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The transition in the field of bone tissue engineering from bone regeneration to in vitro models has come with the challenge of recreating a dense and anisotropic bone-like extracellular matrix (ECM). Although the mechanism by which bone ECM gains its structure is not fully understood, mechanical loading and curvature have been identified as potential contributors. Here, guided by computational simulations, we evaluated cell and bone-like tissue growth and organization in a concave channel with and without directional fluid flow stimulation. Human mesenchymal stromal cells were seeded on donut-shaped silk fibroin scaffolds and osteogenically stimulated for 42 days statically or in a flow perfusion bioreactor. After 14, 28, and 42 days, constructs were investigated for cell and tissue growth and organization. As a result, directional fluid flow was able to improve organic tissue growth but not organization. Cells tended to orient in the tangential direction of the channel, possibly attributed to its curvature. Based on our results, we suggest that organic ECM production but not anisotropy can be stimulated through the application of fluid flow. With this study, an initial attempt in three-dimensions was made to improve the resemblance of in vitro produced bone-like ECM to the physiological bone ECM.

Healthy bone is maintained by the process of bone remodeling. An unbalance in this process can lead to pathologies such as osteoporosis which are often studied with animal models. However, data from animals have limited power in predicting the results that will be obtained in human clinical trials. In search for alternatives to animal models, human in vitro models are emerging as they address the principle of reduction, refinement, and replacement of animal experiments (3Rs). At the moment, no complete in vitro model for bone-remodeling exists. Microfluidic chips offer great possibilities, particularly because of the dynamic culture options, which are crucial for in vitro bone formation. In this study, a scaffold free, fully human, 3D microfluidic coculture model of bone remodeling is presented. A bone-on-a-chip coculture system was developed in which human mesenchymal stromal cells differentiated into the osteoblastic lineage and self-assembled into scaffold free bone-like tissues with the shape and dimensions of human trabeculae. Human monocytes were able to attach to these tissues and to fuse into multinucleated osteoclast-like cells, establishing the coculture. Computational modeling was used to determine the fluid flow induced shear stress and strain in the formed tissue. Furthermore, a set-up was developed allowing for long-term (35 days) on-chip cell culture with benefits including continuous fluid-flow, low bubble formation risk, easy culture medium exchange inside the incubator and live cell imaging options. This on-chip coculture is a crucial advance towards developing in vitro bone remodeling models to facilitate drug testing.

The aim of the present study was to further improve an in vitro 3D osteoblast (OB) – osteoclast (OC) co-culture model of bone by tuning it towards states of formation, resorption, and equilibrium for their future applications in fundamental research, drug development and personalized medicine. This was achieved by varying culture medium composition and monocyte seeding density, the two external parameters that affect cell behavior the most. Monocytes were seeded at two seeding densities onto 3D silk-fibroin constructs pre-mineralized by MSC-derived OBs and were co-cultured in one of three different media (OC stimulating, Neutral and OB stimulating medium) for three weeks. Histology showed mineralized matrix after co-culture and OC markers in the OC medium group. Scanning Electron Microscopy showed large OC-like cells in the OC medium group. Micro-computed tomography showed increased formation in the OB medium group, equilibrium in the Neutral medium group and resorption in the OC medium group. Culture supernatant samples showed high early TRAP release in the OC medium group, a later and lower release in the Neutral medium group, and almost no release in the OB medium group. Increased monocyte seeding density showed a less-than-proportional increase in TRAP release and resorption in OC medium, while it proportionally increased TRAP release in Neutral medium without affecting net resorption. The 3D OB-OC co-culture model was effectively used to show an excess of mineral deposition using OB medium, resorption using OC medium, or an equilibrium using Neutral medium. All three media applied to the model may have their own distinct applications in fundamental research, drug development, and personalized medicine.

Fetal bovine serum (FBS) is a widely used supplement in cell culture medium, despite its known variability in composition which greatly affects cellular function and consequently the outcome of studies. In bone tissue engineering, the deposited mineralized matrix is one of the main outcome parameters, but using different brands of FBS can result in large variations. Alkaline phosphatase (ALP) is present in FBS. Not only is ALP used to judge the osteogenic differentiation of bone cells, it may affect deposition of mineralized matrix. The present study focused on the enzymatic activity of ALP in FBS of different suppliers and its contribution to mineralization in osteogenic differentiation cultures. It was hypothesized that culturing cells in a medium with high intrinsic ALP activity of FBS will lead to higher mineral deposition compared to media with lower ALP activity. The used FBS types were shown to have significant differences in enzymatic ALP activity. Our results indicate that the ALP activity of the medium not only affected the deposited mineralized matrix but also the osteogenic differentiation of cells as measured by a changed cellular ALP activity of human bone marrow derived mesenchymal stromal cells (hBMSC). In media with low inherent ALP activity, the cellular ALP activity was increased and played the major role in the mineralization process; while, in media with high intrinsic ALP activity contribution from the serum, less cellular ALP activity was measured and the ALP activity of the medium also contributed to mineral formation substantially. Our results highlight the diverse effects of ALP activity intrinsic to FBS on osteogenic differentiation and matrix mineralization and how FBS can determine the experimental outcomes, in particular for studies investigating matrix mineralization. Once again, the need to replace FBS with more controlled and known additives is highlighted.Graphical abstract

INTRODUCTION: The development of wound dressing materials that combine healing properties, ability to self-repair the material damages, skin-friendly adhesive nature, and competent mechanical properties have surpassing functional importance in healthcare. Due to their specificity, hydrogels have been recognized as a new gateway in biological materials to treat dysfunctional tissues. The design and creation of injectable hydrogel-based scaffolds have extensively progressed in recent years to improve their therapeutic efficacy and to pave the way for their easy minimally invasive administration. Hence, injectable hydrogel biomaterials have been prepared to eventually translate into minimally invasive therapy and pose a lasting effect on regenerative medicine.AREAS COVERED: This review highlights the recent development of adhesive and injectable hydrogels that have applications in wound healing and wound dressing. Such hydrogel materials are not only expected to improve therapeutic outcomes but also to facilitate the easy surgical process in both wound healing and dressing.EXPERT OPINION: Wound healing seems to be an appealing approach for treating countless life-threatening disorders. With the average increase of life expectancy in human societies, an increase in demand for injectable skin replacements and drug delivery carriers for chronic wound healing is expected.

From a materials perspective, the pillars for the development of clinically translatable scaffold-based strategies for craniomaxillofacial (CMF) bone and periodontal regeneration have included electrospinning and 3D printing (biofabrication) technologies. Here, we offer a detailed analysis of the latest innovations in 3D (bio)printing strategies for CMF bone and periodontal regeneration and provide future directions envisioning the development of advanced 3D architectures for successful clinical translation. First, the principles of electrospinning applied to the generation of biodegradable scaffolds are discussed. Next, we present on extrusion-based 3D printing technologies with a focus on creating scaffolds with improved regenerative capacity. In addition, we offer a critical appraisal on 3D (bio)printing and multitechnology convergence to enable the reconstruction of CMF bones and periodontal tissues. As a future outlook, we highlight future directions associated with the utilisation of complementary biomaterials and (bio)fabrication technologies for effective translation of personalised and functional scaffolds into the clinics.

Periodontitis is a ubiquitous chronic inflammatory, bacteria-triggered oral disease affecting the adult population. If left untreated, periodontitis can lead to severe tissue destruction, eventually resulting in tooth loss. Despite previous efforts in clinically managing the disease, therapeutic strategies are still lacking. Herein, melt electrowriting (MEW) is utilized to develop a compositionally and structurally tailored graded scaffold for regeneration of the periodontal ligament-to-bone interface. The composite scaffolds, consisting of fibers of polycaprolactone (PCL) and fibers of PCL-containing magnesium phosphate (MgP) were fabricated using MEW. To maximize the bond between bone (MgP) and ligament (PCL) regions, we evaluated two different fiber architectures in the interface area. These were a crosshatch pattern at a 0/90° angle and a random pattern. MgP fibrous scaffolds were able to promote in vitro bone formation even in culture media devoid of osteogenic supplements. Mechanical properties after MgP incorporation resulted in an increase of the elastic modulus and yield stress of the scaffolds, and fiber orientation in the interfacial zone affected the interfacial toughness. Composite graded MEW scaffolds enhanced bone fill when they were implanted in an in vivo periodontal fenestration defect model in rats. The presence of an interfacial zone allows coordinated regeneration of multitissues, as indicated by higher expression of bone, ligament, and cementoblastic markers compared to empty defects. Collectively, MEW-fabricated scaffolds having compositionally and structurally tailored zones exhibit a good mimicry of the periodontal complex, with excellent regenerative capacity and great potential as a defect-specific treatment strategy.

Macrophages are amongst the first immune cells that encounter rabies virus (RABV) at virus entry sites. Activation of macrophages is essential for the onset of a potent immune response, but insights into the effects of RABV on macrophage activation are scarce. In this study we performed high-throughput sequencing on RNA extracted from macrophages that were exposed to RABV for 48 hours, and compared their transcriptional profiles to that of non-polarized macrophages (M0), and macrophages polarized towards the canonical M1, M2a and M2c phenotypes. Our analysis revealed that RABV-stimulated macrophages show high expression of several M1, M2a and M2c signature genes. Apart from their partial resemblance to these phenotypes, unbiased clustering analysis revealed that RABV induces a unique and distinct polarization program. Closer examination revealed that RABV induced multiple pathways related to the interferon- and antiviral response, which were not induced under other classical polarization strategies. Surprisingly, our data show that RABV induces an activated rather than a fully suppressed macrophage phenotype, triggering virus-induced activation and polarization. This includes multiple genes with known antiviral (e.g. APOBEC3A, IFIT/OAS/TRIM genes), which may play a role in anti-RABV immunity.

OBJECTIVES: Electrospun scaffolds are a versatile biomaterial platform to mimic fibrillar structure of native tissues extracellular matrix, and facilitate the incorporation of biomolecules for regenerative therapies. Self-assembling peptide P 11-4 has emerged as a promising strategy to induce mineralization; however, P 11-4 application has been mostly addressed for early caries lesions repair on dental enamel. Here, to investigate P 11-4's efficacy on bone regeneration, polymeric electrospun scaffolds were developed, and then distinct concentrations of P 11-4 were physically adsorbed on the scaffolds. METHODS: P 11-4-laden and pristine (P 11-4-free) electrospun scaffolds were immersed in simulated body fluid and mineral precipitation identified by SEM. Functional groups and crystalline phases were analyzed by FTIR and XRD, respectively. Cytocompatibility, mineralization, and gene expression assays were conducted using stem cells from human exfoliated deciduous teeth. To investigate P 11-4-laden scaffolds potential to induce in vivo mineralization, an established rat calvaria critical-size defect model was used. RESULTS: We successfully synthesized nanofibrous (∼ 500 nm fiber diameter) scaffolds and observed that functionalization with P 11-4 did not affect the fibers' diameter. SEM images indicated mineral precipitation, while FTIR and XRD confirmed apatite-like formation and crystallization for P 11-4-laden scaffolds. In addition, P 11-4-laden scaffolds were cytocompatible, highly stimulated cell-mediated mineral deposition, and upregulated the expression of mineralization-related genes compared to pristine scaffolds. P 11-4-laden scaffolds led to enhanced in vivo bone regeneration after 8 weeks compared to pristine PCL. SIGNIFICANCE: Electrospun scaffolds functionalized with P 11-4 are a promising strategy for inducing mineralized tissues regeneration in the craniomaxillofacial complex.

Cells sense and respond to scaffold pore geometry and mechanical stimuli. Many fabrication methods used in bone tissue engineering render structures with poorly controlled pore geometries. Given that cell-scaffold interactions are complex, drawing a conclusion on how cells sense and respond to uncontrolled scaffold features under mechanical loading is difficult. Here, monodisperse templated scaffolds (MTSC) were fabricated and used as a well-defined porous scaffolds to study the effect of dynamic culture conditions on bone-like tissue formation. Human bone marrow derived stromal cells were cultured on MTSC or conventional salt-leached scaffolds (SLSC) for up to 7 weeks, either under static or dynamic conditions (wall shear stress (WSS) using spinner flask bioreactors). The influence of controlled spherical pore geometry of MTSC subjected to static or dynamic conditions on osteoblast cells differentiation, bone-like tissue formation, structure and distribution was investigated. WSS generated within the two idealized geometrical scaffold features was assessed. Distinct response to fluid flow in osteoblast cell differentiation were shown to be dependent on scaffold pore geometry. As revealed by collagen staining and micro-computed tomography images, dynamic conditions promoted a more regular extracellular matrix (ECM) formation and mineral distribution in both scaffold types compared to static conditions. The results showed that regulation of bone-related genes and the amount and the structure of mineralized ECM were dependent on scaffold pore geometry and the mechanical cues provided by the two different culture conditions. Under dynamic conditions, SLSC favored osteoblast cell differentiation and ECM formation, while MTSC enhanced ECM mineralization. The spherical pore shape in MTSC supported a more trabecular bone-like structure under dynamic conditions compared to MTSC statically cultured or to SLSC under either static or dynamic conditions. These results suggest that cell activity and bone-like tissue formation is driven not only by the pore geometry but also by the mechanical environment. This should be taken into account in the future design of complex scaffolds, which should favor cell differentiation while guiding the formation, structure and distribution of the engineered bone tissue. This could help to mimic the anatomical complexity of the bone tissue structure and to adapt to each bone defect needs.Impact statementAging of the human population leads to an increasing need for medical implants with high success rate. We provide evidence that cell activity and the amount and structure of bone-like tissue formation is dependent on the scaffold pore geometry and on the mechanical environment. Fabrication of complex scaffolds comprising concave and planar pore geometries might represent a promising direction towards the tunability and mimicry the structural complexity of the bone tissue. Moreover, the use of fabrication methods that allow a systematic fabrication of reproducible and geometrically controlled structures would simplify scaffold design optimization.

The bone resorbing osteoclasts are a complex type of cell essential for in vivo bone remodeling. There is no consensus on medium composition and seeding density for in vitro osteoclastogenesis, despite the importance thereof on osteoclastic differentiation and activity. The aim of this study was to investigate the relative effect of monocyte or peripheral blood mononuclear cell (PBMC) seeding density, osteoclastic supplement concentration and priming on the in vitro generation of functional osteoclasts, and to explore and evaluate the usefulness of commonly used markers for osteoclast cultures. Morphology and osteoclast formation were analyzed with fluorescence imaging for tartrate resistant acid phosphatase (TRAP) and integrin β3 (Iβ3). TRAP release was analyzed from supernatant samples, and resorption was analyzed from culture on Corning® Osteo Assay plates. In this study, we have shown that common non-standardized culturing conditions of monocyte or PBMCs had a significant effect on the in vitro generation of functional osteoclasts. We showed how increased osteoclastic supplement concentrations supported osteoclastic differentiation and resorption but not TRAP release, while priming resulted in increased TRAP release as well. Increased monocyte seeding densities resulted in more and large TRAP positive bi-nuclear cells, but not directly in more multinucleated osteoclasts, resorption or TRAP release. Increasing PBMC seeding densities resulted in more and larger osteoclasts and more resorption, although resorption was disproportionally low compared to the monocyte seeding density experiment. Exploration of commonly used markers for osteoclast cultures demonstrated that Iβ3 staining was an excellent and specific osteoclast marker in addition to TRAP staining, while supernatant TRAP measurements could not accurately predict osteoclastic resorptive activity. With improved understanding of the effect of seeding density and osteoclastic supplement concentration on osteoclasts, experiments yielding higher numbers of functional osteoclasts can ultimately improve our knowledge of osteoclasts, osteoclastogenesis, bone remodeling and bone diseases.

IntroductionTo study human physiological and pathological bone remodeling while addressing the principle of replacement, reduction and refinement of animal experiments (3Rs), human in vitro bone remodeling models are being developed. Despite increasing safety-, scientific-, and ethical concerns, fetal bovine serum (FBS), a nutritional medium supplement, is still routinely used in these models. To comply with the 3Rs and to improve the reproducibility of such in vitro models, xenogeneic-free medium supplements should be investigated. Human platelet lysate (hPL) might be a good alternative as it has been shown to accelerate osteogenic differentiation of mesenchymal stromal cells (MSCs) and improve subsequent mineralization. However, for a human in vitro bone model, hPL should also be able to adequately support osteoclastic differentiation and subsequent bone resorption. In addition, optimizing co-culture medium conditions in mono-cultures might lead to unequal stimulation of co-cultured cells.MethodsWe compared supplementation with 10% FBS vs. 10%, 5%, and 2.5% hPL for osteoclast formation and resorption by human monocytes (MCs) in mono-culture and in co-culture with (osteogenically stimulated) human MSCs.Results and DiscussionSupplementation of hPL can lead to a less donor-dependent and more homogeneous osteoclastic differentiation of MCs when compared to supplementation with 10% FBS. In co-cultures, osteoclastic differentiation and resorption in the 10% FBS group was almost completely inhibited by MSCs, while the supplementation with hPL still allowed for resorption, mostly at low concentrations. The addition of hPL to osteogenically stimulated MSC mono- and MC-MSC co-cultures resulted in osteogenic differentiation and bone-like matrix formation, mostly at high concentrations.ConclusionWe conclude that hPL could support both osteoclastic differentiation of human MCs and osteogenic differentiation of human MSCs in mono- and in co-culture, and that this can be balanced by the hPL concentration. Thus, the use of hPL could limit the need for FBS, which is currently commonly accepted for in vitro bone remodeling models.

Given the multi-tissue aspects of osteoarthritis (OA) pathophysiology, translation of OA susceptibility genes towards underlying biological mechanism and eventually drug target discovery requires appropriate human in vitro OA models that incorporate both functional bone and cartilage tissue units. Therefore, a microfluidic chip is developed with an integrated fibrous polycaprolactone matrix in which neo-bone and cartilage are produced, that could serve as a tailored human in vitro disease model of the osteochondral unit of joints. The model enables to evaluate OA-related environmental perturbations to (individual) tissue units by controlling environmental cues, for example by adding biochemical agents. After establishing the co-culture in the system, a layer of cartilaginous matrix is deposited in the chondrogenic compartment, while a bone-like matrix is deposited between the fibers, indicated by both histology and gene expression levels of collagen type 2 and osteopontin, respectively. As proof-of-principle, the bone and cartilaginous tissue are exposed to active thyroid hormone, creating an OA disease model. This results in increased expression levels of hypertrophy markers integrin-binding sialoprotein and alkaline phosphatase in both cartilage and bone, as expected. Altogether, this model could contribute to enhanced translation from OA risk genes towards novel OA therapies.

Human in vitro bone models can create the possibility for investigation of physiological bone remodeling while addressing the principle of replacement, reduction and refinement of animal experiments (3R). Current in vitro models lack cell–matrix interactions and their spatiotemporal complexity. To facilitate these analyses, a bone-mimetic template is developed in this study, inspired by bone's extracellular matrix composition and organization. Silk fibroin (SF) is used as an organic matrix, poly-aspartic acid (pAsp) is used to mimic the functionality of noncollagenous proteins, and 10× simulated body fluid serves as mineralization solution. By using pAsp in the mineralization solution, minerals are guided toward the SF material resulting in mineralization inside and as a coating on top of the SF. After cytocompatibility testing, remodeling experiments are performed in which mineralized scaffold remodeling by osteoclasts and osteoblasts is tracked with nondestructive microcomputed tomography and medium analyses over a period of 42 d. The mineralized scaffolds support osteoclastic resorption and osteoblastic mineralization, in the physiological bone remodeling specific sequence. This model could therefore facilitate the investigation of cell–matrix interactions and may thus reduce animal experiments and advance in vitro drug testing for bone remodeling pathologies like osteoporosis, where cell–matrix interactions need to be targeted.

Hydrogels are of interest in cartilage tissue engineering due to their ability to support the encapsulation and chondrogenesis of mesenchymal stromal cells (MSCs). However, features such as hydrogel crosslink density, which can influence nutrient transport, nascent matrix distribution, and the stability of constructs during and after implantation must be considered in hydrogel design. Here, we first demonstrate that more loosely crosslinked (i.e. softer, ∼2 kPa) norbornene-modified hyaluronic acid (NorHA) hydrogels support enhanced cartilage formation and maturation when compared to more densely crosslinked (i.e. stiffer, ∼6–60 kPa) hydrogels, with a >100-fold increase in compressive modulus after 56 d of culture. While soft NorHA hydrogels mature into neocartilage suitable for the repair of articular cartilage, their initial moduli are too low for handling and they do not exhibit the requisite stability needed to withstand the loading environments of articulating joints. To address this, we reinforced NorHA hydrogels with polycaprolactone (PCL) microfibers produced via melt-electrowriting (MEW). Importantly, composites fabricated with MEW meshes of 400 µm spacing increased the moduli of soft NorHA hydrogels by ∼50-fold while preserving the chondrogenic potential of the hydrogels. There were minimal differences in chondrogenic gene expression and biochemical content (e.g. DNA, GAG, collagen) between hydrogels alone and composites, whereas the composites increased in compressive modulus to ∼350 kPa after 56 d of culture. Lastly, integration of composites with native tissue was assessed ex vivo; MSC-laden composites implanted after 28 d of pre-culture exhibited increased integration strengths and contact areas compared to acellular composites. This approach has great potential towards the design of cell-laden implants that possess both initial mechanical integrity and the ability to support neocartilage formation and integration for cartilage repair.

Our goal was to create bioimitated scaffolding materials for biomedical purposes. The guiding idea was that we used an interpenetrating structural hierarchy of natural extracellular matrix as a "pattern" to design hydrogel scaffolds that show favorable properties for tissue regeneration. Polymeric hydrogel scaffolds are made in a simple, environmentally friendly way without additional functionalization. Gelatin and 2-hydroxyethyl methacrylate were selected to prepare interpenetrating polymeric networks and linear alginate chains were added as an interpenetrant to study their influence on the scaffold's functionalities. Cryogelation and porogenation methods were used to obtain the designed scaffolding biomaterials. The scaffold's structural, morphological, and mechanical properties, in vitro degradation, and cell viability properties were assessed to study the effects of the preparation method and alginate loading. Apatite as an inorganic agent was incorporated into cryogelated scaffolds to perform an extensive biological assay. Cryogelated scaffolds possess superior functionalities essential for tissue regeneration: fully hydrophilicity, degradability and mechanical features (2.08-9.75 MPa), and an optimal LDH activity. Furthermore, cryogelated scaffolds loaded with apatite showed good cell adhesion capacity, biocompatibility, and non-toxic behavior. All scaffolds performed equally in terms of metabolic activity and osteoconductivity. Cryogelated scaffolds with/without HAp could represent a new advance to promote osteoconductivity and enhance hard tissue repair. The obtained series of scaffolding biomaterials described here can provide a wide range of potential applications in the area of biomedical engineering., Polymers, 14 (15), ISSN:2073-4360

In recent decades, an increasing number of tissue engineered bone grafts have been developed. However, expensive and laborious screenings in vivo are necessary to assess the safety and efficacy of their formulations. Rodents are the first choice for initial in vivo screens but their size limits the dimensions and number of the bone grafts that can be tested in orthotopic locations. Here, we report the development of a refined murine subcutaneous model for semi-orthotopic bone formation that allows the testing of up to four grafts per mouse one order of magnitude greater in volume than currently possible in mice. Crucially, these defects are also "critical size" and unable to heal within the timeframe of the study without intervention. The model is based on four bovine bone implants, ring-shaped, where the bone healing potential of distinct grafts can be evaluated in vivo. In this study we demonstrate that promotion and prevention of ossification can be assessed in our model. For this, we used a semi-automatic algorithm for longitudinal micro-CT image registration followed by histological analyses. Taken together, our data supports that this model is suitable as a platform for the real-time screening of bone formation, and provides the possibility to study bone resorption, osseointegration and vascularisation.

Advanced in vitro human bone defect models can contribute to the evaluation of materials for in situ bone regeneration, addressing both translational and ethical concerns regarding animal models. In this study, we attempted to develop such a model to study material-driven regeneration, using a tissue engineering approach. By co-culturing human umbilical vein endothelial cells (HUVECs) with human bone marrow-derived mesenchymal stromal cells (hBMSCs) on silk fibroin scaffolds with in vitro critically sized defects, the growth of vascular-like networks and three-dimensional bone-like tissue was facilitated. After a model build-up phase of 28 days, materials were artificially implanted and HUVEC and hBMSC migration, cell-material interactions, and osteoinduction were evaluated 14 days after implantation. The materials physiologically relevant for bone regeneration included a platelet gel as blood clot mimic, cartilage spheres as soft callus mimics, and a fibrin gel as control. Although the in vitro model was limited in the evaluation of immune responses, hallmarks of physiological bone regeneration were observed in vitro. These included the endothelial cell chemotaxis induced by the blood clot mimic and the mineralization of the soft callus mimic. Therefore, the present in vitro model could contribute to an improved pre-clinical evaluation of biomaterials while reducing the need for animal experiments.

Current biomaterial-based strategies explored to treat articular cartilage defects have failed to provide adequate physico-chemical cues in order to guide functional tissue regeneration. Here, it is hypothesized that atmospheric-pressure plasma (APPJ) treatment and melt electrowriting (MEW) will produce microfiber support structures with covalently-immobilized transforming growth factor beta-1 (TGFβ1) that can stimulate the generation of functional cartilage tissue. The effect of APPJ operational speeds to activate MEW polycaprolactone meshes for immobilization of TGFβ1 is first investigated and chondrogenic differentiation and neo-cartilage production are assessed in vitro. All APPJ speeds test enhanced hydrophilicity of the meshes, with the slow treatment speed having significantly less C-C/C-H and more COOH than the untreated meshes. APPJ treatment increases TGFβ1 loading efficiency. Additionally, in vitro experiments highlight that APPJ-based TGFβ1 attachment to the scaffolds is more advantageous than direct supplementation within the medium. After 28 days of culture, the group with immobilized TGFβ1 has significantly increased compressive modulus (more than threefold) and higher glycosaminoglycan production (more than fivefold) than when TGFβ1 is supplied through the medium. These results demonstrate that APPJ activation allows reagent-free, covalent immobilization of TGFβ1 on microfiber meshes and, importantly, that the biofunctionalized meshes can stimulate neo-cartilage matrix formation. This opens new perspectives for guided tissue regeneration.

Bone is a complex natural material with a complex hierarchical multiscale organization, crucial to perform its functions. Ultrastructural analysis of bone is crucial for our understanding of cell to cell communication, the healthy or pathological composition of bone tissue, and its three-dimensional (3D) organization. A variety of techniques has been used to analyze bone tissue. This article describes a combined approach of optical, scanning electron, and transmission electron microscopy for the ultrastructural analysis of bone from the nanoscale to the macroscale, as illustrated by two pathological bone tissues. By following a top-down approach to investigate the multiscale organization of pathological bones, quantitative estimates were made in terms of calcium content, nearest neighbor distances of osteocytes, canaliculi diameter, ordering, and D-spacing of the collagen fibrils, and the orientation of intrafibrillar minerals which enable us to observe the fine structural details. We identify and discuss a series of two-dimensional (2D) and 3D imaging techniques that can be used to characterize bone tissue. By doing so we demonstrate that, while 2D imaging techniques provide comparable information from pathological bone tissues, significantly different structural details are observed upon analyzing the pathological bone tissues in 3D. Finally, particular attention is paid to sample preparation for and quantitative processing of data from electron microscopic analysis.

Introduction Osteoclasts are bone-resorbing cells closely related to bone turnover, whereas different macrophage subtypes contribute to bone fracture healing. As osteoclasts and macrophages share the same hematopoietic origin, the difference between both cell types on osteoblast coupling, crosstalk extent and consequent bone formation remains poorly understood. This study compares the potential of primary cells that are routinely considered as osteoclast and macrophage cultures on their ability to support osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Methods Human Peripheral Blood Mononuclear Cells (hPBMCs) were used to obtain macrophage or osteoclast cultures using appropriate stimulatory factors. With different seeding densities of hPBMCs, conditioned media from macrophage or osteoclast cultures were harvested for comparative evaluation of effects thereof on the osteogenic differentiation of hMSCs. Specific cytological staining was used to qualitatively evaluate macrophage and osteoclast cultures. Additionally, quantitative data on hMSC proliferation, osteogenic differentiation and mineralization were obtained via biochemical assays. Results Conditioned medium from osteoclast cultures obtained via low hPBMCs seeding densities, but not from high hPBMCs seeding densities or macrophages, stimulated hMSC osteogenic differentiation and mineralization. Upon cellular crosstalk, both pre-differentiated osteoclasts and non-polarized macrophages equally supported early hMSC osteogenic differentiation and mineralization, as confirmed by increased alkaline phosphatase levels within 7 days and increased calcium content within 14 days in comparison with undifferentiated controls. Initial hPBMCs seeding density strongly influences osteoclastogenesis and the paracrine effect of the resultant osteoclast population on the osteogenic differentiation of hMSCs. In addition, only in indirect coculture, macrophages provide similar stimulatory effects as pre-differentiated osteoclasts on the osteogenic differentiation of MSCs and mineralization. Conclusion Our results demonstrate stimulatory effects of osteoclast conditioned medium on hMSC osteogenic differentiation, depending on initial hPBMC seeding density. In addition, we show that osteoclast and macrophage cultures contain pools of polarized macrophages, which may be involved in the osteogenic effects. Our data provide insight into bone tissue engineering approaches by using multicellular interactions related to bone remodeling and healing for the in vitro modulation of osteogenic differentiation.

Bioprinting has become an important tool for fabricating regenerative implants and in vitro cell culture platforms. However, until today, extrusion-based bioprinting processes are limited to resolutions of hundreds of micrometers, which hamper the reproduction of intrinsic functions and morphologies of living tissues. This study describes novel hydrogel-based bioinks for cell electrowriting (CEW) of well-organized cell-laden fiber structures with diameters ranging from 5 to 40 μm. Two novel photoresponsive hydrogel bioinks, that is, based on gelatin and silk fibroin, which display distinctly different gelation chemistries, are introduced. The rapid photomediated cross-linking mechanisms, electrical conductivity, and viscosity of these two engineered bioinks allow the fabrication of 3D ordered fiber constructs with small pores (down to 100 μm) with different geometries (e.g., squares, hexagons, and curved patterns) of relevant thicknesses (up to 200 μm). Importantly, the biocompatibility of the gelatin- and silk fibroin-based bioinks enables the fabrication of cell-laden constructs, while maintaining high cell viability post printing. Taken together, CEW and the two hydrogel bioinks open up fascinating opportunities to manufacture microstructured constructs for applications in regenerative medicine and in vitro models that can better resemble cellular microenvironments.

There are currently several commercialized products approved by the Food and Drug Administration and the European Medicines Agency based on the use of recombinant human BMP-2 for the treatment of non-unions long fractures and spinal fusion. However, the adverse effects recorded with the use of BMPs suggest the need for drug delivery carriers that allow reducing the required doses and improve their cost-effectiveness. Herein, we have developed a new osteoconductive scaffold that reduces the required doses of BMP-2 for promoting bone regeneration in an osteoporotic defect model. The composite is, in brief, a gelatin-based 3D scaffold reinforced with either calcium sulfate or hydroxyapatite as an inorganic osteoconductive biomaterial. To this end, the organic/inorganic composite systems showed high hydration capacity and good in vitro degradability. The incorporation of 7.5% (m/v) ceramic compounds resulted in scaffolds with stiffer Young modulus (179 and 75 kPa for CaSO4_7 and HA_7, respectively) than bare gelatin hydrogels (48 kPa). Studies with human bone-marrow derived mesenchymal stem cells (hBM-MSCs) revealed that the 3D scaffolds promote cell adhesion and proliferation along with osteogenic differentiation capabilities. Specifically, downregulation of stemness (Nanog, Oct4) genes and upregulation of osteogenic markers (ALP, Col1a1, Fmod) by two fold were observed over 10 days under basal culture conditions. Promisingly, the sustained in vitro release of BMP-2 observed from the porous reinforced scaffolds allowed us to address the critical-sized osteoporotic mice calvarial defects with a relatively low growth factor doses (600 ng BMP-2/scaffold) compared to conventional doses at 2–15 micrograms. Overall, this study demonstrates the promising potential of osteoconductive gelatin/calcium bioceramics composites as osteogenic growth factors delivery carriers for bone-regeneration via ultra-low growth factor doses.