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 two-dimensional 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, is strongly influenced by the ligand type, but not the ligand density. 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. This article is protected by copyright. All rights reserved.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Magnetic Macroporous Hydrogels as a Novel Approach for Perfused Stem Cell Culture in 3D Scaffolds via Contactless Motion Control.

There is an urgent need for 3D cell culture systems that avoid the oversimplifications and artifacts of conventional culture in 2D. However, 3D culture within the cavities of porous biomaterials or large 3D structures harboring high cell numbers is limited by the needs to nurture cells and to remove growth-limiting metabolites. To overcome the diffusion-limited transport of such soluble factors in 3D culture, mixing can be improved by pumping, stirring or shaking, but this in turn can lead to other problems. Using pumps typically requires custom-made accessories that are not compatible with conventional cell culture disposables, thus interfering with cell production processes. Stirring or shaking allows little control over movement of scaffolds in media. To overcome these limitations, magnetic, macroporous hydrogels that can be moved or positioned within media in conventional cell culture tubes in a contactless manner are presented. The cytocompatibility of the developed biomaterial and the applied magnetic fields are verified for human hematopoietic stem and progenitor cells (HSPCs). The potential of this technique for perfusing 3D cultures is demonstrated in a proof-of-principle study that shows that controlled contactless movement of cell-laden magnetic hydrogels in culture media can mimic the natural influence of differently perfused environments on HSPCs.

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.

3D models of the hematopoietic stem cell niche under steady-state and active conditions.

Hematopoietic stem cells (HSCs) in the bone marrow are able to differentiate into all types of blood cells and supply the organism each day with billions of fresh cells. They are applied to cure hematological diseases such as leukemia. The clinical need for HSCs is high and there is a demand for being able to control and multiply HSCs in vitro. The hematopoietic system is highly proliferative and thus sensitive to anti-proliferative drugs such as chemotherapeutics. For many of these drugs suppression of the hematopoietic system is the dose-limiting toxicity. Therefore, biomimetic 3D models of the HSC niche that allow to control HSC behavior in vitro and to test drugs in a human setting are relevant for the clinics and pharmacology. Here, we describe a perfused 3D bone marrow analog that allows mimicking the HSC niche under steady-state and activated conditions that favor either HSC maintenance or differentiation, respectively, and allows for drug testing.

Fabrication of biofunctionalized, cell-laden macroporous 3D PEG hydrogels as bone marrow analogs for the cultivation of human hematopoietic stem and progenitor cells.

In vitro proliferation of hematopoietic stem cells (HSCs) is yet an unresolved challenge. Found in the bone marrow, HSCs can undergo self-renewing cell division and thereby multiply. Recapitulation of the bone marrow environment in order to provide the required signals for their expansion is a promising approach.Here, we describe a technique to produce biofunctionalized, macroporous poly(ethylene glycol) diacrylate (PEGDA) hydrogels that mimic the spongy 3D architecture of trabecular bones, which host the red, blood-forming bone marrow. After seeding these scaffolds with cells, they can be used as simplified bone marrow analogs for the cultivation of HSCs. This method can easily be conducted with standard laboratory chemicals and equipment. The 3D hydrogels are produced via salt leaching and biofunctionalization of the material is achieved by co-polymerizing the PEGDA with an RGD peptide. Finally, cell seeding and retrieval are described.

Biomimetic macroporous PEG hydrogels as 3D scaffolds for the multiplication of human hematopoietic stem and progenitor cells.

Multiplication of hematopoietic stem cells (HSCs) in vitro with current standard methods is limited and mostly insufficient for clinical applications of these cells. They quickly lose their multipotency in culture because of the fast onset of differentiation. In contrast, HSCs efficiently self-renew in their natural microenvironment (their niche) in the bone marrow. Therefore, engineering artificial bone marrow analogs is a promising biomaterial-based approach for culturing these cells. In the current study, a straight-forward, easy-to-use method for the production of biofunctionalized, macroporous hydrogel scaffolds that mimic the spongy architecture of trabecular bones was developed. As surrogates for cellular components of the niche, mesenchymal stem cells (MSCs) from different sources (bone marrow and umbilical cord) and osteoblast-like cells were tested. MSCs from bone marrow had the strongest pro-proliferative effect on freshly isolated human hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood. Co-culture in the pores of the three-dimensional hydrogel scaffold showed that the positive effect of MSCs on preservation of HSPC stemness was more pronounced in 3D than in standard 2D cell culture systems. Thus, the presented biomimetic scaffolds revealed to meet the basic requirements for creating artificial HSC niches.

Regulation of hematopoietic stem cell behavior by the nanostructured presentation of extracellular matrix components.

Hematopoietic stem cells (HSCs) are maintained in stem cell niches, which regulate stem cell fate. Extracellular matrix (ECM) molecules, which are an essential part of these niches, can actively modulate cell functions. However, only little is known on the impact of ECM ligands on HSCs in a biomimetic environment defined on the nanometer-scale level. Here, we show that human hematopoietic stem and progenitor cell (HSPC) adhesion depends on the type of ligand, i.e., the type of ECM molecule, and the lateral, nanometer-scaled distance between the ligands (while the ligand type influenced the dependency on the latter). For small fibronectin (FN)-derived peptide ligands such as RGD and LDV the critical adhesive interligand distance for HSPCs was below 45 nm. FN-derived (FN type III 7-10) and osteopontin-derived protein domains also supported cell adhesion at greater distances. We found that the expression of the ECM protein thrombospondin-2 (THBS2) in HSPCs depends on the presence of the ligand type and its nanostructured presentation. Functionally, THBS2 proved to mediate adhesion of HSPCs. In conclusion, the present study shows that HSPCs are sensitive to the nanostructure of their microenvironment and that they are able to actively modulate their environment by secreting ECM factors.

Surface Properties of Nanostructured Bio-Active Interfaces: Impacts of Surface Stiffness and Topography on Cell-Surface Interactions.

Due to their ability to confer key functions of the native extracellular matrix (ECM) poly(ethylene glycol) (PEG)-based and PEG-modified materials have been extensively used as biocompatible and biofunctionalized substrate systems to study the influence of environmental parameters on cell adhesion in vitro. Given wide-ranging recent evidence that ECM compliance influences a variety of cell functions, the detailed determination and characterization of the specific PEG surface characteristics including topography, stiffness and chemistry is required. Here, we studied two frequently used bio-active interfaces - PEG-based and PEG-modified surfaces - to elucidate the differences between the physical surface properties, which cells can sense and respond to. For this purpose, two sets of surfaces were synthesized: the first set consisted of nanopatterned glass surfaces containing cRGD-functionalized gold nanoparticles surrounded by a passivated PEG-silane layer and the second set consisted of PEG-diacrylate (PEG-DA) hydrogels decorated with cRGD-functionalized gold nanoparticlesAlthough the two sets of nanostructured materials compared here were highly similar in terms of density and geometrical distribution of the presented bio-ligands as well as in terms of mechanical bulk properties, the topography and mechanical properties of the surfaces were found to be substantially different and are described in detail. In comparison to very stiff and ultrasmooth surface properties of the PEG-passivated glasses, the mechanical properties of PEG-DA surfaces in the biologically relevant stiffness range, together with the increased surface roughness at micro- and nanoscale levels have the potential to affect cell behavior. This potential was verified by studying the adhesive behavior of hematopoietic KG-1a and rat embryonic fibroblast (REF52) cells on both surfaces.

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.