ß-glycerophosphate, not low magnitude fluid shear stress, increases osteocytogenesis in the osteoblast-to-osteocyte cell line IDG-SW3.

AIM: As osteoblasts deposit a mineralized collagen network, a subpopulation of these cells differentiates into osteocytes. Biochemical and mechanical stimuli, particularly fluid shear stress (FSS), are thought to regulate this, but their relative influence remains unclear. Here, we assess both biochemical and mechanical stimuli on long-term bone formation and osteocytogenesis using the osteoblast-osteocyte cell line IDG-SW3. METHODS: Due to the relative novelty and uncommon culture conditions of IDG-SW3 versus other osteoblast-lineage cell lines, effects of temperature and media formulation on matrix deposition and osteocytogenesis were initially characterized. Subsequently, the relative influence of biochemical (ß-glycerophosphate (ßGP) and ascorbic acid 2-phosphate (AA2P)) and mechanical stimulation on osteocytogenesis was compared, with intermittent application of low magnitude FSS generated by see-saw rocker. RESULTS: ßGP and AA2P supplementation were required for mineralization and osteocytogenesis, with 33°C cultures retaining a more osteoblastic phenotype and 37°C cultures undergoing significantly higher osteocytogenesis. ßGP concentration positively correlated with calcium deposition, whilst AA2P stimulated alkaline phosphatase (ALP) activity and collagen deposition. We demonstrate that increasing ßGP concentration also significantly enhances osteocytogenesis as quantified by the expression of green fluorescent protein linked to Dmp1. Intermittent FSS (~0.06 Pa) rocker had no effect on osteocytogenesis and matrix deposition. CONCLUSIONS: This work demonstrates the suitability and ease with which IDG-SW3 can be utilized in osteocytogenesis studies. IDG-SW3 mineralization was only mediated through biochemical stimuli with no detectable effect of low magnitude FSS. Osteocytogenesis of IDG-SW3 primarily occurred in mineralized areas, further demonstrating the role mineralization of the bone extracellular matrix has in osteocyte differentiation.

The Effects of Fucoidan Derived from Sargassum filipendula and Fucus vesiculosus on the Survival and Mineralisation of Osteogenic Progenitors.

Osteosarcoma is a bone cancer primarily affecting teenagers. It has a poor prognosis and diminished quality of life after treatment due to chemotherapy side effects, surgical complications and post-surgical osteoporosis risks. The sulphated polysaccharide fucoidan, derived from brown algae, has been a subject of interest for its potential anti-cancer properties and its impact on bone regeneration. This study explores the influence of crude, low-molecular-weight (LMW, 10-50 kDa), medium-molecular-weight (MMW, 50-100 kDa) and high-molecular-weight (HMW, >100 kDa) fractions from Sargassum filipendula, harvested from the Colombian sea coast, as well as crude fucoidan from Fucus vesiculosus, on a specific human osteoprogenitor cell type, human embryonic-derived mesenchymal stem cells. Fourier transform infrared spectroscopy coupled with attenuated total reflection (FTIR-ATR) results showed the highest sulphation levels and lowest uronic acid content in crude extract from F. vesiculosus. There was a dose-dependent drop in focal adhesion formation, proliferation and osteogenic differentiation of cells for all fucoidan types, but the least toxicity was observed for LMW and MMW. Transmission electron microscopy (TEM), JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimi-dazolylcarbocyanine iodide) staining and cytochrome c analyses confirmed mitochondrial damage, swollen ER and upregulated autophagy due to fucoidans, with the highest severity in the case of F. vesiculosus fucoidan. Stress-induced apoptosis-like cell death by F. vesiculosus fucoidan and stress-induced necrosis-like cell death by S. filipendula fucoidans were also confirmed. LMW and MMW doses of <200 ng/mL were the least toxic and showed potential osteoinductivity. This research underscores the multifaceted impact of fucoidans on osteoprogenitor cells and highlights the delicate balance between potential therapeutic benefits and the challenges involved in using fucoidans for post-surgery treatments in patients with osteosarcoma.

Osteocytes and Primary Cilia.

PURPOSE OF REVIEW: The purpose of this review is to provide a background on osteocytes and the primary cilium, discussing the role it plays in osteocyte mechanosensing. RECENT FINDINGS: Osteocytes are thought to be the primary mechanosensing cells in bone tissue, regulating bone adaptation in response to exercise, with the primary cilium suggested to be a key mechanosensing mechanism in bone. More recent work has suggested that, rather than being direct mechanosensors themselves, primary cilia in bone may instead form a key chemo-signalling nexus for processing mechanoregulated signalling pathways. Recent evidence suggests that pharmacologically induced lengthening of the primary cilium in osteocytes may potentiate greater mechanotransduction, rather than greater mechanosensing. While more research is required to delineate the specific osteocyte mechanobiological molecular mechanisms governed by the primary cilium, it is clear from the literature that the primary cilium has significant potential as a therapeutic target to treat mechanoregulated bone diseases, such as osteoporosis.

Preparation of Interconnected Pickering Polymerized High Internal Phase Emulsions by Arrested Coalescence.

Emulsion templating is a method that enables the production of highly porous and interconnected polymer foams called polymerized high internal phase emulsions (PolyHIPEs). Since emulsions are inherently unstable systems, they can be stabilized either by surfactants or by particles (Pickering HIPEs). Surfactant-stabilized HIPEs form materials with an interconnected porous structure, while Pickering HIPEs typically form closed pore materials. In this study, we describe a system that uses submicrometer polymer particles to stabilize the emulsions. Polymers fabricated from these Pickering emulsions exhibit, unlike traditional Pickering emulsions, highly interconnected large pore structures, and we related these structures to arrested coalescence. We describe in detail the morphological properties of this system and their dependence on different production parameters. This production method might provide an interesting alternative to poly-surfactant-stabilized-HIPEs, in particular where the application necessitates large pore structures.

Bioactive Composite for Orbital Floor Repair and Regeneration.

In the maxillofacial area, specifically the orbital floor, injuries can cause bone deformities in the head and face that are difficult to repair or regenerate. Treatment methodologies include use of polymers, metal, ceramics on their own and in combinations mainly for repair purposes, but little attention has been paid to identify suitable materials for orbital floor regeneration. Polyurethane (PU) and hydroxyapatite (HA) micro- or nano- sized with different percentages (25%, 40% & 60%) were used to fabricate bioactive tissue engineering (TE) scaffolds using solvent casting and particulate leaching methods. Mechanical and physical characterisation of TE scaffolds was investigated by tensile tests and SEM respectively. Chemical and structural properties of PU and PU/HA scaffolds were evaluated by infrared (IR) spectroscopy and Surface properties of the bioactive scaffold were analysed using attenuated total reflectance (ATR) sampling accessory coupled with IR. Cell viability, collagen formed, VEGF protein amount and vascularisation of bioactive TE scaffold were studied. IR characterisation confirmed the integration of HA in composite scaffolds, while ATR confirmed the significant amount of HA present at the top surface of the scaffold, which was a primary objective. The SEM images confirmed the pores' interconnectivity. Increasing the content of HA up to 40% led to an improvement in mechanical properties, and the incorporation of nano-HA was more promising than that of micro-HA. Cell viability assays (using MG63) confirmed biocompatibility and CAM assays confirmed vascularization, demonstrating that HA enhances vascularization. These properties make the resulting biomaterials very useful for orbital floor repair and regeneration.

Fucoidan Inhibition of Osteosarcoma Cells Is Species and Molecular Weight Dependent.

Fucoidan is a brown algae-derived polysaccharide having several biomedical applications. This study simultaneously compares the anti-cancer activities of crude fucoidans from Fucus vesiculosus and Sargassum filipendula, and effects of low (LMW, 10-50 kDa), medium (MMW, 50-100 kDa) and high (HMW, >100 kDa) molecular weight fractions of S. filipendula fucoidan against osteosarcoma cells. Glucose, fucose and acid levels were lower and sulphation was higher in F. vesiculosus crude fucoidan compared to S. filipendula crude fucoidan. MMW had the highest levels of sugars, acids and sulphation among molecular weight fractions. There was a dose-dependent drop in focal adhesion formation and proliferation of cells for all fucoidan-types, but F. vesiculosus fucoidan and HMW had the strongest effects. G1-phase arrest was induced by F. vesiculosus fucoidan, MMW and HMW, however F. vesiculosus fucoidan treatment also caused accumulation in the sub-G1-phase. Mitochondrial damage occurred for all fucoidan-types, however F. vesiculosus fucoidan led to mitochondrial fragmentation. Annexin V/PI, TUNEL and cytochrome c staining confirmed stress-induced apoptosis-like cell death for F. vesiculosus fucoidan and features of stress-induced necrosis-like cell death for S. filipendula fucoidans. There was also variation in penetrability of different fucoidans inside the cell. These differences in anti-cancer activity of fucoidans are applicable for osteosarcoma treatment.

A single short session of media perfusion induces osteogenesis in hBMSCs cultured in porous scaffolds, dependent on cell differentiation stage.

Perfusing culture media through porous cell-seeded scaffolds is now a common approach within many tissue engineering strategies. Human bone-marrow derived mesenchymal stem cells (hBMSC) are a clinically valuable source of osteoprogenitors that respond to mechanical stimuli. However, the optimal mechanical conditions for their osteogenic stimulation in vitro have not been defined. Whereas the effects of short durations of media fluid flow have been studied in monolayers of osteoblastic cells, in 3D culture continuous or repeated perfusion is usually applied. Here, we investigated whether a short, single perfusion session applied to hBMSCs cultured in 3D would enhance their osteogenesis in vitro. We cultured hBMSCs on gelatine-coated, porous polyurethane scaffolds with osteogenic supplements and stimulated them with a single 2-h session of unidirectional, steady, 2.5 mL/min media perfusion, at either early or late stages of culture in 3D. Some cells were pre-treated in monolayer with osteogenic supplements to advance cell differentiation, followed by 3D culture also with the osteogenic supplements. We report that this single, short session of media perfusion can markedly enhance the expression of bone-related transcription and growth factors, and matrix components, by hBMSCs but that it is more effective when cells reach the pre-osteoblast or osteoblast differentiation stage. These findings could aid in the optimization of 3D culture protocols for efficient bone tissue engineering. Biotechnol. Bioeng. 2016;113: 1814-1824. © 2016 Wiley Periodicals, Inc.

Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts.

Bone turnover in vivo is regulated by mechanical forces such as shear stress originating from interstitial oscillatory fluid flow (OFF), and bone cells in vitro respond to mechanical loading. However, the mechanisms by which bone cells sense mechanical forces, resulting in increased mineral deposition, are not well understood. The aim of this study was to investigate the role of the primary cilium in mechanosensing by osteoblasts. MLO-A5 murine osteoblasts were cultured in monolayer and subjected to two different OFF regimens: 5 short (2 h daily) bouts of OFF followed by morphological analysis of primary cilia; or exposure to chloral hydrate to damage or remove primary cilia and 2 short bouts (2 h on consecutive days) of OFF. Primary cilia were shorter and there were fewer cilia per cell after exposure to periods of OFF compared with static controls. Damage or removal of primary cilia inhibited OFF-induced PGE2 release into the medium and mineral deposition, assayed by Alizarin red staining. We conclude that primary cilia are important mediators of OFF-induced mineral deposition, which has relevance for the design of bone tissue engineering strategies and may inform clinical treatments of bone disorders causes by load-deficiency.

Controlling surface topology and functionality of electrospun fibers on the nanoscale using amphiphilic block copolymers to direct mesenchymal progenitor cell adhesion.

Surface patterning in three dimensions is of great importance in biomaterials design for controlling cell behavior. A facile one-step functionalization of biodegradable PDLLA fibers using amphiphilic diblock copolymers is demonstrated here to systematically vary the fiber surface composition. The copolymers comprise a hydrophilic poly[oligo(ethylene glycol) methacrylate] (POEGMA), poly[(2-methacryloyloxy)ethyl phosphorylcholine] (PMPC), or poly[2-(dimethylamino)ethyl methacrylate)] (PDMAEMA) block and a hydrophobic poly(l-lactide) (PLA) block. The block copolymer-modified fibers have increased surface hydrophilicity compared to that of PDLLA fibers. Mixtures of PLA-PMPC and PLA-POEGMA copolymers are utilized to exploit microphase separation of the incompatible hydrophilic PMPC and POEGMA blocks at the fiber surface. Conjugation of an RGD cell-adhesive peptide to one hydrophilic block (POEGMA) using thiol-ene chemistry produces fibers with domains of cell-adhesive (POEGMA) and cell-inert (PMPC) sites, mimicking the adhesive properties of the extracellular matrix (ECM). Human mesenchymal progenitor cells (hES-MPs) showed much better adhesion to the fibers with surface-adhesive heterogeneity compared to that to fibers with only adhesive or only inert surface chemistries.

Surfactant-free gelatin-stabilised biodegradable polymerised high internal phase emulsions with macroporous structures.

High internal phase emulsion (HIPE) templating is a well-established method for the generation of polymeric materials with high porosity (>74%) and degree of interconnectivity. The porosity and pore size can be altered by adjusting parameters during emulsification, which affects the properties of the resulting porous structure. However, there remain challenges for the fabrication of polyHIPEs, including typically small pore sizes (∼20-50 µm) and the use of surfactants, which can limit their use in biological applications. Here, we present the use of gelatin, a natural polymer, during the formation of polyHIPE structures, through the use of two biodegradable polymers, polycaprolactone-methacrylate (PCL-M) and polyglycerol sebacate-methacrylate (PGS-M). When gelatin is used as the internal phase, it is capable of stabilising emulsions without the need for an additional surfactant. Furthermore, by changing the concentration of gelatin within the internal phase, the pore size of the resulting polyHIPE can be tuned. 5% gelatin solution resulted in the largest mean pore size, increasing from 53 µm to 80 µm and 28 µm to 94 µm for PCL-M and PGS-M respectively. In addition, the inclusion of gelatin further increased the mechanical properties of the polyHIPEs and increased the period an emulsion could be stored before polymerisation. Our results demonstrate the potential to use gelatin for the fabrication of surfactant-free polyHIPEs with macroporous structures, with potential applications in tissue engineering, environmental and agricultural industries.

Porous biomaterials for tissue engineering: a review.

The field of biomaterials has grown rapidly over the past decades. Within this field, porous biomaterials have played a remarkable role in: (i) enabling the manufacture of complex three-dimensional structures; (ii) recreating mechanical properties close to those of the host tissues; (iii) facilitating interconnected structures for the transport of macromolecules and cells; and (iv) behaving as biocompatible inserts, tailored to either interact or not with the host body. This review outlines a brief history of the development of biomaterials, before discussing current materials proposed for use as porous biomaterials and exploring the state-of-the-art in their manufacture. The wide clinical applications of these materials are extensively discussed, drawing on specific examples of how the porous features of such biomaterials impact their behaviours, as well as the advantages and challenges faced, for each class of the materials.

Nanoscopic mechanical anisotropy in hydrogel surfaces.

The bulk mechanical properties of soft materials have been studied widely, but it is unclear to what extent macroscopic behavior is reflected in nanomechanics. Using an atomic force microscopy (AFM) imaging method called force spectroscopy mapping (FSM), it is possible to map the nanoscopic spatial distribution of Young's modulus, i.e. "stiffness," and determine if soft or stiff polymer domains exist to correlate nano- and macro-mechanics. Two model hydrogel systems typically used in cell culture and polymerized by a free radical polymerization process, i.e. poly (vinyl pyrrolidone) (PVP) and poly(acrylamide) (PAam) hydrogels, were found to have significantly different nanomechanical behavior despite relatively similar bulk stiffness and roughness. PVP gels contained a large number of soft and stiff nanodomains, and their size was inversely related to crosslinking density and changes in crosslinking efficiency within the hydrogel. In contrast, PAam gels displayed small nanodomains occuring at low frequency, indicating relatively uniform polymerization. Given the responsiveness of cells to changes in gel stiffness, inhomogeneities found in the PVP network indicate that careful nanomechanical characterization of polymer substrates is necessary to appreciate complex cell behavior.

Novel electrospun polyurethane/gelatin composite meshes for vascular grafts.

Novel polymeric micro-nanostructure meshes as blood vessels substitute have been developed and investigated as a potential solution to the lack of functional synthetic small diameter vascular prosthesis. A commercial elastomeric polyurethane (Tecoflex EG-80A) and a natural biopolymer (gelatin) were successfully co-electrospun from different spinnerets on a rotating mandrel to obtain composite meshes benefiting from the mechanical characteristics of the polyurethane and the natural biopolymer cytocompatibility. Morphological analysis showed a uniform integration of micrometric (Tecoflex) and nanometric (gelatin) fibers. Exposure of the composite meshes to vapors of aqueous glutaraldehyde solution was carried out, to stabilize the gelatin fibers in an aqueous environment. Uniaxial tensile testing in wet conditions demonstrated that the analyzed Tecoflex-Gelatin specimens possessed higher extensibility and lower elastic modulus than conventional synthetic grafts, providing a closer matching to native vessels. Biological evaluation highlighted that, as compared with meshes spun from Tecoflex alone, the electrospun composite constructs enhanced endothelial cells adhesion and proliferation, both in terms of cell number and morphology. Results suggest that composite Tecoflex-Gelatin meshes could be promising alternatives to conventional vascular grafts, deserving of further studies on both their mechanical behaviour and smooth muscle cell compatibility.

Boosting the Osteogenic and Angiogenic Performance of Multiscale Porous Polycaprolactone Scaffolds by In Vitro Generated Extracellular Matrix Decoration.

Tissue engineering (TE)-based bone grafts are favorable alternatives to autografts and allografts. Both biochemical properties and the architectural features of TE scaffolds are crucial in their design process. Synthetic polymers are attractive biomaterials to be used in the manufacturing of TE scaffolds, due to various advantages, such as being relatively inexpensive, enabling precise reproducibility, possessing tunable mechanical/chemical properties, and ease of processing. However, such scaffolds need modifications to improve their limited interaction with biological tissues. Structurally, multiscale porosity is advantageous over single-scale porosity; therefore, in this study, we have considered two key points in the design of a bone repair material; (i) manufacture of multiscale porous scaffolds made of photocurable polycaprolactone (PCL) by a combination of emulsion templating and three-dimensional (3D) printing and (ii) decoration of these scaffolds with the in vitro generated bone-like extracellular matrix (ECM) to create biohybrid scaffolds that have improved biological performance compared to PCL-only scaffolds. Multiscale porous scaffolds were fabricated, bone cells were cultured on them, and then they were decellularized. The biological performance of these constructs was tested in vitro and in vivo. Mesenchymal progenitors were seeded on PCL-only and biohybrid scaffolds. Cells not only showed improved attachment on biohybrid scaffolds but also exhibited a significantly higher rate of cell growth and osteogenic activity. The chick chorioallantoic membrane (CAM) assay was used to explore the angiogenic potential of the biohybrid scaffolds. The CAM assay indicated that the presence of the in vitro generated ECM on polymeric scaffolds resulted in higher angiogenic potential and a high degree of tissue infiltration. This study demonstrated that multiscale porous biohybrid scaffolds present a promising approach to improve bioactivity, encourage precursors to differentiate into mature bones, and to induce angiogenesis.

Comparison of the Anabolic Effects of Reported Osteogenic Compounds on Human Mesenchymal Progenitor-derived Osteoblasts.

There is variability in the reported effects of compounds on osteoblasts arising from differences in experimental design and choice of cell type/origin. This makes it difficult to discern a compound's action outside its original study and compare efficacy between compounds. Here, we investigated five compounds frequently reported as anabolic for osteoblasts (17ß-estradiol (oestrogen), icariin, lactoferrin, lithium chloride, and menaquinone-4 (MK-4)) on human mesenchymal progenitors to assess their potential for bone tissue engineering with the aim of identifying a potential alternative to expensive recombinant growth factors such as bone morphogenetic protein 2 (BMP-2). Experiments were performed using the same culture conditions to allow direct comparison. The concentrations of compounds spanned two orders of magnitude to encompass the reported efficacious range and were applied continuously for 22 days. The effects on the proliferation (resazurin reduction and DNA quantification), osteogenic differentiation (alkaline phosphatase (ALP) activity), and mineralised matrix deposition (calcium and collagen quantification) were assessed. Of these compounds, only 10 µM MK-4 stimulated a significant anabolic response with 50% greater calcium deposition. Oestrogen and icariin had no significant effects, with the exception of 1 µM icariin, which increased the metabolic activity on days 8 and 22. 1000 µg/mL of lactoferrin and 10 mM lithium chloride both significantly reduced the mineralised matrix deposition in comparison to the vehicle control, despite the ALP activity being higher in lithium chloride-treated cells at day 15. This demonstrates that MK-4 is the most powerful stimulant of bone formation in hES-MPs of the compounds investigated, highlighting its potential in bone tissue engineering as a method of promoting bone formation, as well as its prospective use as an osteoporosis treatment.

Characterization of a composite polylactic acid-hydroxyapatite 3D-printing filament for bone-regeneration.

Autologous cancellous-bone grafts are the current gold standard for therapeutic interventions in which bone-regeneration is desired. The main limitations of these implants are the need for a secondary surgical site, creating a wound on the patient, the limited availability of harvest-safe bone, and the lack of structural integrity of the grafts. Synthetic, resorbable, bone-regeneration materials could pose a viable treatment alternative, that could be implemented through 3D-printing. We present here the development of a polylactic acid-hydroxyapatite (PLA-HAp) composite that can be processed through a commercial-grade 3D-printer. We have shown that this material could be a viable option for the development of therapeutic implants for bone regeneration. Biocompatibility in vitro was demonstrated through cell viability studies using the osteoblastic MG63 cell-line, and we have also provided evidence that the presence of HAp in the polymer matrix enhances cell attachment and osteogenicity of the material. We have also provided guidelines for the optimal PLA-HAp ratio for this application, as well as further characterisation of the mechanical and thermal properties of the composite. This study encompasses the base for further research on the possibilities and safety of 3D-printable, polymer-based, resorbable composites for bone regeneration.

Combined Porogen Leaching and Emulsion Templating to produce Bone Tissue Engineering Scaffolds.

Bone has a hierarchy of porosity that is often overlooked when creating tissue engineering scaffolds where pore sizes are typically confined to a single order of magnitude. High internal phase emulsion (HIPE) templating produces polymerized HIPEs (polyHIPEs): highly interconnected porous polymers which have two length scales of porosity covering the 1-100 µm range. However, additional larger scales of porosity cannot be introduced in the standard emulsion formulation. Researchers have previously overcome this by additively manufacturing emulsions; fabricating highly microporous struts into complex macroporous geometries. This is time consuming and expensive; therefore, here we assessed the feasibility of combining porogen leaching with emulsion templating to introduce additional macroporosity. Alginate beads between 275 and 780 µm were incorporated into the emulsion at 0, 50, and 100 wt%. Once polymerized, alginate was dissolved leaving highly porous polyHIPE scaffolds with added macroporosity. The compressive modulus of the scaffolds decreased as alginate porogen content increased. Cellular performance was assessed using MLO-A5 post-osteoblasts. Seeding efficiency was significantly higher and mineralized matrix deposition was more uniformly deposited throughout porogen leached scaffolds compared to plain polyHIPEs. Deep cell infiltration only occurred in porogen leached scaffolds as detected by histology and lightsheet microscopy. This study reveals a quick, low cost and simple method of producing multiscale porosity scaffolds for tissue engineering.

Design and Evaluation of an Osteogenesis-on-a-Chip Microfluidic Device Incorporating 3D Cell Culture.

Microfluidic-based tissue-on-a-chip devices have generated significant research interest for biomedical applications, such as pharmaceutical development, as they can be used for small volume, high throughput studies on the effects of therapeutics on tissue-mimics. Tissue-on-a-chip devices are evolving from basic 2D cell cultures incorporated into microfluidic devices to complex 3D approaches, with modern designs aimed at recapitulating the dynamic and mechanical environment of the native tissue. Thus far, most tissue-on-a-chip research has concentrated on organs involved with drug uptake, metabolism and removal (e.g., lung, skin, liver, and kidney); however, models of the drug metabolite target organs will be essential to provide information on therapeutic efficacy. Here, we develop an osteogenesis-on-a-chip device that comprises a 3D environment and fluid shear stresses, both important features of bone. This inexpensive, easy-to-fabricate system based on a polymerized High Internal Phase Emulsion (polyHIPE) supports proliferation, differentiation and extracellular matrix production of human embryonic stem cell-derived mesenchymal progenitor cells (hES-MPs) over extended time periods (up to 21 days). Cells respond positively to both chemical and mechanical stimulation of osteogenesis, with an intermittent flow profile containing rest periods strongly promoting differentiation and matrix formation in comparison to static and continuous flow. Flow and shear stresses were modeled using computational fluid dynamics. Primary cilia were detectable on cells within the device channels demonstrating that this mechanosensory organelle is present in the complex 3D culture environment. In summary, this device aids the development of 'next-generation' tools for investigating novel therapeutics for bone in comparison with standard laboratory and animal testing.

Effect of cell sample size in atomic force microscopy nanoindentation.

Single-cell technologies are powerful tools to evaluate cell characteristics. In particular, Atomic Force Microscopy (AFM) nanoindentation experiments have been widely used to study single cell mechanical properties. One important aspect related to single cell techniques is the need for sufficient statistical power to obtain reliable results. This aspect is often overlooked in AFM experiments were sample sizes are arbitrarily set. The aim of the present work was to propose a tool for sample size estimation in the context of AFM nanoindentation experiments of single cell. To this aim, a retrospective approach was used by acquiring a large dataset of experimental measurements on four bone cell types and by building saturation curves for increasing sample sizes with a bootstrap resampling method. It was observed that the coefficient of variation (CV%) decayed with a function of the form y = axb with similar parameters for all samples tested and that sample sizes of 21 and 83 cells were needed for the specific cells and protocol employed if setting a maximum threshold on CV% of 10% or 5%, respectively. The developed tool is made available as an open-source repository and guidelines are provided for its use for AFM nanoindentation experimental design.

A Novel Bilayer Polycaprolactone Membrane for Guided Bone Regeneration: Combining Electrospinning and Emulsion Templating.

Guided bone regeneration is a common dental implant treatment where a barrier membrane (BM) is used between epithelial tissue and bone or bone graft to prevent the invasion of the fast-proliferating epithelial cells into the defect site to be able to preserve a space for infiltration of slower-growing bone cells into the periodontal defect site. In this study, a bilayer polycaprolactone (PCL) BM was developed by combining electrospinning and emulsion templating techniques. First, a 250 µm thick polymerised high internal phase emulsion (polyHIPE) made of photocurable PCL was manufactured and treated with air plasma, which was shown to enhance the cellular infiltration. Then, four solvent compositions were investigated to find the best composition for electrospinning a nanofibrous PCL barrier layer on PCL polyHIPE. The biocompatibility and the barrier properties of the electrospun layer were demonstrated over four weeks in vitro by histological staining. Following in vitro assessment of cell viability and cell migration, cell infiltration and the potential of PCL polyHIPE for supporting blood vessel ingrowth were further investigated using an ex-ovo chick chorioallantoic membrane assay. Our results demonstrated that the nanofibrous PCL electrospun layer was capable of limiting cell infiltration for at least four weeks, while PCL polyHIPE supported cell infiltration, calcium and mineral deposition of bone cells, and blood vessel ingrowth through pores.