Innervation has proven to be critical in bone homeostasis/regeneration due to the effect of soluble factors, produced by nerve fibers, associated with changes in the activity of bone cells. Thus, in this study, we have established and characterized a coculture system comprising sensory neurons and osteoblasts to mimic the in vivo scenario where nerve fibers can be found in a bone microenvironment. Embryonic or adult primary dorsal root ganglion (DRG) and MC3T3-E1 osteoblastic cells were cocultured in compartmentalized microfluidic platforms and morphological and functional tests were performed. The time of adhesion and readout of axonal outgrowth were improved by the alignment of DRG with the axis of microgrooves, which showed to be a crucial step for the designed experiments. Cocultures of entire DRG from adult origin with osteoblasts were performed, showing extended DRG projections towards the axonal compartment, reaching osteoblastic cells. Immunocytochemistry showed that the neurites present within the osteoblastic compartment were immunoreactive to synapsin and calcitonin gene-related peptide suggesting the presence of specialized structures involved in this crosstalk. This evidence was further confirmed by electron microscopy where varicosities were detected as well as electron dense structures in neurite membranes. Aiming to mimic the properties of tissue extracellular matrices, MC3T3-E1 cells were seeded in the axonal side upon laminin, collagen or within 3D functionalized alginate matrices and axonal outgrowth was clearly observed. In order to analyze and quantify data with reproducible image analysis, a semi-automated algorithm was also developed. The collagen and laminin substrates displayed a higher amount of axons reaching the axonal side. Overall, the established method revealed to be a suitable tool to study the interaction between the peripheral nervous system and bone cells in different contexts mimicking the in vivo scenario.

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Cellular activities in 3D are differentially affected by several matrix-intrinsic and extrinsic factors. This study highlights the relevance of optimizing initial cell densities when establishing 3D cultures for specific applications. Independently of the entrapping density, MSCs cultured within RGD-alginate hydrogels showed steady-state levels of metabolic activity and were in a nearly non-proliferative state, but recovered "normal" activity levels when retrieved from 3D matrices and re-cultured as monolayers. Importantly, high-densities promoted the establishment of cell-cell contacts with formation of multicellular clusters stabilized by endogenous ECM, and also stimulated MSCs osteogenic differentiation. These MSC-ECM microtissues may be used as building blocks for tissue engineering.

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Strontium (Sr) has been described as having beneficial influence in bone strength and architecture. However, negative systemic effects have been reported on oral administration of Sr ranelate, leading to strict restrictions in clinical application. We hypothesized that local delivery of Sr improves osteogenesis without eliciting detrimental side effects. Therefore, the in vivo response to an injectable Sr-hybrid system composed of RGD-alginate hydrogel cross-linked in situ with Sr and reinforced with Sr-doped hydroxyapatite microspheres, was investigated. The system was injected in a critical-sized bone defect model and compared to a similar Sr-free material. Micro-CT results show a trend towards higher new bone formed in Sr-hybrid group and major histological differences were observed between groups. Higher cell invasion was detected at the center of the defect of Sr-hybrid group after 15 days with earlier bone formation. Higher material degradation with increase of collagen fibers and bone formation in the center of the defect after 60 days was observed as opposed to bone formation restricted to the periphery of the defect in the control. These histological findings support the evidence of an improved response with the Sr enriched material. Importantly, no alterations were observed in the Sr levels in systemic organs or serum.

In the tissue engineering field dynamic culture systems, such as spinner flasks, are widely used due to their ability to improve mass transfer in suspension cell cultures. However, this culture system is often unsuitable to culture cells in three-dimensional (3D) scaffolds. To address this drawback, we designed a multicompartment holder for 3D cell culture, easily adaptable to spinner flasks. Here, the device was tested with human mesenchymal stem cells (MSCs) seeded in 3D porous chitosan scaffolds that were maintained in spinner flasks under dynamic conditions (50 rpm). Standard static culture conditions were used as control. The dynamic conditions were shown to significantly increase MSCs proliferation over 1 week (approximately 6-fold) and to improve cell distribution within the scaffold. Moreover, they also promoted osteogenic differentiation of MSCs, inducing an earlier peak in alkaline phosphatase (ALP) activity, and a more homogenous ALP staining and matrix mineralization in the whole scaffolds, but particularly in the center. Overall, this study shows a new multicompartment holder to culture 3D scaffolds that can broaden the application of spinner flasks.

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Electrospun (ESP) scaffolds are a promising type of tissue engineering constructs for large defects with limited depth. To form new functional tissue, the scaffolds need to be infiltrated with cells, which will deposit extracellular matrix. However, due to dense fiber packing and small pores, cell and tissue infiltration of ESP scaffolds is limited. Here, we combine two established methods, increasing fiber diameter and co-spinning sacrificial fibers, to create a porous ESP scaffold that allows robust tissue infiltration. Full cell infiltration across 2 mm thick scaffolds is seen 3 weeks after subcutaneous implantation in rats. After 6 weeks, the ESP scaffolds are almost fully filled with de novo tissue. Cell infiltration and tissue formation in vivo in this thickness has not been previously achieved. In addition, we propose a novel method for in vitro cell seeding to improve cell infiltration and a model to study 3D migration through a fibrous mesh. This easy approach to facilitate cell infiltration further improves previous efforts and could greatly aid tissue engineering approaches utilizing ESP scaffolds. Statement of significanceElectrospinning creates highly porous scaffolds with nano-to micrometer sized fibers and are a promising candidate for a variety of tissue engineering applications. However, smaller fibers also create small pores which are difficult for cells to penetrate, restricting cells to the top layers of the scaffolds. Here, we have improved the cell infiltration by optimizing fiber diameter and by co-spinning a sacrificial polymer. We developed novel culture technique that can be used to improve cell seeding and to study cytokine driven 3D migration through fibrous meshes. After subcutaneous implantation, infiltration of tissue and cells was observed up to throughout up to 2 mm thick scaffolds. This depth of infiltration in vivo had not yet been reported for electrospun scaffolds. The scaffolds we present here can be used for in vitro studies of migration, and for tissue engineering in defects with a large surface area and limited depth. IntroductionElectrospun (ESP) scaffolds are highly porous and consist of nano-or micrometer sized fibers of natural or synthetic polymers, mimicking the fibrous composition of tissue extra cellular matrix (ECM) [1][2][3] . ESP scaffolds provide more mechanical support than hydrogels and are more flexible than scaffolds produced by additive manufacturing, making them interesting for tissue engineering approaches [4] . Large ESP mats are easily produced but are often limited to a thickness of several mm due to delamination and charge distribution. This makes ESP scaffolds particularly interesting for defects with a large surface area, but limited depth. This includes skin patches [5] , corneal repair [6] , cartilage regeneration [7] , vascular grafts [8] and nerve guides [9] , among others. However, due to dense fiber packing and small pores, deep cell infiltration in ESP scaffolds remains a challenge [10, 11] . To create new fully functional tissue, ESP...