The authors would like to thank Riccardo Urbanet for technical assistance with the liquid handling devices and Rodi Odabasi for support with the epifluorescence microscopy. This work was financed by the Swiss National Science Foundation (grant numbers 310030E_202429 and 205321_204318) and Ectica Technologies AG.

3D Co-Culture Assay: Vascularized Osteogenic Niches for Cancer Drug Screening

<ol>
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Ectica Technologies AG is the manufacturer of the 3DProSeed hydrogel well plate and has commercial interests. Benjamin R. Simona and Martin Ehrbar are shareholders of Ectica Technologies AG.

Here, we describe a protocol for the establishment of an in vitro model of highly vascularized bone and bone marrow niches in a fully synthetic and controllable 3D PEG-based matrix, which has a variety of applications in bone and bone marrow biology research, tissue engineering, and cancer research. This model builds on a synthetic PEG-based hydrogel that is functionalized with RGD peptides and MMP cleavage sites and is cast with an in-depth density gradient on glass-bottom 96-well imaging plates30. This plug-and-play platform was shown to allow the establishment of highly interconnected 3D cellular networks without the need to encapsulate cells into the hydrogel. Similar to the earlier-described cell encapsulation protocol, in this work, we show the remodeling of the substrate by a cell-inherent ECM28 to create a cell type-specific microenvironment. Thus, with this method, drug screening assays and high-content analyses can be easily performed under highly reproducible, organotypic 3D culture conditions. The glass-bottom 96-well plates and the optically transparent hydrogels render the platform compatible with liquid handling automation and high-throughput microscopy.
The first step in generating an osteogenic vascular bone marrow niche is the pre-culture of hBM-MSCs on the PEG hydrogel for at least 3 days. During this time, they attach to the hydrogel, penetrate it, and start establishing cell-cell contacts and ECM deposition. Before seeding the hBM-MSCs, the storage buffer must be removed. As the hydrogel is situated inside an inner well within the standard well of the 96-well imaging plate, it is safe to insert the aspiration tip along the side of the well until it touches the inner well ring. A vacuum pump can be used for aspiration if it is set at the lowest possible suction force. Alternatively, an automated plate washer with the nozzle height adjusted to at least 0.8 mm above the inner well ring can be used to aspirate the buffer from the hydrogel plate. Using automation for liquid handling can minimize damage to the hydrogel surface and lead to higher reproducibility of the resulting cultures. Small defects on the hydrogel surface become visible once the cells settle on the hydrogel and appear on a lower focus plane in defective hydrogel areas. Therefore, acquiring reference images on day 0 serves as a good quality control for the cell seeding homogeneity and hydrogel surface integrity. While small hydrogel surface defects do not preclude the further use of the well, the cells tend to cluster on the defective areas and may grow into non-representative patterns or more quickly reach the bottom glass, where they grow into a monolayer. These artifacts must be noted when using/evaluating these wells. Similar considerations apply for any medium changes performed during the entire duration of the assay.
The second step of the protocol involves the addition of GFP-HUVECs to the pre-formed hBM-MSC monoculture (day 0 of co-culture). The ECM deposited by the hBM-MSC provides a great scaffold for the growth of endothelial cells, which in this work, even in the presence of hBM-MSC-conditioned medium, could only form round cell clusters on the hydrogels (not shown). Upon seeding&#160;on the hBM-MSC cultures, the HUVECs integrate and form microvessel-like structures comparable to the ones observed in co-cultures generated by cell encapsulation27,28. Typically, well-developed 3D microvascular-like networks form within 4 days of co-culture, and this can be longitudinally monitored by the use of GFP-labeled HUVECs. These structures can be maintained for at least 7 days in culture, meaning there is sufficient time to follow changes in the vascular network organization in response to treatments, such as for the screening of anti-angiogenic drugs. The morphological elements of the endothelial network can be quantified in batch mode by segmenting the GFP images using well-established tools, such as the Angiogenesis Analyzer plugin of ImageJ33, and their parameters can be used to evaluate, for example, drug efficacy and pharmacodynamics.
One significant advantage of the described cellular model for many potential applications is its plasticity. Simply supplementing the culture medium with different growth factors can change the appearance of the co-culture. For example, the presence of BMP-2 throughout the mono- and co-culture period creates an osteogenic vascular niche, showing increased ALP activity, extracellular calcium deposition, as well as ECM assembly and deposition. On the contrary, in the presence of FGF-2, the osteogenic markers are absent, and the co-culture forms fewer lateral cell associations but shows more pronounced 3D cell growth. The fact that FGF-2 suppresses ALP activity while BMP-2 elicits stronger ALP activity compared to no growth factor treatment is in accordance with previous observations27. Yet, despite these big differences in the hBM-MSC stromal component, the extent of the microvascular network was very similar for the two growth factor-treated conditions in this work. In the control cultures, only a few short vascular networks formed, representing perhaps a poorly vascularized bone marrow niche. This suggests that by simply changing the type, concentration, and timing of the growth factors added to the culture medium, a range of well-defined vascularized bone marrow niches, as would be required for comparative studies, could be produced. However, to ensure reproducible results, it is important to note that the culture progression and morphology may vary depending on the history of the cells used (e.g., the passage number and detachment method used during routine culture maintenance), and it is advisable to control for such factors during the assay design.
Here, as a first application of this model, we demonstrate the sensitivity of the engineered microvascular networks to treatment with 10 &#181;g/mL bevacizumab. Notably, it is important to confirm that the algorithm used can accurately recognize the endothelial network, as artifacts are often generated in images with poorly developed networks. If this is the case, the parameters used for image processing (before and during segmentation) need to be fine-tuned, often on a trial-and-error basis.
As a second application, we present an advanced co-culture model formed by the sequential seeding of mesenchymal, endothelial, and cancer cells. This model allows for studying the interactions between cancer cells, the stroma, and the vasculature of the bone marrow, which may be important factors during metastasis. Additionally, this model could be used for drug screening applications and testing compounds with targets beyond angiogenesis.
In 2D cultures, cells do not receive physiologic microenvironmental signals, do not acquire naturally occurring cell morphologies, and consequently, differentiate differently compared to cells in native 3D environments35. When grown in engineered 3D hydrogels, the cells deposit an inherent ECM early on, which provides adhesion sites and can be actively remodeled28,36. Here, to establish a simplified 3D model for screening applications, vessel-forming cells were seeded onto the surface of engineered hydrogels and allowed to establish vascular networks in the absence of perfusion. The imaging-based evaluations were conducted on 2D projections of the endothelial cells contributing to vascular structures. However, only confocal images revealed the less pronounced ingrowth of the 3D vascular networks in the BMP-2-stimulated samples when compared to the FGF-2-stimulated samples. This suggests that the length of the formed vascular structures was underestimated, while their connectivity was overestimated. Additionally, interactions between perivascular and endothelial cells and vascular lumen formation have not been investigated. These aspects, especially in terms of drug treatment responses, will require further attention. Finally, refined protocols to first establish extensive 3D vascular networks and only then induce their osteogenic differentiation would be desirable to generate more physiological bone and bone marrow models.
Overall, the model presented here is highly versatile and can be easily tailored toward specific applications. For example, mesenchymal and endothelial cells from different sources could be used. It is known that adipose tissue MSCs and umbilical cord MSCs express different angiogenic factors compared to BM-MSCs, and they can easily be substituted as an alternative stromal component37. Endothelial cells isolated from already defined bone marrow niches could also be used instead of HUVECs. One could also establish the co-culture with patient-derived, matching bone marrow mesenchymal and endothelial cells for personalized medicine applications, as has recently been suggested for vascularized muscle co-cultures38. Additionally, the design of the hydrogel plate allows the longitudinal monitoring of the culture with both bright-field and fluorescence microscopy, thus offering the user the possibility to shorten or extend the culture time depending on the application. Alternatively, the cell densities used for seeding could be adjusted accordingly to accelerate or delay the formation of the cell network if shorter or longer observation times are needed than those in this protocol. In any case, caution is needed to avoid cell overgrowth into sheet-like structures, which can lead to the contraction of the hydrogel and eventual cell detachment.
Finally, a broad range of assays can be performed using this model. In addition to immunofluorescence and microscopy performed in live or fixed cultures, the 3D cultures may be enzymatically digested, and the cells can be retrieved and subjected to any type of biochemical assay. Here, we demonstrate the determination of ALP activity and DNA content quantification in cell lysates using colorimetric/fluorometric assays, but the system is compatible with many other techniques, including PCR, RNAseq, and proteomics. If the sensitivity of the desired assay is not very high, one can pool samples from more than one well to increase the amount of sample available for the assay. If the desired application requires faster gel dissolution, orbital shaking of the plate could be applied in combination with smaller volumes of the digestive solution to ensure vortex formation in the wells, assuming that all wells on the plate will be used in this manner (living cultures are sensitive to such harsh handling). In summary, we present here a protocol that, if used as described, guarantees the generation of an in vitro model that recapitulates the key aspects of osteogenic vascular niches but is also versatile enough to be modified for tailor-made applications.

The bone and bone marrow are structurally and functionally complex organs central to human health. This is reflected by the existence of distinct niches that regulate hematopoiesis and bone maintenance1. It is now widely accepted that in healthy bone marrow, the maintenance and expansion of hematopoietic and skeletal stem cells, as well as their progeny, are controlled by distinct niches. These niches comprise various cell types, including osteolineage cells, mesenchymal stem cells, endothelial and perivascular cells, neuronal and glial cells, adipocytes, osteoclasts, macrophages, and neutrophils2. Not surprisingly, these mostly vasculature-associated niches are also involved in the development of various types of leukemia3 and are the site of metastasis for different cancers4. Owing to its specific roles in bone formation, remodeling, and bone (marrow) maintenance, the bone-associated vasculature has distinct specialized structures different from the vasculature found elsewhere in the body5,6,7. Thus, anti-angiogenic or vasculature-modulating drugs applied systemically may have different effects within these specialized environments8. Therefore, models to investigate the molecular mechanisms involved in maintaining the bone and bone marrow physiological properties, bone and bone marrow regeneration, and responses to therapeutic treatments are highly desirable.
Classical two-dimensional (2D) tissue cultures and in vivo investigations using animal models have provided invaluable insight into the roles of different cells and molecular players involved in the development of bone and bone marrow9,10. Models that allow for high-throughput experiments with relevant human cells could improve our understanding of how to modulate selected parameters in these highly complex systems.
In the past decade, principles derived from tissue engineering have been employed to generate 3D tissue models11,12. These have mostly relied on the encapsulation of tissue-relevant cells into biomaterials to establish 3D mono- or co-cultures13. Among the most used biomaterials are fibrin14, collagen15, and Matrigel16,17, all of which are highly biocompatible and provide appropriate conditions for the growth of many cell types. These biomaterials have the ability to generate in vitro models that recapitulate key aspects of the different vascular niches found in vivo18. Moreover, the use of microfluidic devices to generate perfused vascular bone and bone marrow models has contributed to the generation of in vitro models of higher complexity19,20,21,22.
The difficulty in controlling the composition and engineering the properties of naturally occurring biomaterials has inspired the development of synthetic analogs that can be rationally designed with predictable physical, chemical, and biological properties23,24. We have developed fully synthetic factor XIII (FXIII) cross-linked poly(ethylene glycol) (PEG)-based hydrogels, which are functionalized with RGD peptides and matrix metalloprotease (MMP) cleavage sites to facilitate cell attachment and remodeling25,26. The modular design of these biomaterials has been successfully used to optimize conditions for the formation of 3D vascularized bone and bone marrow models27,28.
For the testing of larger numbers of different culture conditions and new therapeutics, models with a higher throughput capability are required. We have recently shown that the FXIII cross-linking of our PEG hydrogel can be controlled through an electrochemical process such that an in-depth hydrogel stiffness gradient is formed29. When cells are added on top of such hydrogels, they migrate toward the interior and gradually develop into highly interconnected 3D cellular networks30. The elimination of the need to encapsulate cells into the hydrogel, which is usually present with other 3D scaffolds, not only simplifies the experimental design but also allows the sequential addition of different cell types at different time points to generate complex co-culture systems. These hydrogels are available pre-cast onto glass-bottom 96-well imaging plates, thus making the establishment of 3D cultures achievable by manual as well as automated cell seeding protocols. The optical transparency of the PEG hydrogels renders the platform compatible with microscopy.
Here, we present a straightforward method for the generation and characterization of vascularized osteogenic niches within this&#160;ready-to-use, synthetic plug-and-play platform. We show that the development of vascular networks can be stimulated with a growth factor commonly used for inducing in vitro osteogenesis, bone morphogenetic protein-2 (BMP-2), while osteogenic differentiation can be prevented by supplementation of fibroblast growth factor 2 (FGF-2)27,31. The networks formed are different when compared to FGF-2-stimulated networks in terms of the overall appearance, as well as the cell and ECM distribution. Moreover, we monitored the osteogenic induction using alkaline phosphatase as a marker. We demonstrate the increased expression of this marker over time and compare the expression to that in FGF-2 stimulated networks using qualitative and quantitative methods. Finally, we demonstrate the suitability of the generated niches of this model for two potential applications. Firstly, we performed a proof-of-concept drug sensitivity assay by adding bevacizumab to pre-formed niches and monitoring the degradation of the vascular networks in its presence. Secondly, we added MDA-MB-231 breast cancer and U2OS osteosarcoma cells to pre-formed osteogenic niches, showing that the niches can be used to study interactions between cancer cells and their environment.

<table><tbody><tr><td>0.25% Trypsin-EDTA</td><td>Gibco</td><td>25200-072</td><td></td></tr><tr><td>2 mL microtubes</td><td>Eppendorf</td><td>30120094</td><td></td></tr><tr><td>2-Amino-2-methyl-1-propanol</td><td>Sigma</td><td>A9199</td><td></td></tr><tr><td>3DProSeed hydrogel well plate</td><td>Ectica Technologies</td><td>ECT.PS1.001.096</td><td></td></tr><tr><td>4-Nitrophenyl phosphate disodium salt hexahydrate</td><td>Sigma</td><td>71768</td><td></td></tr><tr><td>Alizarin Red S</td><td>Sigma</td><td>A5533</td><td></td></tr><tr><td>Anti-Collagen IV antibody</td><td>Abcam</td><td>ab6311</td><td></td></tr><tr><td>Anti-Laminin 1+2 antibody</td><td>Abcam</td><td>ab7463</td><td></td></tr><tr><td>Automated plate washer</td><td>Agilent Biotek</td><td>EL&amp;chi;50</td><td></td></tr><tr><td>Automated washer/dispenser</td><td>Agilent Biotek</td><td>MULTIFLO FX equipped with a peristaltic pump 5uL cassette</td><td></td></tr><tr><td>Bevacizumab</td><td>Evidentic</td><td>ID PS-E07-2019-00119 A009</td><td></td></tr><tr><td>BMP-2</td><td>Peprotech</td><td>120-02C</td><td></td></tr><tr><td>BSA</td><td>AppliChem</td><td>A1391</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5415 R</td><td>To centrifuge 2 mL tubes at 16100 x g during ALP analysis</td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica</td><td>Stellaris 5</td><td></td></tr><tr><td>Conical 50 mL centrifuge tubes</td><td>TPP</td><td>91050</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td></td></tr><tr><td>DyLight 649 Donkey anti-rabbit IgG (minimal x-reactivity) Antibody</td><td>Biolegend</td><td>406406</td><td></td></tr><tr><td>DyLight 649 Goat anti-mouse IgG (minimal x-reactivity) Antibody</td><td>Biolegend</td><td>405312</td><td></td></tr><tr><td>EGM-2</td><td>Lonza</td><td>CC-3162</td><td></td></tr><tr><td>Epifluorescence microscope</td><td>Leica</td><td>DMI6000B</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10500-064</td><td></td></tr><tr><td>FGF-2</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>Fibronectin (IST-9)</td><td>Santa Cruz</td><td>sc-59826</td><td></td></tr><tr><td>GFP-HUVECs</td><td>PELOBiotech</td><td>PB-CAP-0001GFP</td><td></td></tr><tr><td>hBM-MSCs</td><td>-</td><td>-</td><td>Isolated at University Hospital Basel; Papadimitropoulos A, Piccinini E, Brachat S, et al. Expansion of human mesenchymal stromal cells from fresh bone marrow in a 3D scaffold-based system under direct perfusion. PLoS One. 2014;9(7):e102359</td></tr><tr><td>Inverted microscope</td><td>Zeiss</td><td>200M</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>MDA-MB-231 breast cancer cell line</td><td>-</td><td></td><td>Kindly obtained from J Massagu&amp;eacute; at the Memorial Sloan-Kettering Cancer Center</td></tr><tr><td>MEM&amp;alpha;</td><td>Gibco</td><td>22571-038</td><td></td></tr><tr><td>Multimode imaging reader</td><td>Agilent Biotek</td><td>Cytation 1</td><td>For automated imaging</td></tr><tr><td>Multimode imaging reader - fluorescence and absorbance</td><td>Agilent Biotek</td><td>Cytation 5</td><td>For measuring absorbance and fluorescence intensity duing ALP analysis</td></tr><tr><td>Paraformaldehyde</td><td>Artechemis</td><td>US 040</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010-015</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Phalloidin-rhodamine</td><td>Invitrogen</td><td>R415</td><td></td></tr><tr><td>Picro-Sirius Red Solution</td><td>Abcam</td><td>ab246832</td><td></td></tr><tr><td>Quant-iT PicoGreen dsDNA Assay kit</td><td>ThermoFisher Scientific</td><td>P7589</td><td></td></tr><tr><td>Recombinant Anti-Collagen I antibody</td><td>Abcam</td><td>ab260043</td><td></td></tr><tr><td>SIGMAFAST BCIP/NBT</td><td>Sigma</td><td>B5655-25TAB</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma</td><td>1064981000</td><td></td></tr><tr><td>Sodium phosphate dibasic, anhydrous</td><td>Sigma</td><td>S-0876</td><td></td></tr><tr><td>Sodium phosphate monobasic, monohydrate</td><td>Merck</td><td>1.06346</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Tween20</td><td>AppliChem</td><td>A4974</td><td></td></tr><tr><td>U2OS osteosarcoma cell line</td><td>-</td><td></td><td>Kindly obtained from J Snedeker at the Institute for Biomechanics, Zurich&amp;nbsp;</td></tr><tr><td>&amp;alpha;-trehalose dihydrate</td><td>Sigma</td><td>90208</td><td></td></tr></tbody></table>

simple establishment of a vascularized osteogenic bone marrow niche using pre-cast poly(ethylene glycol) (peg) hydrogels in an imaging microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise&#160;would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

Vascular niche cultures were established by sequentially seeding hBM-MSCs and GFP-HUVECs onto pre-cast PEG-based hydrogels with a stiffness gradient within a 96-well imaging plate (Figure 1). The cultures were monitored longitudinally via live epifluorescence microscopy and further characterized at selected time points. The extracellular compartment was assessed via direct color stainings and antibody-based stainings. ALP&#160;activity was quantified after retrieving and lysing the cells from the generated niches. Furthermore, we demonstrate the suitability of this platform for anti-angiogenic drug sensitivity assays and as a basis for cancer co-culture models.
Co-cultures of hBM-MSCs and GFP-expressing HUVECs were established by seeding 3 x 104 cells/well in the absence of growth factors or in the presence of either FGF-2 or BMP-2 at 50 ng/mL, as described in the protocol. From an early time point, differences between the cultures could be observed both from the bright-field and fluorescence images showing only the GFP-HUVECs (Figure 2A). By observing the same areas longitudinally, differences in the development of the cultures could be noted, such as a faster development in the presence of FGF-2. Generally, the cultures appeared less developed in the absence of any growth factor, with fewer cells of either type spreading and the presence of acellular areas. In contrast, the bright-field images showed the most dense cultures in the presence of BMP-2. However, vascular-like networks formed in both growth factor-containing conditions, and the most extensive and interconnected networks were formed with FGF-2. These observed differences could also be quantified using Angiogenesis Analyzer for ImageJ. Indeed, the total network length was highest in the presence of FGF-2 and lowest in the absence of growth factors (Figure 2B). The number of junctions indicating branching points in the networks followed the same trend as the total length (Figure 2C). Conversely, both growth factor-containing conditions featured significantly fewer isolated segments, indicating higher interconnectivity, than the condition without any growth factor (Figure 2D). Additionally, 3D confocal imaging revealed stronger endothelial cell penetration in terms of depth in the FGF-2-stimulated condition (Figure 2E).
hBM-MSC/GFP-HUVEC co-cultures were maintained for 7 days in the presence of FGF-2 or BMP-2 before being fixed and stained for ECM components. Immunocytochemical stainings followed by confocal laser scanning microscopy showed striking differences in culture morphology depending on the type of growth factor supplementation (Figure 3A). With FGF-2, the culture was organized into condensed microvascular-like structures, which were dense in both endothelial and mesenchymal cells, whereas in the presence of BMP-2, the hBM-MSCs spanned a much larger area, as evident from the more extensive F-actin-positive and GFP-negative areas. The ECM proteins fibronectin and collagen I were localized in a similar manner, while laminin and collagen IV were more concentrated around the endothelial structures. However, this increased concentration around the endothelial structures was much more pronounced in the presence of FGF-2 than in the presence of BMP-2. In addition to the antibody-based stainings, direct color stainings were performed to assess the overall fibrotic state of the ECM (Picrosirius Red staining; Figure 3B), as well as the deposition of Ca on the ECM (Alizarin Red staining; Figure 3C) of the formed niches. The Picrosirius Red staining was stronger and more extensive in the niches cultured with BMP-2, and the Alizarin Red staining followed the same trend.
Next, the cultures were characterized in terms of their osteogenic potential by assessing ALP activity as an early osteogenic marker. On day 4 and day 7 of co-culture, cells were retrieved from the niches by digesting the hydrogels with Trypsin. To quantify the ALP activity, the retrieved cells were lysed, and a pNPP assay was performed. The values obtained were normalized against the total DNA for each sample to account for potential differences in the cell numbers across conditions. Indeed, small differences between the DNA content of the conditions could be observed, and the fewest cells were retrieved from the condition without any growth factor (not shown). The normalized ALP activity, however, varied greatly across conditions, with a trend of increasing activity with higher concentrations of BMP-2 and a plateau at 100 ng/mL (Figure 4A,B). The lowest activity levels were identified for the condition containing 50 ng/mL FGF-2. While similar trends could be observed for both of the time points assessed, all the values significantly increased over time in culture from day 4 to day 7. In addition to the quantitative assay, the ALP activity could be qualitatively visualized using direct color staining based on the BCIP/NBT substrate conversion. More extensive and more intense purple staining was observed in the presence of BMP-2 as compared to FGF-2 (Figure 4C).
To demonstrate two potential applications of the characterized osteogenic niches, a proof-of-concept drug sensitivity study and cancer co-cultures were performed. For the drug sensitivity assay, bevacizumab or a control solution consisting of the diluent of the bevacizumab formulation was added to the culture medium containing fresh BMP-2. On day 4, when established networks were formed in the presence of 50 ng/mL BMP-2, either the control solution or bevacizumab-containing medium was added during the regular medium change, and the cultures were monitored using fluorescence imaging. The addition of 10 &#181;g/mL bevacizumab led to the retraction or ablation of the previously formed network, while the control condition still featured extensive networks 2 days after the medium change (Figure 5A,B). These changes could also be quantified by tracking the total length of the networks using Angiogenesis Analyzer for ImageJ on fluorescence images acquired daily (Figure 5C). Alternatively, bevacizumab or any other compound could also be added from the beginning of the co-culture to assess their influence on the formation of the networks. In the case of bevacizumab, this completely inhibited the formation of endothelial networks (not shown).
For the second application, MDA-MB-231 breast cancer or U2OS osteosarcoma cells were added to day 4 co-cultures at a density of 1.5 x 103 cells/well in fresh culture medium containing 50 ng/mL BMP-2. To distinguish them from the GFP-labelled HUVECs and the non-labeled hBM-MSCs, the cancer cells were incubated with CellTrace FarRed just before the seeding onto the osteogenic niches. The cultures were monitored by fluorescence microscopy; at the beginning, most of the cancer cells localized near the surface of the substrate, but after 2 days, they could be found in closer proximity to the layers containing the vascular co-cultures. Thus, day 2 was selected as the starting point for time-lapse microscopy to show the dynamics of the interactions between the cancer cells and the cells within the vascular niche. Interestingly, the MDA-MB-231 cells could be seen both approaching and moving away from endothelial structures and, thus, were possibly probing or remodeling their environment (Figure 5D). Using CellTrace FarRed as a label for the cancer cells, GFP as a label for HUVECs, and additional staining for F-actin, all the cell types could be distinguished using confocal laser scanning microscopy (Figure 5E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65413/65413fig01.jpg" /> 
Figure 1:&#160;A simple approach for the reliable generation of vascularized, osteogenic niches. Pre-cast synthetic hydrogels with an in-depth stiffness gradient allow for the generation of 3D cultures via sequential cell seeding without the need for direct encapsulation. hBM-MSCs are pre-cultured for 3 days before GFP-expressing HUVECs are added. The cultures are monitored by acquiring bright-field and GFP signals longitudinally. At select time points, the niches are further evaluated for their ECM deposition and osteogenic state. Alkaline phosphatase activity is assessed via direct color staining and by retrieving cells from the niches and performing a pNPP assay on the cell lysates. <a href="https://www.jove.com/files/ftp_upload/65413/65413fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65413/65413fig02.jpg" /> 
Figure 2: Stimulation of vascular-like networks with FGF-2 or BMP-2. (A) Bright-field and fluorescence (GFP) images were acquired on day 2, day 4, and day 6 of the co-culture of cells grown in the absence of growth factors or in the presence of FGF-2 or BMP-2, both at 50 ng/mL. Scale bar: 400 &#181;m. (B-D) Quantified parameters of the GFP-HUVEC-networks imaged on day 4 of co-culture, analyzed using Angiogenesis Analyzer for ImageJ. The data are represented as mean &#177; standard deviation. The statistical analysis was performed using GraphPad Prism 9.5.1. An ordinary one-way ANOVA with Dunnett&#39;s multiple comparisons test was performed with n &#8805; 4; * P &#60; 0.05; ** P &#60; 0.01; *** P &#60; 0.001. (E) Three-dimensional reconstructions generated from confocal stacks (total height: 547.5 &#181;m; z-step: 2.5 &#181;m) of the GFP and F-actin signals for the FGF-2- (top row) and BMP-2- (bottom row) stimulated conditions. Scale bar: 300 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65413/65413fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65413/65413fig03.jpg" /> 
Figure 3: Induction of de-localized hBM-MSC spreading and ECM deposition by BMP-2. (A-C) The 7 day co-cultures grown in the presence of either FGF-2 or BMP-2 were subjected to (A) immunofluorescence or (B,C) direct color staining. (A) The cultures were stained for F-actin and the ECM proteins fibronectin, laminin, collagen I, and collagen IV. The images depict the maximum intensity projections of the confocal stacks (total height: 100 &#181;m; z-step: 5 &#181;m). Scale bar: 200 &#181;m. (B,C) The cultures were stained using (B) Picrosirius Red and (C) Alizarin Red. The top row depicts stitched, whole-well overviews of the images acquired at 2.5x magnification (scale bar: 1,000 &#181;m), while the bottom row depicts one field of view acquired at 5x magnification (scale bar: 400 &#181;m). <a href="https://www.jove.com/files/ftp_upload/65413/65413fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65413/65413fig04.jpg" /> 
Figure 4: Biochemical assessment of BMP-2-induced ALP activity through direct color staining. (A,B) The ALP activity was determined in the cell lysates of cultures grown in the absence of growth factors or in the presence of BMP-2 at different concentrations or FGF-2 at 50 ng/mL for (A) 4 days or (B) 7 days of co-culture. The ALP activity is shown normalized to the DNA content of each lysate sample. The data are represented as mean &#177; standard deviation. The statistical analysis was performed using GraphPad Prism 9.5.1. An ordinary one-way ANOVA with Dunnett&#39;s multiple comparisons test was performed with n = 5;&#160;**** P &#60; 0.0001. (C) Direct color staining of the ALP activity in niches grown in the presence of 50 ng/mL FGF-2 or BMP-2 for 7 days of co-culture. The images on the left-hand side depict stitched whole-well overviews of images acquired at 2.5x magnification (scale bar: 1,000 &#181;m), while the images on the right-hand side depict one field of view acquired at 5x magnification (scale bar: 400 &#181;m). <a href="https://www.jove.com/files/ftp_upload/65413/65413fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65413/65413fig05.jpg" /> 
Figure 5: Employment of osteogenic, vascularized niches in advanced cancer models. (A) The GFP signals of cultures grown in the presence of BMP-2 were imaged on day 4 of co-culture before a control solution (left) or bevacizumab at 10 &#181;g/mL (right) were added for 2 days, after which the cultures were imaged again (day 6 of co-culture). Scale bar: 600 &#181;m. (B) Higher magnification images of the bevacizumab-treated cultures are shown in A. Scale bar: 250 &#181;m.&#160;(C) The total length of the endothelial networks as shown in A&#160;were quantified using Angiogenesis Analyzer for ImageJ from images acquired daily; n &#8805; 3. (D) CellTrace FarRed-labeled MDA-MB-231 breast cancer cells were added to the 4 day co-cultures grown in the presence of BMP-2, and time-lapse images were acquired starting 2 days after the cancer cell addition. Scale bar: 100 &#181;m. (E) Maximum intensity projections of confocal stacks (total height: 70 &#181;m; z-step: 2.4 &#181;m) of triple co-cultures generated as described in D and fixed and stained for F-actin. Left: niches featuring MDA-MB-231 breast cancer cells; right: niches featuring U2OS osteosarcoma cells. Scale bars for images on the left: 200 &#181;m; scale bars for images on the right: 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65413/65413fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast PolyEthylene Glycol PEG Hydrogels in an Imaging Microplate at JoVE.com

Author Spotlight: Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(Ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate  

An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...

simple-establishment-vascularized-osteogenic-bone-marrow-niche-using

Nanyang Technological University

Research

JoVE Journal

Bioengineering

2.4K Views.  University Hospital Zurich, University of Zurich. An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

Video: Author Spotlight: Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(Ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate  

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Developmental Biology

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized matrix. This organization is challenging to replicate due to the heterogeneous environment of bone. Ceramic omnidirectional bioprinting in cell-suspensions (COBICS) is a microgel-based bioprinting technique that uniquely replicates the mineral and cellular structure of bone. COBICS prints complex, biologically relevant constructs without the need for sacrificial support materials or harsh postprocessing steps (e.g., radiation and high-temperature sintering), which are two of the biggest challenges in the additive manufacturing of bone mimetic constructs. This technique is enabled via the freeform extrusion of a novel calcium phosphate-based ink within a gelatin-based microgel suspension. The yield-stress properties of the suspension allow deposition and support the printed bone structure. UV crosslinking and nanoprecipitation then "lock" it in place. The ability to print nanostructured bone-mimetic ceramics within cell-laden biomaterials provides spatiotemporal control over macro- and micro-architecture and facilitates the real-time fabrication of complex bone constructs in clinical settings.

This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized ...

Osteo-Organoid-Derived Cells for Effective HSCT in Irradiated Mice

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

A Human Bone Marrow 3D Model to Investigate the Dynamics and Interactions Between Resident Cells in Physiological or Tumoral Contexts

The medullary niche is a complex ecosystem that is essential to maintain homeostasis for resident cells. Indeed, the bone marrow, which includes a complex extracellular matrix and various cell types, such as mesenchymal stem cells, osteoblasts, and endothelial cells, is deeply involved in hematopoietic stem cell regulation through direct cell-cell interactions, as well as cytokine production. To closely mimic this in vivo structure and conduct experiments reflecting the responses of the human bone marrow, several 3D models have been created based on biomaterials, relying primarily on primary stromal cells. Here, a protocol is described to obtain a minimal and standardized system that is easy to set up and provides features of bone marrow-like structure, which combines different cell populations including endothelial cells, and reflects the heterogeneity of in vivo bone marrow tissue. This 3D bone marrow-like structure-assembled using calcium phosphate-based particles and human cell lines, representative of the bone marrow microenvironment-allows the monitoring of a wide variety of biological processes by combining or replacing different primary cell populations within the system. The final 3D structures can then either be harvested for image analysis after fixation, paraffin-embedding, and histological/immunohistochemical staining for cell localization within the system, or dissociated to collect each cellular component for molecular or functional characterization.

Here, we describe an easy-to-implement, standardized, microphysiological system that reflects the complexity of the human bone marrow's in vivo structure, providing a pertinent model to finely study a broad range of normal and pathological events.

The medullary niche is a complex ecosystem that is essential to maintain homeostasis for resident cells. Indeed, the bone marrow, which includes a complex ...

Biology

Artificial Cartilage Implants for Bone Regeneration via Endochondral Ossification

Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it does not have a mechanism to induce angiogenesis. Implanting artificial cartilage tissue fabricated from MSCs induces angiogenesis and bone formation in vivo via endochondral ossification (ECO). Therefore, this ECO-mediated approach may be a promising bone regeneration therapy in the future. An important aspect of the clinical application of this ECO-mediated approach is establishing a protocol for preparing enough cartilage to be implanted to repair the bone defect. It is especially not practical to design a single mass of grafted cartilage of a size that conforms to the shape of the actual bone defect. Therefore, the cartilage to be transplanted must have the property of forming bone integrally when multiple pieces are implanted. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification to meet clinical requirements. Although many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo, the optimal scaffold material to meet the needs of clinical applications has yet to be determined. Hyaluronic acid (HA) is a crucial component of the cartilage extracellular matrix and is a biodegradable and biocompatible polysaccharide. Here, we show that HA hydrogels have excellent properties to support in vitro differentiation of MSC-based cartilage tissue and promote endochondral bone formation in vivo.

Author Spotlight: Enhancing Bone Regeneration with Vascularized Artificial Cartilage Integration 

Bone therapy via endochondral ossification by implanting artificial cartilage tissue produced from mesenchymal stem cells has the potential to circumvent the drawbacks of conventional therapies. Hyaluronic acid hydrogels are effective in scaling up uniformly differentiated cartilage grafts as well as creating integrated bone with vascularization between fused grafts in vivo.

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it ...

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

Summary

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Chapters in this video

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Use of Human Perivascular Stem Cells for Bone Regeneration

Generation and Recovery of β-cell Spheroids From Step-growth PEG-peptide Hydrogels

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Engineered Vascularized Muscle Flap

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

A Human Bone Marrow 3D Model to Investigate the Dynamics and Interactions Between Resident Cells in Physiological or Tumoral Contexts

Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells