological traits. Exposure to the inflammatory factors led to increased network formation by endothelial cells, reminiscent of the abnormal angiogenesis found in OA subchondral bone, demineralization of the constructs, and increased collagen production, signs of OA related bone sclerosis. Furthermore, inflammation led to augmented expression of osteogenic (alkaline phosphatase (ALP) and osteocalcin (OCN)) and angiogenic (vascular endothelial growth factor (VEGF)) genes. The treatment, with a conditioned medium from the mechanically-induced OA engineered microcartilage, also caused increased demineralization and expression of ALP, OCN, ADAMTS5, and VEGF; however, changes in network formation by endothelial cells were not observed in this second case, suggesting a possible different mechanism of action in inducing OA-like phenotypes. We propose that this vascularized bone model could represent a first step for the in vitro study of bone changes under OA mimicking conditions and possibly serve as a tool in testing anti-OA drugs.

The Added Value of the “Co” in Co-Culture Systems in Research on Osteoarthritis Pathology and Treatment Development

“…However, addition of the MSOD was required to maintain the network. Stimulation also induced demineralization and increase of collagen synthesis similar to changes in OA bone (Pirosa et al, 2021). Moreover, the cytokineinduced increased expression of alkaline phosphatase (ALP) and vascular endothelial growth factor (VEGF), markers for osteogenesis and angiogenesis, respectively, was more pronounced in co-culture.…”

“…In order to create an OA model for subchondral bone and its vasculature, a photo cross-linked gelatin methacrylate (GelMA)-based co-culture of a mix of immortalized mesenchymal stromal cell line (MSOD) and HUVEC cells was utilized. MSOD cells underwent osteogenic differentiation by the presence of the HUVEC cells ( Pirosa et al, 2021 ) MSOD mono and co-culture showed signs of mineralization. Cytokine stimulation (IL-1β, TNF-α and IL-6, all at concentrations found in synovial fluid of OA patients) increased endothelial network formation, similar to what is found in OA, in both HUVEC and MSOD-HUVEC culture.…”

“…Moreover, the cytokine-induced increased expression of alkaline phosphatase (ALP) and vascular endothelial growth factor (VEGF), markers for osteogenesis and angiogenesis, respectively, was more pronounced in co-culture. The co-culture was also stimulated with conditioned medium of a cartilage-on-a-chip model which consisted of chondrocytes that were embedded in a PEG-based hydrogel loaded onto a compressible PDMS device ( Pirosa et al, 2021 ). Cells in the chip were supra-physiologically compressed to induce an osteoarthritic phenotype ( Occhetta et al, 2019 ).…”

“…Cells in the chip were supra-physiologically compressed to induce an osteoarthritic phenotype ( Occhetta et al, 2019 ). Stimulation of the MSOD-HUVEC co-culture with the conditioned medium induced demineralization but prevented endothelial network formation, suggesting the pathways triggered were distinctly different from those induced by the abovementioned cytokine panel ( Pirosa et al, 2021 ). Taken together co-culture could clearly demonstrate crosstalk between both cell types and also indicates that bone-like cells influence the phenotype of endothelial cells and vice versa.…”

Diversity of Vascular Niches in Bones and Joints During Homeostasis, Ageing, and Diseases

“…This cartilage on a chip method includes coculture studies of primary endothelial cells with mesenchymal cell lines and investigates the osteogenic differentiation and tubes formation. Exposure of inflammatory cytokines to this model is able to attain the OA characteristics by depicting increased expression of MMP13 and ADAMTS5 (130).…”