Biology subjects

de Wildt, B. W. M.

Publications and source records attributed to de Wildt, B. W. M..

8 recordsLinked to original sources

bioRxiv2022

The impact of culture variables on 3D human in vitro bone remodeling; a design of experiments approach

Human in vitro bone remodeling models, using osteoclast-osteoblast co-cultures, could facilitate the investigation of human healthy (i.e., balanced) and pathological (i.e., unbalanced) bone remodeling while reducing the need for animal experiments. Although current in vitro osteoclast-osteoblast co-cultures have improved our understanding of bone remodeling, they lack culture method and outcome measurement standardization, which is hampering reproducibility and translatability. Therefore, in vitro bone remodeling models could benefit from a thorough evaluation of the impact of culture variables on functional and translatable outcome measures, with the aim to reach healthy balanced osteoclast and osteoblast activity. Using a resolution III fractional factorial design, we identified the main effects of commonly used culture variables on bone turnover markers in a robust in vitro human bone remodeling model. Our model was able to capture physiological quantitative resorption - formation coupling along all conditions, whereby remodeling could be enhanced by external stimuli. Especially culture conditions of two runs showed promising results, where conditions of one run could be used as a high bone turnover system and conditions of another run as a self-regulating system as the addition of osteoclastic and osteogenic differentiation factors was not required for remodeling. The results generated with our in vitro model allow for better translation between in vitro studies and towards in vivo studies, for improved preclinical bone remodeling drug development. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/516134v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1068cc1org.highwire.dtl.DTLVardef@102d788org.highwire.dtl.DTLVardef@1942864org.highwire.dtl.DTLVardef@1e7d8b7_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

A dialysis medium refreshment cell culture set-up for an osteoblast-osteoclast coculture

Culture medium exchange leads to loss of valuable auto- and paracrine factors produced by the cells. However, frequent renewal of culture medium is necessary for nutrient supply and to prevent waste product accumulation. Thus it remains the gold standard in cell culture applications. The use of dialysis as a medium refreshment method could provide a solution as low molecular weight molecules such as nutrients and waste products could easily be exchanged, while high molecular weight components such as growth factors, used in cell interactions, could be maintained in the cell culture compartment. This study investigates a dialysis culture approach for an in vitro bone remodeling model. In this model, both the differentiation of human mesenchymal stromal cells (MSCs) into osteoblasts and monocytes (MCs) into osteoclasts is studied. A custom-made simple dialysis culture system with a commercially available cellulose dialysis insert was developed. The data reported here revealed increased osteoblastic and osteoclastic activity in the dialysis groups compared to the standard non-dialysis groups, mainly shown by significantly higher alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activity, respectively. This simple culture system has the potential to create a more efficient microenvironment allowing for cell interactions via secreted factors in mono- and cocultures and could be applied for many other tissues.

bioengineering↗

bioRxiv2022

Characterization of three-dimensional bone-like tissue growth and organization under influence of curvature and directional fluid flow

The transition in the field of bone tissue engineering from bone regeneration to three-dimensional in vitro models has come with the challenge of recreating a dense and anisotropic bone-like extracellular matrix with cell culture. The creation of such an organized bone-like extracellular matrix has received little attention thus far. Although the mechanism by which bone extracellular matrix gains its structure is not fully understood, curvature (especially concavities), mechanical loading due to deformations or directional fluid flow, and osteocyte signaling have been identified as potential contributors. Here, guided by computational simulations, we evaluated three-dimensional cell and bone-like tissue growth and organization in a concave channel with and without directional fluid flow stimulation. Human bone-marrow derived mesenchymal stromal cells were seeded on donut-shaped silk fibroin scaffolds and stimulated to undergo osteogenic differentiation for 42 days statically or in a flow perfusion bioreactor. Constructs were investigated for cell distribution, and tissue growth and organization on day 14, 28, and 42. As a result, directional fluid flow was able to improve bone-like tissue growth but not organization. After 28 days of culture, when osteogenic differentiation was likely accomplished, cells tended to have a small preference for orientation in the tangential (i.e., circumferential) direction of the channel. Based on our results, we suggest that three-dimensional bone-like tissue anisotropy might be guided by curvature, while extracellular matrix production can be increased through the application of fluid shear stress. With this study, an initial attempt in three-dimensions was made to improve the resemblance of in vitro produced bone-like extracellular matrix to the physiological bone extracellular matrix.

bioengineering↗

bioRxiv2022

Evaluating material-driven regeneration in a tissue engineered human in vitro bone defect model

Advanced in vitro human bone defect models can contribute to the evaluation of materials for in situ bone regeneration, addressing both translational and ethical concerns regarding animal models. In this study, we attempted to develop such a model to study material-driven regeneration, using a tissue engineering approach. By co-culturing human umbilical vein endothelial cells (HUVECs) with human bone marrow-derived mesenchymal stromal cells (hBMSCs) on silk fibroin scaffolds with in vitro critically sized defects, the growth of vascular-like networks and three-dimensional bone-like tissue was facilitated. After a model build-up phase of 28 days, materials were artificially implanted and HUVEC and hBMSC migration, cell-material interactions, and osteoinduction were evaluated 14 days after implantation. The materials physiologically relevant for bone regeneration included a platelet gel as blood clot mimic, cartilage spheres as soft callus mimics, and a fibrin gel as control. Although the in vitro model was limited in the evaluation of immune responses, hallmarks of physiological bone regeneration were observed in vitro. These included the endothelial cell chemotaxis induced by the blood clot mimic and the mineralization of the soft callus mimic. Therefore, the present in vitro model could contribute to an improved pre-clinical evaluation of biomaterials while reducing the need for animal experiments. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/502914v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@ff8a7borg.highwire.dtl.DTLVardef@dcbddcorg.highwire.dtl.DTLVardef@34c865org.highwire.dtl.DTLVardef@15084f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Tuning the resorption-formation balance in an in vitro 3D osteoblast-osteoclast co-culture model of bone

The aim of the present study was to further improve an in vitro 3D osteoblast (OB) - osteoclast (OC) co-culture model of bone by tuning it towards states of formation, resorption, and equilibrium for their future applications in fundamental research, drug development and personalized medicine. This was achieved by varying culture medium composition and monocyte seeding density, the two external parameters that affect cell behavior the most. Monocytes were seeded at two seeding densities onto 3D silk-fibroin constructs pre-mineralized by MSC-derived OBs and were co-cultured in one of three different media (OC stimulating, Neutral and OB stimulating medium) for three weeks. Histology showed mineralized matrix after co-culture and OC markers in the OC medium group. Scanning Electron Microscopy showed large OC-like cells in the OC medium group. Micro-computed tomography showed increased formation in the OB medium group, equilibrium in the Neutral medium group and resorption in the OC medium group. Culture supernatant samples showed high early TRAP release in the OC medium group, a later and lower release in the Neutral medium group, and almost no release in the OB medium group. Increased monocyte seeding density showed a less-than-proportional increase in TRAP release and resorption in OC medium, while it proportionally increased TRAP release in Neutral medium without affecting net resorption. The 3D OB-OC co-culture model was effectively used to show an excess of mineral deposition using OB medium, resorption using OC medium, or an equilibrium using Neutral medium. All three media applied to the model may have their own distinct applications in fundamental research, drug development, and personalized medicine.

bioengineering↗

bioRxiv2022

Bioinspired Silk Fibroin Mineralization for Advanced In Vitro Bone Remodeling Models

Human in vitro bone models can create the possibility for investigation of physiological bone remodeling while addressing the principle of replacement, reduction and refinement of animal experiments (3R). Current in vitro models lack cell-matrix interactions and their spatiotemporal complexity. To facilitate these analyses, a bone-mimetic template was developed in this study, inspired by bones extracellular matrix composition and organization. Silk fibroin (SF) was used as an organic matrix, poly-aspartic acid (pAsp) was used to mimic the functionality of non-collagenous proteins, and 10x simulated body fluid served as mineralization solution. By using pAsp in the mineralization solution, minerals were guided towards the SF material resulting in mineralization inside and as a coating on top of the SF. After cytocompatibility testing, remodeling experiments were performed in which mineralized scaffold remodeling by osteoclasts and osteoblasts was tracked with non-destructive micro-computed tomography and medium analyses over a period of 42 days. The mineralized scaffolds supported osteoclastic resorption and osteoblastic mineralization, in the physiological bone remodeling specific sequence. This model could therefore facilitate the investigation of cell-matrix interactions and may thus reduce animal experiments and advance in vitro drug testing for bone remodeling pathologies like osteoporosis, where cell-matrix interactions need to be targeted.

bioengineering↗

bioRxiv2022

Human Platelet Lysate as Alternative of Fetal Bovine Serum for Enhanced Human in vitro Bone Resorption and Remodeling

To study human physiological and pathological bone remodeling while addressing the principle of replacement, reduction and refinement of animal experiments (3Rs), human in vitro bone remodeling models are being developed. Despite increasing safety-, scientific-, and ethical concerns, fetal bovine serum (FBS), a nutritional medium supplement, is still routinely used in these models. To comply with the 3Rs and to improve the reproducibility of such in vitro models, xenogeneic-free medium supplements should be investigated. Human platelet lysate (hPL) might be a good alternative as it has been shown to accelerate osteogenic differentiation of mesenchymal stromal cells (MSCs) and improve subsequent mineralization. However, for a human in vitro bone model, hPL should also be able to adequately support osteoclastic differentiation and subsequent bone resorption. In addition, optimizing co-culture medium conditions in mono-cultures might lead to unequal stimulation of co-cultured cells. Here, we compared supplementation with 10% FBS vs. 10%, 5%, and 2.5% hPL for osteoclast formation and resorption by human monocytes (MCs) in mono-culture and in co-culture with (osteogenically stimulated) human MSCs. Our results indicate that the supplementation of hPL can lead to a less donor-dependent and more homogeneous osteoclastic differentiation of MCs when compared to supplementation with 10% FBS. In co-cultures, osteoclastic differentiation and resorption in the 10% FBS group was almost completely inhibited by MSCs, while the supplementation with hPL still allowed for resorption, mostly at low concentrations. The addition of hPL to osteogenically stimulated MSC mono- and MC-MSC co-cultures resulted in osteogenic differentiation and bone-like matrix formation, mostly at high concentrations. Therefore, we conclude that hPL could support both osteoclastic differentiation of human MCs and osteogenic differentiation of human MSCs in mono- and in co-culture, and that this can be balanced by the hPL concentration. Thus, the use of hPL could limit the need for FBS, which is currently commonly accepted for in vitro bone remodeling models.

immunology↗

bioRxiv2021

Osteoblast-osteoclast co-cultures: a systematic review and map of available literature

Drug research with animal models is expensive, time-consuming and translation to clinical trials is often poor, resulting in a desire to replace, reduce, and refine the use of animal models. One approach to replace and reduce the use of animal models in research is using in vitro cell-culture models. To study bone physiology, bone diseases and drugs, many studies have been published using osteoblast-osteoclast co-cultures. The use of osteoblast-osteoclast co-cultures is usually not clearly mentioned in the title and abstract, making it difficult to identify these studies without a systematic search and thorough review. As a result, researchers are all developing their own methods from the ground up, leading to conceptually similar studies with many methodological differences and, as a direct consequence, incomparable results. The aim of this study was to systematically review existing osteoblast-osteoclast co-culture studies published up to 6 January 2020, and to give an overview of their methods, predetermined outcome measures (formation and resorption, and ALP and TRAP quantification as surrogate markers for formation and resorption, respectively), and other useful parameters for analysis. Information regarding these outcome measures was extracted and collected in a database, and each study was further evaluated on whether both the osteoblasts and osteoclasts were analyzed using relevant outcome measures. From these studies, additional details on methods, cells and culture conditions were extracted into a second database to allow searching on more characteristics. The two databases presented in this publication provide an unprecedented amount of information on cells, culture conditions and analytical techniques for using and studying osteoblast-osteoclast cocultures. They allow researchers to identify publications relevant to their specific needs and allow easy validation and comparison with existing literature. Finally, we provide the information and tools necessary for others to use, manipulate and expand the databases for their needs.

cell biology↗