now clear that architectures that reproduce tissue organization in 3D are favored for studying function as first shown by Bissell and co-workers nearly 30 years ago. [5,6] Another emerging concept is the capacity for multiple cell types to auto-organize when given the appropriate mechanical cues. [7] A large variety of 3D models have been developed, with the major subtypes being spheroids/organoids, multilayered tissue like models, and scaffold models. [8] 3D tissues are frequently constructed as spheroids using hydrogel-like matrices such as Matrigel, [9] or PuraMatrix, [10] with a highly desirable degree of mechanical softness, however, there can be problems related to cost and inhomogeneity of the materials. [11] Multilayer models hold a lot of promise for applications such as skin toxicology measurements, [12] however, they cannot be easily applied for more complex tissue or organ constructs including vasculature. Considerable attention has thus focused on the development of biocompatible scaffold materials for hosting cells in 3D. A variety of synthetic and bio-derived polymers (some resorbable, some not) have been used to mimic the ECM or connective tissue. In terms of technology integration, the major advances so far for 3D cell biology have been related to materials and methods used for scaffold preparation, and integration of microfabrication techniques, for example for fluidics. As might be expected, a challenge of 3D culture over 2D is associated with the difficulty of oxygenation of tissues in the absence of vasculature. Microfluidics have gained favor for a number of reasons, including for perfusion, reduction in reagent volumes, and the fact that flow induced stress This work reports the design of a live-cell monitoring platform based on a macroporous scaffold of a conducting polymer, poly(3,4-ethylene dioxythiophene):poly(styrenesulfonate). The conducting polymer scaffolds support 3D cell cultures due to their biocompatibility and tissue-like elasticity, which can be manipulated by inclusion of biopolymers such as collagen. Integration of a media perfusion tube inside the scaffold enables homogenous cell spreading and fluid transport throughout the scaffold, ensuring long term cell viability. This also allows for co-culture of multiple cell types inside the scaffold. The inclusion of cells within the porous architecture affects the impedance of the electrically conducting polymer network and, thus, is utilized as an in situ tool to monitor cell growth. Therefore, while being an integral part of the 3D tissue, the conducting polymer is an active component, enhancing the tissue function, and forming the basis for a bioelectronic device with integrated sensing capability.

-Pottuz, et al.. Accurate characterization of pure silicon-substituted hydroxyapatite powders synthesized by a new precipitation route. Acta Materialia, Elsevier, 2013, 9 (6)

The present study aimed at elucidating the effect of local pH in the extracellular microenvironment of tissue-engineered (TE) constructs on bone cell functions pertinent to new tissue formation. To this aim, we evaluated the osteogenicity process associated with bone constructs prepared from human Bone marrow-derived mesenchymal stem cells (hBMSC) combined with 45S5 bioactive glass (BG), a material that induces alkalinization of the external medium. The pH measured in cell-containing BG constructs was around 8.0, that is, 0.5 U more alkaline than that in two other cell-containing materials (hydroxyapatite/tricalcium phosphate [HA/TCP] and coral) constructs tested. When implanted ectopically in mice, there was no de novo bone tissue in the BG cell-containing constructs, in contrast to results obtained with either HA/TCP or coral ceramics, which consistently promoted the formation of ectopic bone. In addition, the implanted 50:50 composites of both HA/TCP:BG and coral:BG constructs, which displayed a pH of around 7.8, promoted 20-30-fold less amount of bone tissue. Interestingly, hBMSC viability in BG constructs was not affected compared with the other two types of material constructs tested both in vitro and in vivo. Osteogenic differentiation (specifically, the alkaline phosphatase [ALP] activity and gene expression of RUNX2, ALP, and BSP) was not affected when hBMSC were maintained in moderate alkaline pH (≤7.90) external milieu in vitro, but was dramatically inhibited at higher pH values. The formation of mineralized nodules in the extracellular matrix of hBMSC was fully inhibited at alkaline (>7.54) pH values. Most importantly, there is a pH range (specifically, 7.9-8.27) at which hBMSC proliferation was not affected, but the osteogenic differentiation of these cells was inhibited. Altogether, these findings provided evidence that excessive alkalinization in the microenvironment of TE constructs (resulting, for example, from material degradation) affects adversely the osteogenic differentiation of osteoprogenitor cells.

Most of our knowledge of bone cell physiology is derived from experiments carried out in vitro on polystyrene substrates. However, these traditional monolayer cell cultures do not reproduce the complex and dynamic three-dimensional (3D) environment experienced by cells in vivo. Thus, there is a growing interest in the use of 3D culture systems as tools for understanding bone biology. These in-vitro-engineered systems, less complex than in vivo models, should ultimately recapitulate and control the main biophysical, biochemical, and biomechanical cues that define the in vivo bone environment, while allowing their monitoring. This review focuses on state-of-the-art and the current advances in the development of 3D culture systems for bone biology research. It describes more specifically advantages related to the use of such systems, and details main characteristics and challenges associated with its three main components, that is, scaffold, cells, and perfusion bioreactor systems. Finally, future challenges for noninvasive imaging technologies are addressed.