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Mesenchymal stromal cells from the bone marrow (BMSCs) exhibit a functional decline during aging. We previously found that extracellular matrix (ECM) engineered from human induced pluripotent stem cell-derived mesenchymal progenitors enhances the osteogenic capacity of BMSCs. In the current study, we investigated how this ECM affects the three-lineage differentiation and secretory activity of BMSCs. BMSCs were seeded on the ECM layer and osteogenic, adipogenic and chondrogenic lineages were induced in monolayer or micromass cultures. Differentiation responses were compared to controls on tissue culture plastic after 21 days, and secretion of interleukin 6 was evaluated after 3 and 21 days of culture. We found a significant increase in BMSC growth on the ECM in all three differentiation media compared with controls. Osteogenic cultures on the ECM resulted in significantly higher alkaline phosphatase activity, osteogenic gene expression, collagen deposition, and matrix mineralization. In adipogenic cultures, a significant decline in adipocyte formation was found on the ECM. Chondrogenic induction on the ECM resulted in significantly increased chondrogenic gene expression, glycosaminoglycans deposition and collagen type II deposition, and no significant increase in collagen type X gene expression compared to control. Secretion of interleukin 6 was modulated by the three differentiation media and culture surface, and was reduced after 21 days of osteogenic and chondrogenic induction on the ECM. Together, our data suggest that engineered ECM modulates BMSCs trilineage differentiation toward enhanced osteogenesis and chondrogenesis, and reduced adipogenesis. Thus, our ECM might provide a bioactive component for enhancing osteochondral regeneration in older patients.

Background: Human induced pluripotent stem cells represent a scalable source of youthful tissue progenitors and secretomes for regenerative therapies. The aim of our study was to investigate the potential of conditioned medium (CM) from hiPSC-mesenchymal progenitors (hiPSC-MPs) to stimulate osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (MSCs). We also investigated whether prolonged cultivation or osteogenic pre-differentiation of hiPSC-MPs could enhance the stimulatory activity of CM., Methods: MSCs were isolated from 13 donors (age 20-90 years). CM derived from hiPSC-MPs was added to the MSC cultures and the effects on proliferation and osteogenic differentiation were examined after 14 days and 6 weeks. The stimulatory activity of hiPSC-MP-CM was compared with the activity of MSC-derived CM and with the activity of CM prepared from hiPSC-MPs pre-cultured in growth or osteogenic medium for 14 days. Comparative proteomic analysis of CM was performed to gain insight into the molecular components responsible for the stimulatory activity., Results: Primary bone marrow-derived MSC exhibited variability, with a tendency towards lower proliferation and tri-lineage differentiation in older donors. hiPSC-MP-CM increased the proliferation and alkaline phosphatase activity of MSC from several adult/aged donors after 14 days of continuous supplementation under osteogenic conditions. However, CM supplementation failed to improve the mineralization of MSC pellets after 6 weeks under osteogenic conditions. hiPSC-MP-CM showed greater enhancement of proliferation and ALP activity than CM derived from bone marrow-derived MSCs. Moreover, 14-day cultivation but not osteogenic pre-differentiation of hiPSC-MPs strongly enhanced CM stimulatory activity. Quantitative proteomic analysis of d14-CM revealed a distinct profile of components that formed a highly interconnected associations network with two clusters, one functionally associated with binding and organization of actin/cytoskeletal components and the other with structural constituents of the extracellular matrix, collagen, and growth factor binding. Several hub proteins were identified that were reported to have functions in cell-extracellular matrix interaction, osteogenic differentiation and development., Conclusions: Our data show that hiPSC-MP-CM enhances early osteogenic differentiation of human bone marrow-derived MSCs and that prolonged cultivation of hiPSC-MPs enhances CM-stimulatory activity. Proteomic analysis of the upregulated protein components provides the basis for further optimization of hiPSC-MP-CM for bone regenerative therapies., Competing Interests: Declarations Ethics approval and consent to participate The study was conducted with informed consent for the research use of tissue remaining after surgery and with full ethical approval by the Ethics Committee for the AUVA Hospitals (“Ethikkomission der AUVA”, Vienna, Austria)”, approval No. 1/2005, title “Tracking and improving cartilage regeneration in a human cartilage defect in vivo model” (“Vervolgung und Verbesserung der Knorpelregeneration in einem humanen Knorpeldefekt in vivo Modell”), approved February 9th 2006, and amendment from September 28th 2009, to include “The extraction of demineralized bone, analytical determinations in the spongiosa area, and isolation of osteoblasts and bone marrow” (“Die Gewinnung von demineralisiertem Knochen, Analytische Bestimmungen im Spongiosabereich, isolierung von Osteoblasten und Knochenmark”). The patients provided written informed consent for the use of samples. Consent for publication All authors confirm their consent for publication. Competing interests Authors of the study declare no competing interests., (© 2024. The Author(s).)

Objectives: Bone remodelling is a highly dynamic process dependent on the precise coordination of osteoblasts and haematopoietic-cell derived osteoclasts. Changes in core metabolic pathways during osteoclastogenesis, however, are largely unexplored and it is unknown whether and how these processes are involved in bone homeostasis., Methods: We metabolically and transcriptionally profiled cells during osteoclast and osteoblast generation. Individual gene expression was characterised by quantitative PCR and western blot. Osteoblast function was assessed by Alizarin red staining. immunoresponsive gene 1 ( Irg1 ) - deficient mice were used in various inflammatory or non-inflammatory models of bone loss. Tissue gene expression was analysed by RNA in situ hybridisation., Results: We show that during differentiation preosteoclasts rearrange their tricarboxylic acid cycle, a process crucially depending on both glucose and glutamine. This rearrangement is characterised by the induction of Irg1 and production of itaconate, which accumulates intracellularly and extracellularly. While the IRG1-itaconate axis is dispensable for osteoclast generation in vitro and in vivo, we demonstrate that itaconate stimulates osteoblasts by accelerating osteogenic differentiation in both human and murine cells. This enhanced osteogenic differentiation is accompanied by reduced proliferation and altered metabolism. Additionally, supplementation of itaconate increases bone formation by boosting osteoblast activity in mice. Conversely, Irg1- deficient mice exhibit decreased bone mass and have reduced osteoproliferative lesions in experimental arthritis., Conclusion: In summary, we identify itaconate, generated as a result of the metabolic rewiring during osteoclast differentiation, as a previously unrecognised regulator of osteoblasts., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Additive and lithographic manufacturing technologies using photopolymerisation provide a powerful tool for fabricating multiscale structures, which is especially interesting for biomimetic scaffolds and biointerfaces. However, most resins are tailored to one particular fabrication technology, showing drawbacks for versatile use. Hence, we used a resin based on thiol-ene chemistry, leveraging its numerous advantages such as low oxygen inhibition, minimal shrinkage and high monomer conversion. The resin is tailored to applications in additive and lithographic technologies for future biofabrication where fast curing kinetics in the presence of oxygen are required, namely 3D inkjet printing, digital light processing and nanoimprint lithography. These technologies enable us to fabricate scaffolds over a span of six orders of magnitude with a maximum of 10 mm and a minimum of 150 nm in height, including bioinspired porous structures with controlled architecture, hole-patterned plates and micro/submicro patterned surfaces. Such versatile properties, combined with noncytotoxicity, degradability and the commercial availability of all the components render the resin as a prototyping material for tissue engineers.

Regeneration of bone defects is often limited due to compromised bone tissue physiology. Previous studies suggest that engineered extracellular matrices enhance the regenerative capacity of mesenchymal stromal cells. In this study, we used human-induced pluripotent stem cells, a scalable source of young mesenchymal progenitors (hiPSC-MPs), to generate extracellular matrix (iECM) and test its effects on the osteogenic capacity of human bone-marrow mesenchymal stromal cells (BMSCs). iECM was deposited as a layer on cell culture dishes and into three-dimensional (3D) silk-based spongy scaffolds. After decellularization, iECM maintained inherent structural proteins including collagens, fibronectin and laminin, and contained minimal residual DNA. Young adult and aged BMSCs cultured on the iECM layer in osteogenic medium exhibited a significant increase in proliferation, osteogenic marker expression, and mineralization as compared to tissue culture plastic. With BMSCs from aged donors, matrix mineralization was only detected when cultured on iECM, but not on tissue culture plastic. When cultured in 3D iECM/silk scaffolds, BMSCs exhibited significantly increased osteogenic gene expression levels and bone matrix deposition. iECM layer showed a similar enhancement of aged BMSC proliferation, osteogenic gene expression, and mineralization compared with extracellular matrix layers derived from young adult or aged BMSCs. However, iECM increased osteogenic differentiation and decreased adipocyte formation compared with single protein substrates including collagen and fibronectin. Together, our data suggest that the microenvironment comprised of iECM can enhance the osteogenic activity of BMSCs, providing a bioactive and scalable biomaterial strategy for enhancing bone regeneration in patients with delayed or failed bone healing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hanetseder, Levstek, Teuschl-Woller, Frank, Schaedl, Redl and Marolt Presen.)

Reactive oxygen species (ROS) have recently been recognized as important signal transducers, particularly regulating proliferation and differentiation of cells. Diphenyleneiodonium (DPI) is known as an inhibitor of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) and is also affecting mitochondrial function. The aim of this study was to investigate the effect of DPI on ROS metabolism and mitochondrial function in human amniotic membrane mesenchymal stromal cells (hAMSCs), human bone marrow mesenchymal stromal cells (hBMSCs), hBMSCs induced into osteoblast-like cells, and osteosarcoma cell line MG-63. Our data suggested a combination of a membrane potential sensitive fluorescent dye, tetramethylrhodamine methyl ester (TMRM), and a ROS-sensitive dye, CM-H2DCFDA, combined with a pretreatment with mitochondria-targeted ROS scavenger MitoTEMPO as a good tool to examine effects of DPI. We observed critical differences in ROS metabolism between hAMSCs, hBMSCs, osteoblast-like cells, and MG-63 cells, which were linked to energy metabolism. In cell types using predominantly glycolysis as the energy source, such as hAMSCs, DPI predominantly interacted with NOX, and it was not toxic for the cells. In hBMSCs, the ROS turnover was influenced by NOX activity rather than by the mitochondria. In cells with aerobic metabolism, such as MG 63, the mitochondria became an additional target for DPI, and these cells were prone to the toxic effects of DPI. In summary, our data suggest that undifferentiated cells rather than differentiated parenchymal cells should be considered as potential targets for DPI.