Bioinstructive Coatings for Hematopoietic Stem Cell Expansion Based on Chemical Vapor Deposition Copolymerization.

We report the chemical vapor deposition (CVD) of dual-functional polymer films for the specific and orthogonal immobilization of two biomolecules (notch ligand delta-like 1 (DLL1) and an RGD-peptide) that govern the fate of hematopoietic stem and progenitor cells. The composition of the CVD polymer and thus the biomolecule ratio can be tailored to investigate and optimize the influence of the relative surface concentrations of biomolecules on stem cell behavior. Prior to cell experiments, all surfaces were characterized by infrared reflection adsorption spectroscopy, time-of-flight secondary ion mass spectrometry, and X-ray photoelectron spectroscopy to confirm the presence of both biomolecules. In a proof-of-principle stem cell culture study, we show that all polymer surfaces are cytocompatible and that the proliferation of the hematopoietic stem and progenitor cells is predominantly influenced by the surface concentration of immobilized DLL1.

Impact of substrate elasticity on human hematopoietic stem and progenitor cell adhesion and motility.

In the bone marrow, hematopoietic stem cells (HSCs) reside in endosteal and vascular niches. The interactions with the niches are essential for the maintenance of HSC number and properties. Although the molecular nature of these interactions is well understood, little is known about the role of physical parameters such as matrix elasticity. Osteoblasts, the major cellular component of the endosteal HSC niche, flatten during HSC mobilization. We show that this process is accompanied by osteoblast stiffening, demonstrating that not only biochemical signals but also mechanical properties of the niche are modulated. HSCs react to stiffer substrates with increased cell adhesion and migration, which could facilitate the exit of HSCs from the niche. These results indicate that matrix elasticity is an important factor in regulating the retention of HSCs in the endosteal niche and should be considered in attempts to propagate HSCs in vitro for clinical applications.

A Multifunctional Nanostructured Hydrogel as a Platform for Deciphering Niche Interactions of Hematopoietic Stem and Progenitor Cells.

For over half a century, hematopoietic stem cells (HSCs) have been used for transplantation therapy to treat severe hematologic diseases. Successful outcomes depend on collecting sufficient donor HSCs as well as ensuring efficient engraftment. These processes are influenced by dynamic interactions of HSCs with the bone marrow niche, which can be revealed by artificial niche models. Here, a multifunctional nanostructured hydrogel is presented as a 2D platform to investigate how the interdependencies of cytokine binding and nanopatterned adhesive ligands influence the behavior of human hematopoietic stem and progenitor cells (HSPCs). The results indicate that the degree of HSPC polarization and motility, observed when cultured on gels presenting the chemokine SDF-1α and a nanoscale-defined density of a cellular (IDSP) or extracellular matrix (LDV) α4ß1 integrin binding motif, are differently influenced on hydrogels functionalized with the different ligand types. Further, SDF-1α promotes cell polarization but not motility. Strikingly, the degree of differentiation correlates negatively with the nanoparticle spacing, which determines ligand density, but only for the cellular-derived IDSP motif. This mechanism potentially offers a means of predictably regulating early HSC fate decisions. Consequently, the innovative multifunctional hydrogel holds promise for deciphering dynamic HSPC-niche interactions and refining transplantation therapy protocols.

On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field.

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established.

Osteoblast-secreted factors enhance the expression of dysadherin and CCL2-dependent migration of renal carcinoma cells.

Renal cell carcinoma (RCC) frequently metastasizes to the bone marrow. These metastases are characterized by extensive osteolytic lesions. The mechanism, however, by which RCC cells metastasize to bone marrow remains poorly understood. To unravel the role of bone marrow cells in this context, we performed cell adhesion and migration assays using human RCC cell lines to analyze the influence of resident bone marrow cells on renal tumor cells. The strongest adhesion of RCC cells was observed to osteoblasts. Moreover, conditioned medium of osteoblasts (OB-CM) significantly increased RCC cell migration. By gene expression analysis dysadherin was identified as a transcript whose expression could be elevated more than twofold in RCC cells when exposed to OB-CM. Suppression of dysadherin expression in RCC cells by siRNA reduced their ability to migrate in the presence of OB-CM. Furthermore, the RCC cells secreted high amounts of the chemokine CCL2 when tumor cells migrated under the influence of osteoblast-secreted factors. CCL2 neutralization strongly reduced the migratory ability of the RCC cells. Silencing the expression of dysadherin in RCC cells resulted in a twofold reduction of CCL2 protein expression indicating a dysadherin-dependent expression of the chemokine. Taken together, our data show that osteoblasts are the major cell type of the bone marrow that affect RCC cells by secreting factors that increase the expression of dysadherin and CCL2 in the tumor cells leading to enhanced cell migration. These data suggest an osteoblast-induced autocrine mechanism for a facilitated homing of RCC cells to the bone marrow.

Artificial niches: biomimetic materials for hematopoietic stem cell culture.

Hematopoietic stem cells (HSCs) are indispensable for the treatment of patients with hematological disorders such as leukemia. However, the amount of available transplantable HSCs is limited. Therefore, new approaches to multiply HSCs in the laboratory are needed. Promising biomimetic technologies for HSC expansion are currently developed. This feature article gives an insight into the significance of this approach and introduces the essential building blocks (cells, matrix, and scaffolds) of biomimetic materials. Some recent strategies are highlighted and the challenges and possible applications of such materials are discussed.

The extracellular matrix of hematopoietic stem cell niches.

Hematopoietic stem cells (HSCs) are the life-long source of all types of blood cells. Their function is controlled by their direct microenvironment, the HSC niche in the bone marrow. Although the importance of the extracellular matrix (ECM) in the niche by orchestrating niche architecture and cellular function is widely acknowledged, it is still underexplored. In this review, we provide a comprehensive overview of the ECM in HSC niches. For this purpose, we first briefly outline HSC niche biology and then review the role of the different classes of ECM molecules in the niche one by one and how they are perceived by cells. Matrix remodeling and the emerging importance of biophysics in HSC niche function are discussed. Finally, the application of the current knowledge of ECM in the niche in form of artificial HSC niches for HSC expansion or targeted differentiation as well as drug testing is reviewed.

Flow-induced glycocalyx formation and cell alignment of HUVECs compared to iPSC-derived ECs for tissue engineering applications.

The relevance of cellular in vitro models highly depends on their ability to mimic the physiological environment of the respective tissue or cell niche. Static culture conditions are often unsuitable, especially for endothelial models, since they completely neglect the physiological surface shear stress and corresponding reactions of endothelial cells (ECs) such as alignment in the direction of flow. Furthermore, formation and maturation of the glycocalyx, the essential polysaccharide layer covering all endothelial surfaces and regulating diverse processes, is highly dependent on applied fluid flow. This fragile but utterly important macromolecular layer is hard to analyze, its importance is often underestimated and accordingly neglected in many endothelial models. Therefore, we exposed human umbilical vein ECs (HUVECs) and human induced pluripotent stem cell-derived ECs (iPSC-ECs) as two relevant EC models in a side-by-side comparison to static and physiological dynamic (6.6 dyn cm-2) culture conditions. Both cell types demonstrated an elongation and alignment along the flow direction, some distinct changes in glycocalyx composition on the surface regarding the main glycosaminoglycan components heparan sulfate, chondroitin sulfate or hyaluronic acid as well as an increased and thereby improved glycocalyx thickness and functionality when cultured under homogeneous fluid flow. Thus, we were able to demonstrate the maturity of the employed iPSC-EC model regarding its ability to sense fluid flow along with the general importance of physiological shear stress for glycocalyx formation. Additionally, we investigated EC monolayer integrity with and without application of surface shear stress, revealing a comparable existence of tight junctions for all conditions and a reorganization of the cytoskeleton upon dynamic culture leading to an increased formation of focal adhesions. We then fabricated cell sheets of EC monolayers after static and dynamic culture via non-enzymatic detachment using thermoresponsive polymer coatings as culture substrates. In a first proof-of-concept we were able to transfer an aligned iPSC-EC sheet to a 3D-printed scaffold thereby making a step in the direction of vascular modelling. We envision these results to be a valuable contribution to improvements of in vitro endothelial models and vascular engineering in the future.

A parallelized, perfused 3D triculture model of leukemia forin vitrodrug testing of chemotherapeutics.

Leukemia patients undergo chemotherapy to combat the leukemic cells (LCs) in the bone marrow. During therapy not only the LCs, but also the blood-producing hematopoietic stem and progenitor cells (HSPCs) may be destroyed. Chemotherapeutics targeting only the LCs are urgently needed to overcome this problem and minimize life-threatening side-effects. Predictivein vitrodrug testing systems allowing simultaneous comparison of various experimental settings would enhance the efficiency of drug development. Here, we present a three-dimensional (3D) human leukemic bone marrow model perfused using a magnetic, parallelized culture system to ensure media exchange. Chemotherapeutic treatment of the acute myeloid leukemia cell line KG-1a in 3D magnetic hydrogels seeded with mesenchymal stem/stromal cells (MSCs) revealed a greater resistance of KG-1a compared to 2D culture. In 3D tricultures with HSPCs, MSCs and KG-1a, imitating leukemic bone marrow, HSPC proliferation decreased while KG-1a cells remained unaffected post treatment. Non-invasive metabolic profiling enabled continuous monitoring of the system. Our results highlight the importance of using biomimetic 3D platforms with proper media exchange and co-cultures for creatingin vivo-like conditions to enablein vitrodrug testing. This system is a step towards drug testing in biomimetic, parallelizedin vitroapproaches, facilitating the discovery of new anti-leukemic drugs.

Fused Deposition Modeling of Microfluidic Chips in Transparent Polystyrene.

Polystyrene (PS) is one of the most commonly used thermoplastic materials worldwide and plays a ubiquitous role in today's biomedical and life science industry and research. The main advantage of PS lies in its facile processability, its excellent optical and mechanical properties, as well as its biocompatibility. However, PS is only rarely used in microfluidic prototyping, since the structuring of PS is mainly performed using industrial-scale replication processes. So far, microfluidic chips in PS have not been accessible to rapid prototyping via 3D printing. In this work, we present, for the first time, 3D printing of transparent PS using fused deposition modeling (FDM). We present FDM printing of transparent PS microfluidic channels with dimensions as small as 300 µm and a high transparency in the region of interest. Furthermore, we demonstrate the fabrication of functional chips such as Tesla-mixer and mixer cascades. Cell culture experiments showed a high cell viability during seven days of culturing, as well as enabling cell adhesion and proliferation. With the aid of this new PS prototyping method, the development of future biomedical microfluidic chips will be significantly accelerated, as it enables using PS from the early academic prototyping all the way to industrial-scale mass replication.

Rebuilding the hematopoietic stem cell niche: Recent developments and future prospects.

Hematopoietic stem cells (HSCs) have proven their clinical relevance in stem cell transplantation to cure patients with hematological disorders. Key to their regenerative potential is their natural microenvironment - their niche - in the bone marrow (BM). Developments in the field of biomaterials enable the recreation of such environments with increasing preciseness in the laboratory. Such artificial niches help to gain a fundamental understanding of the biophysical and biochemical processes underlying the interaction of HSCs with the materials in their environment and the disturbance of this interplay during diseases affecting the BM. Artificial niches also have the potential to multiply HSCs in vitro, to enable the targeted differentiation of HSCs into mature blood cells or to serve as drug-testing platforms. In this review, we will introduce the importance of artificial niches followed by the biology and biophysics of the natural archetype. We will outline how 2D biomaterials can be used to dissect the complexity of the natural niche into individual parameters for fundamental research and how 3D systems evolved from them. We will present commonly used biomaterials for HSC research and their applications. Finally, we will highlight two areas in the field of HSC research, which just started to unlock the possibilities provided by novel biomaterials, in vitro blood production and studying the pathophysiology of the niche in vitro. With these contents, the review aims to give a broad overview of the different biomaterials applied for HSC research and to discuss their potentials, challenges and future directions in the field. STATEMENT OF SIGNIFICANCE: Hematopoietic stem cells (HSCs) are multipotent cells responsible for maintaining the turnover of all blood cells. They are routinely applied to treat patients with hematological diseases. This high clinical relevance explains the necessity of multiplication or differentiation of HSCs in the laboratory, which is hampered by the missing natural microenvironment - the so called niche. Biomaterials offer the possibility to mimic the niche and thus overcome this hurdle. The review introduces the HSC niche in the bone marrow and discusses the utility of biomaterials in creating artificial niches. It outlines how 2D systems evolved into sophisticated 3D platforms, which opened the gateway to applications such as, expansion of clinically relevant HSCs, in vitro blood production, studying niche pathologies and drug testing.

Limited Potential or Unfavorable Manipulations? Strategies Toward Efficient Mesenchymal Stem/Stromal Cell Applications.

Despite almost 50 years of research and over 20 years of preclinical and clinical studies, the question of curative potential of mesenchymal stem/stromal cells (MSCs) is still widely discussed in the scientific community. Non-reproducible treatment outcomes or even absence of treatment effects in comparison to control groups challenges the potential of these cells for routine application both in tissue engineering and in regenerative medicine. One of the reasons of such outcomes is non-standardized and often disadvantageous ex vivo manipulation of MSCs prior therapy. In most cases, clinically relevant cell numbers for MSC-based therapies can be only obtained by in vitro expansion of isolated cells. In this mini review, we will discuss point by point possible pitfalls in the production of human MSCs for cell therapies, without consideration of material-based applications. Starting with cell source, choice of donor and recipient, as well as isolation methods, we will then discuss existing expansion protocols (two-/three-dimensional cultivation, basal medium, medium supplements, static/dynamic conditions, and hypoxic/normoxic conditions) and influence of these strategies on the cell functionality after implantation. The role of potency assays will also be addressed. The final aim of this mini review is to illustrate the heterogeneity of current strategies for gaining MSCs for clinical applications with their strengths and weaknesses. Only a careful consideration and standardization of all pretreatment processes/methods for the different applications of MSCs will ensure robust and reproducible performance of these cell populations in the different experimental and clinical settings.

Nitric Oxide in the Control of the in vitro Proliferation and Differentiation of Human Hematopoietic Stem and Progenitor Cells.

Hematopoietic stem and progenitor cell (HSPC) transplantation is the best-studied cellular therapy and successful in vitro control of HSPCs has wide clinical implications. Nitric oxide (NO) is a central signaling molecule in vivo and has been implicated in HSPC mobilization to the blood stream in mice. The influence of NO on HSPC behavior in vitro is, however, largely obscure due to the variety of employed cell types, NO administration systems, and used concentration ranges in the literature. Additionally, most studies are based on murine cells, which do not necessarily mimic human HSPC behavior. Thus, the aim of the present study was the systematic, concentration-dependent evaluation of NO-mediated effects on human HSPC behavior in vitro. By culture in the presence of the long-term NO donor diethylenetriamine/nitric oxide adduct (DETA/NO) in a nontoxic concentration window, a biphasic role of NO in the regulation of HSPC behavior was identified: Low DETA/NO concentrations activated classical NO signaling, identified via increased intracellular cyclic guanosine monophosphate (cGMP) levels and proteinkinases G (PKG)-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation and mediated a pro-proliferative response of HSPCs. In contrast, elevated NO concentrations slowed cell proliferation and induced HSPC differentiation. At high concentrations, s-nitrosylation levels were elevated, and myeloid differentiation was increased at the expense of lymphoid progenitors. Together, these findings hint at a central role of NO in regulating human HSPC behavior and stress the importance and the potential of the use of adequate NO concentrations for in vitro cultures of HSPCs, with possible implications for clinical application of in vitro expanded or differentiated HSPCs for cellular therapies.

Iron Nanoparticle Composite Hydrogels for Studying Effects of Iron Ion Release on Red Blood Cell In Vitro Production.

Growing numbers of complex surgical interventions increase the need for blood transfusions, which cannot be fulfilled by the number of donors. Therefore, the interest in producing erythrocytes from their precursors-the hematopoietic stem and progenitor cells (HSPCs)-in laboratories is rising. To enable this, in vitro systems are needed, which allow analysis of the effects of essential factors such as iron on erythroid development. For this purpose, iron ion-releasing systems based on poly(ethylene glycol) (PEG)-iron nanocomposites are developed to assess if gradual iron release improves iron bioavailability during in vitro erythroid differentiation. The nanocomposites are synthesized using surfactant-free pulsed laser ablation of iron directly in the PEG solution. The iron concentrations released from the material are sufficient to influence in vitro erythropoiesis. In this way, the production of erythroid cells cultured on flat PEG-iron nanocomposite hydrogel pads can be enhanced. In contrast, erythroid differentiation is not enhanced in the biomimetic macroporous 3D composite scaffolds, possibly because of local iron overload within the pores of the system. In conclusion, the developed iron nanoparticle-PEG composite hydrogel allows constant iron ion release and thus paves the way (i) to understand the role of iron during erythropoiesis and (ii) toward the development of biomaterials with a controlled iron release for directing erythropoiesis in culture.

Monitoring matrix remodeling in the cellular microenvironment using microrheology for complex cellular systems.

Multiple particle tracking (MPT) microrheology was employed for monitoring the development of extracellular matrix (ECM) mechanical properties in the direct microenvironment of living cells. A customized setup enabled us to overcome current limitations: (i) Continuous measurements were enabled using a cell culture chamber, with this, matrix remodeling by fibroblasts in the heterogeneous environment of macroporous scaffolds was monitored continuously. (ii) Employing tracer laden porous scaffolds for seeding human mesenchymal stem cells (hMSCs), we followed conventional differentiation protocols. Thus, we were, for the first time able to study the massive alterations in ECM elasticity during hMSC differentiation. (iii) MPT measurements in 2D cell cultures were enabled using a long distance objective. Exemplarily, local mechanical properties of the ECM in human umbilical vein endothelial cell (HUVEC) cultures, that naturally form 2D layers, were investigated scaffold-free. Using our advanced setup, we measured local, apparent elastic moduli G0,app in a range between 0.08 and 60 Pa. For fibroblasts grown in collagen-based scaffolds, a continuous decrease of local matrix elasticity resulted during the first 10 hours after seeding. The osteogenic differentiation of hMSC cells cultivated in similar scaffolds, led to an increase of G0,app by 100 %, whereas after adipogenic differentiation it was reduced by 80 %. The local elasticity of ECM that was newly secreted by HUVECs increased significantly upon addition of protease inhibitor and in high glucose conditions even a twofold increase in G0,app was observed. The combination of these advanced methods opens up new avenues for a broad range of investigations regarding cell-matrix interactions and the propagation of ECM mechanical properties in complex biological systems. STATEMENT OF SIGNIFICANCE: Cells sense the elasticity of their environment on a micrometer length scale. For studying the local elasticity of extracellular matrix (ECM) in the direct environment of living cells, we employed an advanced multipleparticle tracking microrheology setup. MPT is based on monitoring the Brownian motion oftracer particles, which is restricted by the surrounding network. Network elasticity can thusbe quantified. Overcoming current limitations, we realized continuous investigations of ECM elasticityduring fibroblast growth. Furthermore, MPT measurements of stem cell ECM showed ECMstiffening during osteogenic differentiation and softening during adipogenic differentiation.Finally, we characterized small amounts of delicate ECM newly secreted in scaffold-freecultures of endothelial cells, that naturally form 2D layers.

Migration Assay for Leukemic Cells in a 3D Matrix Toward a Chemoattractant.

In leukemia, leukemic cells hijack the hematopoietic stem cell (HSC) microenvironment in the bone marrow-the so-called stem cell niche-by flooding the niche with clonal progeny of leukemic cells. They can exploit signaling pathways which are critical for HSC development to support their own survival, homing, and maintenance. These interactions of leukemic cells with the microenvironment have an impact on therapy progress and patient outcome. Therefore, signals for homing and anchorage of leukemic cells to the bone marrow have to be investigated by using tools that allow the migration of cells toward critical signals. Here, we describe an in vitro migration assay for leukemic cells toward a chemoattractant in a 3D environment exemplified by migration of the cell line OCI-AML3 to a CXC motif chemokine ligand 12 (CXCL12) gradient. For this purpose, a chemotaxis slide is filled with a hydrogel system mimicking the extracellular matrix in vivo. The cells are encapsulated into the hydrogel network during polymerization, and a CXCL12 gradient is introduced in the enclosed chambers to trigger migration. Cell migration in the 3D network of the hydrogel is monitored by time-lapse microscopy. We describe the experimental setup and the tools for cell tracking and data analysis.

Microfluidic Shear Force Assay to Determine Cell Adhesion Forces.

Cell adhesion is implicated in many physiological settings such as the retention of hematopoietic stem cells (HSCs) in their bone marrow niches or their migration into the bloodstream. During HSC mobilization these adhesion sites are cleaved and have to be newly formed during HSC homing and engraftment. To determine the adhesive properties of HSCs on different extracellular matrix (ECM) molecules, we present a microfluidic shear force assay, where a laminar flow is used to detach a semi-adherent cell population, the HSC model cell line KG-1a, from an ECM protein-coated substrate. This technique combines the high throughput of population-based assays with the ability to observe cell detachment in real time. Additionally, it is suitable for weakly adherent cells, as the setup allows cell incubation on various substrates and application of shear stress ranging several orders of magnitude in one setup without additional washing or transfer steps. As a measure for the adhesion strength of the studied cell population on the substrate, the critical shear force τ50 is determined which is required to remove 50% of the initially adherent cell fraction.

3D models of the bone marrow in health and disease: yesterday, today and tomorrow.

The complex interaction between hematopoietic stem cells (HSCs) and their microenvironment in the human bone marrow ensures a life-long blood production by balancing stem cell maintenance and differentiation. This so-called HSC niche can be disturbed by malignant diseases. Investigating their consequences on hematopoiesis requires deep understanding of how the niches function in health and disease. To facilitate this, biomimetic models of the bone marrow are needed to analyse HSC maintenance and hematopoiesis under steady-state and diseased conditions. Here, 3D bone marrow models, their fabrication methods (including 3D bioprinting) and implementations recapturing bone marrow functions in health and diseases, are presented.

Potential of electrospun cationic BSA fibers to guide osteogenic MSC differentiation via surface charge and fibrous topography.

Large or complex bone fractures often need clinical treatments for sufficient bone repair. New treatment strategies have pursued the idea of using mesenchymal stromal cells (MSCs) in combination with osteoinductive materials to guide differentiation of MSCs into bone cells ensuring complete bone regeneration. To overcome the challenge of developing such materials, fundamental studies are needed to analyze and understand the MSC behavior on modified surfaces of applicable materials for bone healing. For this purpose, we developed a fibrous scaffold resembling the bone/bone marrow extracellular matrix (ECM) based on protein without addition of synthetic polymers. With this biomimetic in vitro model we identified the fibrous structure as well as the charge of the material to be responsible for its effects on MSC differentiation. Positive charge was introduced via cationization that additionally supported the stability of the scaffold in cell culture, and acted as nucleation point for mineralization during osteogenesis. Furthermore, we revealed enhanced focal adhesion formation and osteogenic differentiation of MSCs cultured on positively charged protein fibers. This pure protein-based and chemically modifiable, fibrous ECM model allows the investigation of MSC behavior on biomimetic materials to unfold new vistas how to direct cells' differentiation for the development of new bone regenerating strategies.

Biomimetic 3D in vitro model of biofilm triggered osteomyelitis for investigating hematopoiesis during bone marrow infections.

In this work, we define the requirements for a human cell-based osteomyelitis model which overcomes the limitations of state of the art animal models. Osteomyelitis is a severe and difficult to treat infection of the bone that develops rapidly, making it difficult to study in humans. We have developed a 3D in vitro model of the bone marrow, comprising a macroporous material, human hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs). Inclusion of biofilms grown on an implant into the model system allowed us to study the effects of postoperative osteomyelitis-inducing bacteria on the bone marrow. The bacteria influenced the myeloid differentiation of HSPCs as well as MSC cytokine expression and the MSC ability to support HSPC maintenance. In conclusion, we provide a new 3D in vitro model which meets all the requirements for investigating the impact of osteomyelitis. STATEMENT OF SIGNIFICANCE: Implant-associated osteomyelitis is a persistent bacterial infection of the bone which occurs in many implant patients and can result in functional impairments or even entire loss of the extremity. Nevertheless, surprisingly little is known on the triangle interaction between implant material, bacterial biofilm and affected bone tissue. Closing this gap of knowledge would be crucial for the fundamental understanding of the disease and the development of novel treatment strategies. For this purpose, we developed the first biomaterial-based system that is able to mimic implant-associated osteomyelitis outside of the body, thus, opening the avenue to study this fatal disease in the laboratory.