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Osteoclasts, the bone resorbing cells of hematopoietic origin formed by macrophage fusion, are essential in bone health and disease. However, in vitro research on osteoclasts remains challenging due to heterogeneous cultures that only contain a few multinucleated osteoclasts. Indeed, a strategy to generate homogeneous populations of multinucleated osteoclasts in a scalable manner has remained elusive. Here, the investigation focuses on whether microencapsulation of human macrophages in microfluidically generated hollow, sacrificial tyramine-conjugated dextran (Dex-TA) microgels could facilitate macrophage precursor aggregation and formation of multinucleated osteoclasts. Therefore, human mononuclear cells are isolated from buffy coats and differentiated toward macrophages. Macrophages are encapsulated in microgels using flow focus microfluidics and outside-in enzymatic oxidative phenolic crosslinking, and differentiated toward osteoclasts. Morphology, viability, and osteoclast fusion of microencapsulated cells are assessed. Furthermore, microgels are degraded to allow cell sorting of released cells based on osteoclastic marker expression. The successful encapsulation and osteoclast formation of human macrophages in Dex-TA microgels are reported for the first time using high-throughput droplet microfluidics. Intriguingly, osteoclast formation within these 3D microenvironments occurs at a significantly higher level compared to the conventional 2D culture system. Furthermore, the feasibility of establishing a pure osteoclast culture from cell transfer and release from degradable microgels is demonstrated., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

Mesenchymal stromal/stem cell derived-extracellular vesicles (MSC-EVs) have gained interest as drug delivery nanoparticles, having immunoregulatory and potentiating tissue repair property. To maintain growth of MSCs and obtain pure MSC-derived EVs, the culture media should contain fetal bovine serum (FBS) devoid of EVs, as the presence of FBS EVs confounds the properties of MSC-EVs. Therefore, we tested three methods: 18h ultracentrifugation (UC) and ultrafiltration (UF), which are common FBS EV depletion methods in current MSC-EV research, and polyethylene glycol (PEG) precipitation to obtain three EV depleted FBS (EVdFBS) batches, and compared them to FBS and commercial (Com) EVdFBS on human adipose stem cell (hADSC) growth, differentiation, enrichment of EVs in hADSC supernatant and their biological function on collagen metabolism. Our comparative study showed UC and UF vary in terms of depletion efficiency and do not completely deplete EVs and affects the growth-promoting quality of FBS. Specifically, FBS EV depletion was comparable between PEG (95.6%) and UF (96.6%) but less by UC (82%), as compared to FBS. FBS protein loss was markedly different among PEG (47%), UF (87%), and UC (51%), implying the ratio of EV depletion over protein loss was PEG (2.03), UF (1.11), and UC (1.61). A significant decrease of TGFβ/Smad signaling, involving in MSC growth and physiology, was observed by UF. After 96 hours of exposure to 5% FBS or 5% four different EVdFBS cell growth media, the osteogenesis ability of hADSCs was not impaired but slightly lower mRNA expression level of Col2a observed in EVdFBS media during chondrogenesis. In consistent with low confluency of hADSCs observed by optical microscope, cell proliferation in response to 5% UF EVdFBS media was inhibited significantly. Importantly, more and purer ADSCs EVs were obtained from ADSCs cultured in 5% PEG EVdFBS media, and they retained bioactive as they upregulated the expression of Col1a1, TIMP1 of human knee synovial fibroblast. Taken together, this study showed that PEG precipitation is the most efficient method to obtain EV depleted FBS for growth of MSCs, and to obtain MSC EVs with minimal FBS EV contamination., Competing Interests: NO, (Copyright: © 2023 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Stromal vascular fraction (SVF) is the primary isolate obtained after enzymatic digestion of adipose tissue that contains various cell types. Its successful application for cell-based construct preparation in an intra-operative setting for clinical bone augmentation and regeneration has been previously reported. However, the performance of SVF-based constructs compared with traditional ex vivo expanded adipose tissue-derived mesenchymal stromal cells (ATMSCs) remains unclear and direct comparative analyses are scarce. Consequently, we here aimed at comparing the in vitro osteogenic differentiation capacity of donor-matched SVF versus ATMSCs as well as their osteoinductive capacity. Human adipose tissue from nine different donors was used to isolate SVF, which was further purified via plastic-adherence to obtain donor-matched ATMSCs. Both cell populations were immunophenotypically characterized for mesenchymal stromal cell, endothelial, and hematopoietic markers after isolation and immunocytochemical staining was used to identify different cell types during prolonged cell culture. Based on normalization using plastic-adherence fraction determination, SVF and ATMSCs were seeded and cultured in osteogenic differentiation medium for 28 days. Further, SVF and ATMSCs were seeded onto devitalized bovine bone granules and subcutaneously implanted into nude mice. After 42 days of implantation, granules were retrieved, histologically processed, and stained with hematoxylin and eosin (HE) to assess ectopic bone formation. The ATMSCs were shown to be a homogenous cell population during cell culture, whereas SVF cultures consisted of multiple cell types. All donor-matched comparisons showed either accelerated or stronger mineralization for SVF cultures in vitro . However, neither SVF nor ATMSCs loaded on devitalized bone granules induced ectopic bone formation on subcutaneous implantation, as opposed to control granules loaded with bone morphogenetic protein-2 (BMP-2), which triggered ectopic bone formation with 100% incidence. Despite the observed lack of osteoinduction, our findings provide important in vitro evidence on the osteogenic superiority of intra-operatively available SVF as compared with donor-matched ATMSCs. Consequently, further studies should focus on optimizing the efficacy of these cell populations for implementation in orthotopic bone fracture or defect treatment.

Copper (Cu)-loaded electrospun membranes were tailored for guided bone regeneration (GBR), targeting the stimulation of innate cells active in bone growth and the prevention of bacterial infections. Functional GBR membranes were produced via an electrospinning set-up using a silk-based solution associated with polyethylene oxide (Silk/PEO - control). Experimental groups were loaded with copper oxide using varying weight percentages (0.05 % to 1 % of CuO). The morphological, structural, chemical, and mechanical properties of membranes were evaluated. Direct and indirect in vitro cytocompatibility experiments were performed with primary human bone mesenchymal stem cells and primary human umbilical vein endothelial cells. The antibacterial potential of membranes was tested with Staphylococcus aureus and Fusobacterium nucleatum biofilm. CuO was successfully incorporated into membranes as clusters without compromising their mechanical properties for clinical applicability. Increased Cu concentrations generated membranes with thinner nanofibers, greater pore areas, and stronger antimicrobial effect (p < 0.01). Cu 2+ ion was released from the nanofiber membranes during 1 week, showing higher release in acidic conditions. CuO 0.1 % and CuO 0.05 % membranes were able to support and stimulate cell adhesion and proliferation (p < 0.05), and favor angiogenic responses of vascular cells. In addition, detailed quantitative and qualitative analysis determined that amount of the attached biofilm was reduced on the tailored functional Cu 2+ -loaded GBR membrane. Importantly, these qualities represent a valuable strategy to improve the bone regeneration process and diminish the risk of bacterial infections., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Osteoclasts are multinucleated cells derived from the hematopoietic monocyte/macrophage lineage that possess the unique capacity to resorb bone. Due to the crucial role of osteoclasts in maintaining bone homeostasis and pathologies, this cell type is pivotal in multiple research areas dedicated to bone physiology in health and disease. Although numerous methods for generation of human osteoclasts are already available, those rely either on cell labeling-based purification or an intermediate adhesion step after which cells are directly differentiated toward osteoclasts. While the former requires additional reagents and equipment, the latter harbors the risk of variable osteoclast formation due to varying numbers of osteoclast precursors available for different donors. In this study, we report a facile and reliable three-step method for the generation of human osteoclasts from blood-derived precursor cells. Monocytes were obtained after adhering peripheral blood-derived mononuclear cells to plastic substrates followed by macrophage induction and proliferation resulting in a homogeneous population of osteoclast precursors. Finally, macrophages were seeded into suitable culture vessels and differentiated toward osteoclasts. Osteoclastogenesis was monitored longitudinally using nondestructive techniques, while the functionality of mature osteoclasts was confirmed after 14 days of culture by analysis of functional (e.g., elevated tartrate-resistant acid phosphatase [TRAP]-activity, resorption) and morphological (e.g., presence of TRAP, actin ring, and integrin β 3 ) characteristics. Furthermore, we propose to use combinatory staining of three morphological osteoclast markers, rather than previously reported staining of a single or maximal two markers, to clearly distinguish osteoclasts from undifferentiated mononuclear cells. Impact statement Research related to bone biology requires a standardized and reliable method for in vitro generation of human osteoclasts. We here describe such a procedure which avoids shortcomings of previously published protocols. Further, we report on nondestructive methods to qualitatively and quantitatively monitor osteoclastogenesis longitudinally, and on analysis of osteoclast generation and functionality after 14 days. Specifically, we recommend assessment of morphological human osteoclast characteristics using combinatory staining of three markers to confirm successful osteoclast generation.

The effects of immune cells, in particular macrophages, on the behaviour of mesenchymal stromal cells (MSCs) have recently gained much attention for MSCs-based tissue-engineered constructs. This study aimed to evaluate the effect of monocytes/macrophages on the osteogenic differentiation of adipose-derived mesenchymal stromal cells (ADMSCs) in three-dimensional (3D) cocultures. For this, we cocultured THP-1 monocytes, M1 macrophages, or M2 macrophages with ADMSCs on 3D poly(lactic-co-glycolic) acid (PLGA)/polycaprolactone (PCL) scaffolds using osteogenic medium for up to 42 days. We found that osteogenic differentiation of ADMSCs was inhibited by monocytes and both macrophage subtypes in 3D scaffolds. Furthermore, coculture of monocytes/macrophages with ADMSCs resulted in downregulated secretion of oncostatin M (OSM) and bone morphogenetic protein 2 (BMP-2) and inhibited expression of osteogenic markers alkaline phosphatase (ALP), bone sialoprotein (BSP), and runt-related transcription factor 2 (RUNX2). Compared with both macrophage subtypes, monocytes inhibited osteogenic differentiation of ADMSCs more significantly. These data suggest that the mutual interactions between monocytes/macrophages and ADMSCs negatively affect MSC osteogenic differentiation and thus possibly bone healing capacity, which highlights the importance of the micro-environment in influencing cell-based constructs to treat bone defects and the potential to improve their performance by resolving the inflammation ahead of treatment., (© 2019 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.)

Stem cell-based bone tissue engineering based on the combination of a scaffold and expanded autologous mesenchymal stem cells (MSCs) represents the current state-of-the-art treatment for bone defects and fractures. However, the procedure of such construct preparation requires extensive ex vivo manipulation of patient's cells to achieve enough stem cells. Therefore, it is impractical and not cost-effective compared to other therapeutic interventions. For these reasons, a more practical strategy circumventing any ex vivo manipulation and an additional surgery for the patient would be advantageous. Intraoperative concept-based bone tissue engineering, where constructs are prepared with easily accessible autologous cells within the same surgical procedure, allows for such a simplification. In this study, we discuss the concept of intraoperative construct preparation for bone tissue engineering and summarize the available cellular options for intraoperative preparation. Furthermore, we propose methods to prepare intraoperative constructs, and review data of currently available preclinical and clinical studies using intraoperatively prepared constructs for bone regenerative applications. We identify several obstacles hampering the application of this emerging approach and highlight perspectives of technological innovations to advance the future developments of intraoperative construct preparation.