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Abstract The presence of CD8+ T cells in the cytoplasm of biliary epithelial cells (BEC) has been correlated with biliary damage associated with primary biliary cholangitis (PBC). Here, we characterise the mechanism of CD8+ T cell invasion into BEC. CD8+ T cells observed within BEC were large, eccentric, and expressed E-cadherin, CD103 and CD69. They were also not contained within secondary vesicles. Internalisation required cytoskeletal rearrangements which facilitated contact with BEC. Internalised CD8+ T cells were observed in both non-cirrhotic and cirrhotic diseased liver tissues but enriched in PBC patients, both during active disease and at the time of transplantation. E-cadherin expression by CD8+ T cells correlated with frequency of internalisation of these cells into BEC. E-cadherin+ CD8+ T cells formed β-catenin-associated interactions with BEC, were larger than E-cadherin- CD8+ T cells and invaded into BEC more frequently. Overall, we unveil a distinct cell-in-cell structure process in the liver detailing the invasion of E-cadherin+ CD103+ CD69+ CD8+ T cells into BEC.

Skin inflammation associated with chronic diseases involves a direct role of keratinocytes in its immunopathogenesis, triggering a cascade of immune responses. Despite this, highly targeted treatments remain elusive, highlighting the need for more specific therapeutic strategies. In this study, nanocapsules containing quinizarin (QZ/NC) were developed and evaluated in an in vitro model of keratinocyte-mediated inflammation, incorporating the action of photodynamic therapy (PDT) and analyzing permeation in a 3D skin model. Comprehensive physicochemical, stability, cytotoxicity, and permeation analyses of the nanomaterials were conducted. The nanocapsules demonstrated desirable physicochemical properties, remained stable throughout the analysis period, and exhibited no spectroscopic alterations. Cytotoxicity tests revealed no toxicity at the lowest concentrations of QZ/NC. Permeation and cellular uptake studies confirmed QZ/NC permeation in 3D skin models, along with intracellular incorporation and internalization of the drug, thereby enhancing its efficacy in drug delivery. The developed model for inducing the inflammatory process in vitro yielded promising results, particularly when the synthesized nanomaterial was combined with PDT, showing a reduction in cytokine levels. These findings suggest a potential new therapeutic approach for treating inflammatory skin diseases.

Abstract Heterotopic ossification (HO), the pathological formation of bone in soft tissues, is a debilitating condition, as well as one of the few instances of de novo bone formation in adults. Chemical mapping of HO tissue showed distinct islands of calcium phosphate within phosphate-deficient, calcium-rich regions, suggesting a transition to apatitic bone mineral from a non-phosphatic precursor. The transition of amorphous calcium carbonate (ACC), a generally suggested bone-mineral precursor, in physiological conditions was thus investigated. Here, we show that adenosine triphosphate (ATP), present in high amounts in forming bone, stabilised ACC for weeks in physiological conditions and that enzymatic degradation of ATP triggered rapid crystallisation into apatite, through an amorphous calcium phosphate phase. It is suggested that this localised enzymatic degradation could explain the chemical heterogeneity seen in HO and may also represent a pathway to physiological bone mineralisation.

Fibrotic diseases account for 45% of all deaths in the developed world, and progressive scarring conditions, such as dystrophic epidermolysis bullosa (EB), result in a significant reduction in quality of life. There is thus a need for novel approaches in fibrosis prevention. Herein, it is shown that iota carrageenan, a low‐cost, regulatory approved polysaccharide, is effective at preventing scarring by inhibiting collagen fibrillogenesis, abrogating the transforming growth factor beta 1 (TGFβ1) pathway, and lubricating the epithelium. It is demonstrated that iota carrageenan, and other anionic polysaccharides, can prevent collagen fibril formation, a key step in scar formation. It also binds TGFβ1 to prevent myofibroblast transdifferentiation, evidenced by a significant reduction in both transcription and translation of collagen 1 and alpha smooth muscle actin. Iota carrageenan may be formulated as a microparticle suspension, to allow spraying for even coverage on hard‐to‐reach anatomical sites, like the oral mucosa. This formulation provides good lubrication, to reduce the shear forces that initiate the progressive oral scarring in EB. This study not only provides a novel therapy for fibrosis prevention, but also demonstrates the potential of self‐delivering immunomodulatory polysaccharides as a safe, cost‐effective, and stable platform, which can be more easily translated for clinical benefit.

IntroductionFor decades, functional primary human osteocyte cultures have been crucially needed for understanding their role in bone anabolic processes and in endocrine phosphate regulation via the bone-kidney axis. Mature osteocyte proteins (sclerostin, DMP1, Phex and FGF23) play a key role in various systemic diseases and are targeted by successful bone anabolic drugs (anti-sclerostin antibody and teriparatide (PTH1-34)). However, cell lines available to study osteocytes produce very little sclerostin and low levels of mature osteocyte markers. We have developed a primary human 3D organotypic culture system that replicates the formation of mature osteocytes in bone.MethodsPrimary human osteoblasts were seeded in a fibrinogen / thrombin gel around 3D-printed hanging posts. Following contraction of the gel around the posts, cells were cultured in osteogenic media and conditioned media was collected for analysis of secreted markers of osteocyte formation.ResultsThe organoids were viable for at least 6 months, allowing co-culture with different cell types and testing of bone anabolic drugs. Bulk RNAseq data displayed the developing marker trajectory of ossification and human primary osteocyte formation in vitro over an initial 8- week period. Vitamin D3 supplementation increased mineralization and sclerostin secretion, while hypoxia and PTH1-34 modulated sclerostin. Our culture system also secreted FGF23, enabling the future development of a bone-kidney-parathyroid-vascular multi-organoid or organ-on-a-chip system to study disease processes and drug effects using purely human cells.DiscussionThis 3D organotypic culture system provides a stable, long-lived, and regulated population of mature human primary osteocytes for a variety of research applications.

Abstract The World Health Organisation has called for a 40% increase in personal protective equipment manufacturing worldwide, recognising that frontline workers need effective protection during the COVID-19 pandemic. Current devices suffer from high fit-failure rates leaving significant proportions of users exposed to risk of viral infection. Driven by non-contact, portable, and widely available 3D scanning technologies, a workflow is presented whereby a user’s face is rapidly categorised using relevant facial parameters. Device design is then directed down either a semi-customised or fully-customised route. Semi-customised designs use the extracted eye-to-chin distance to categorise users in to pre-determined size brackets established via a cohort of 200 participants encompassing 87.5% of the cohort. The user’s nasal profile is approximated to a Gaussian curve to further refine the selection in to one of three subsets. Flexible silicone provides the facial interface accommodating minor mismatches between true nasal profile and the approximation, maintaining a good seal in this challenging region. Critically, users with outlying facial parameters are flagged for the fully-customised route whereby the silicone interface is mapped to 3D scan data. These two approaches allow for large scale manufacture of a limited number of design variations, currently nine through the semi-customised approach, whilst ensuring effective device fit. Furthermore, labour-intensive fully-customised designs are targeted as those users who will most greatly benefit. By encompassing both approaches, the presented workflow balances manufacturing scale-up feasibility with the diverse range of users to provide well-fitting devices as widely as possible. Novel flow visualisation on a model face is presented alongside qualitative fit-testing of prototype devices to support the workflow methodology.

Chemobrionic materials with applications in tissue engineering can be grown at the interface of calcium and phosphate solutions, however, controlling their dimensions and purity can be difficult. Herein, a flow device allows control of the height and in situ purification of chemical gardens, which can be encased in a photoset hydrogel for subsequent use.

Abstract The treatment of spinal cord injury (SCI) is a complex challenge in regenerative medicine, complicated by the low intrinsic capacity of CNS neurons to regenerate their axons and the heterogeneity in size, shape and extent of human injuries. For example, some contusion injuries do not compromise the dura mater and in such cases implantation of preformed scaffolds or drug delivery systems may cause further damage. Injectable in situ thermosensitive scaffolds are therefore a less invasive alternative. In this study, we report the development of a novel, flowable, thermosensitive, injectable drug delivery system comprising bovine collagen (BC) and fibrinogen (FB) that forms a solid BC/FB gel (Gel) immediately upon exposure to physiological conditions and can be used to deliver reparative drugs, such as the naturally occurring anti-inflammatory, anti-scarring agent Decorin, into adult rat spinal cord lesion sites. In dorsal column lesions of adult rats treated with the Gel + Decorin, cavitation was completely suppressed and instead lesion sites became filled with injury-responsive cells and extracellular matrix materials, including collagen and laminin. Decorin increased the intrinsic potential of dorsal root ganglion neurons (DRGN) by increasing their expression of regeneration associated genes (RAGs), enhanced local axon regeneration/sprouting, as evidenced both histologically and by improved electrophysiological, locomotor and sensory function recovery. These results suggest that this drug formulated, injectable hydrogel has the potential to be further studied and translated into the clinic.

Abstract Bone is a highly responsive organ, which continuously adapts to the environment it is subjected to in order to withstand metabolic demands. These events are difficult to study in this particular tissue in vivo, due to its rigid, mineralised structure and inaccessibility of the cellular component located within. This manuscript presents the development of a micron-scale bone organoid prototype, a concept that can allow the study of bone processes at the cell-tissue interface. The model is constructed with a combination of primary female osteoblastic and osteoclastic cells, seeded onto femoral head micro-trabeculae, where they recapitulate relevant phenotypes and functions. Subsequently, constructs are inserted into a simulated microgravity bioreactor (NASA-Synthecon) to model a pathological state of reduced mechanical stimulation. In these constructs, we detected osteoclastic bone resorption sites, which were different in morphology in the simulated microgravity group compared to static controls. Once encapsulated in human fibrin and exposed to analogue microgravity for 5 days, masses of bone can be observed being lost from the initial structure, allowing to simulate the bone loss process further. Constructs can function as multicellular, organotypic units. Large osteocytic projections and tubular structures develop from the initial construct into the matrix at the millimetre scale. Micron-level fragments from the initial bone structure are detected travelling along these tubules and carried to sites distant from the native structure, where new matrix formation is initiated. We believe this model allows the study of fine-level physiological processes, which can shed light into pathological bone loss and imbalances in bone remodelling.

Abstract Prophylactic antibiotic bone cements are extensively used in orthopaedics. However, the development of antimicrobial resistance to antibiotics, demonstrates a need to find alternative treatments. Herein, an antimicrobial honey (SurgihoneyRO-SHRO) has been successfully incorporated into a calcium sulphate (CS) based cement to produce a hard tissue scaffold with the ability to inhibit bacterial growth. Antimicrobial properties elicited from SHRO are predominantly owed to the water-initiated production of reactive oxygen species (ROS). As an alternative to initially loading CS cement with SHRO, in order to prevent premature activation, SHRO was added into the already developing cement matrix, locking available water into the CS crystal structure before SHRO addition. Promisingly, this methodology produced > 2.5 times (715.0 ± 147.3 μM/mL/g) more ROS over 24 h and exhibited a compressive strength (32.2 ± 5.8 MPa) comparable to trabecular bone after 3 weeks of immersion. In-vitro the SHRO loaded CS scaffolds were shown to inhibit growth of clinically relevant organisms, Staphylococcus aureus and Pseudomonas aeruginosa, with comparable potency to equivalent doses of gentamicin. Encouragingly, formulations did not inhibit wound healing or induce an inflammatory response from osteoblasts. Overall this study highlights the prophylactic potential of CS-SHRO cements as an alternative to traditional antibiotics.

Abstract Background The cornea is innervated with a rich supply of sensory nerves that play important roles in ocular surface health. Any injury or pathology of the corneal nerves increases the risk of dry eye disease and infection. This study aims to evaluate the therapeutic potential of topical decorin to improve corneal nerve regeneration in a mouse model of sterile epithelial abrasion injury. Methods Bilateral central corneal epithelial abrasions (2-mm, Alger Brush) were performed on young C57BL/6 J mice to remove the corneal sensory nerves. Decorin, or vehicle, was applied topically, three times per day for 1 week or every 2 h for 6 h. Spectral-domain optical coherence tomography was performed to measure the abrasion area and corneal thickness. Wholemount immunofluorescence staining was used to assess sensory nerve regeneration (β-tubulin III) and immune cell density (CD45, Iba1, CD11c). To investigate the specific role of dendritic cells (DCs), Cx3cr1gfp/gfp mice, which spontaneously lack resident corneal epithelial DCs, were also investigated. The effect of prophylactic topical administration of recombinant human decorin (applied prior to the abrasion) was also investigated. Nerve tracing (NeuronJ software) was performed to compare recovery of basal nerve axons and superficial nerve terminals in the central and peripheral cornea. Results At 6 h after injury, topical decorin application was associated with greater intraepithelial DC recruitment but no change in re-epithelialisation or corneal thickness, compared to the vehicle control. One week after injury, sub-basal nerve plexus and superficial nerve terminal density were significantly higher in the central cornea in the decorin-treated eyes. The density of corneal stromal macrophages in the decorin-treated eyes and their contralateral eyes was significantly lower compared to saline-treated corneas. No significant improvement in corneal nerve regeneration was observed in Cx3cr1gfp/gfp mice treated with decorin. Conclusions Decorin promotes corneal epithelial nerve regeneration after injury. The neuroregenerative effect of topical decorin was associated with a higher corneal DC density during the acute phase, and fewer macrophages at the study endpoint. The corneal neuroregenerative effects of decorin were absent in mice lacking intraepithelial DCs. Together, these findings support a role for decorin in DC-mediated neuroregeneration following corneal abrasion injury.

Skin exhibits a complex structure consisting of three predominant layers (epidermis, dermis, and hypodermis). Extensive trauma may result in the loss of these structures and poor repair, in the longer term, forming scarred tissue and associated reduction in function. Although a number of skin replacements exist, there have been no solutions that recapitulate the chemical, mechanical, and biological roles that exist within native skin. This study reports the use of suspended layer additive manufacturing to produce a continuous tri-layered implant, which closely resembles human skin. Through careful control of the bioink composition, gradients (chemical and cellular) were formed throughout the printed construct. Culture of the model demonstrated that over 21 days, the cellular components played a key role in remodeling the supporting matrix into architectures comparable with those of healthy skin. Indeed, it has been demonstrated that even at seven days post-implantation, the integration of the implant had occurred, with mobilization of the adipose tissue from the surrounding tissue into the construct itself. As such, it is believed that these implants can facilitate healing, commencing from the fascia, up toward the skin surface—a mechanism recently shown to be key within deep wounds.

Structured fluid biomaterials, including gels, creams, emulsions and particle suspensions, are used extensively across many industries, including great interest within the medical field as controlled release vehicles to improve the therapeutic benefit of delivered drugs and cells. Colloidal forces within these materials create multiscale cohesive interactions, giving rise to intricate microstructures and physical properties, exemplified by increasingly complex mathematical descriptions. Yield stresses and viscoelasticity, typically arising through the material microstructure, vastly improve site-specific retention, and protect valuable therapeutics during application. One powerful application route is spraying, a convenient delivery method capable of applying a thin layer of material over geometrically uneven surfaces and hard-to-reach anatomical locations. The process of spraying is inherently disruptive, breaking a bulk fluid in successive steps into smaller elements, applying multiple forces over several length scales. Historically, spray research has focused on simple, inviscid solutions and dispersions, far from the complex microstructures and highly viscoelastic properties of concentrated colloidal biomaterials. The cohesive forces in colloidal biomaterials appear to conflict with the disruptive forces that occur during spraying. This review explores the physical bass and mathematical models of both the multifarious material properties engineered into structured fluid biomaterials and the disruptive forces imparted during the spray process, in order to elucidate the challenges and identify opportunities for rational design of sprayable, structured fluid biomaterials.

The COVID-19 global pandemic, as well as the widespread persistence of influenza and the common cold, create the need for new medical devices such as nasal sprays to prevent viral infection and transmission. Carrageenan, a sulfated polysaccharide, has a broad, non-pharmacological antiviral capacity, however it performs poorly in two key areas; spray coverage and mucoadhesion. Therefore gellan, another polysaccharide, was investigated as an excipient to improve these properties. It was found that viscoelastic relaxation time was the key predictor of spray coverage, and by reducing this value from 2.5 to 0.25 s, a mix of gellan and carrageenan gave more than four times the coverage of carrageenan alone (p < 0.0001). Gellan also demonstrated enhanced adhesion to a mucus analog that increased significantly with time (p < 0.0001), suggesting the development of specific gellan–mucin interactions. This property was conferred to carrageenan on mixing the two polymers. Together, this data suggests that gellan is a promising excipient to improve both sprayability and mucoadhesion of carrageenan for use in antiviral nasal sprays.

Glaucoma is a leading cause of irreversible blindness globally, with primary open angle glaucoma (POAG) being the most common subset. Raised intraocular pressure is an important risk factor for POAG and is caused by a reduction in aqueous humour (AqH) outflow due to dysfunctional cellular and matrix dynamics in the eye’s main drainage site, the trabecular meshwork (TM) and Schlemm’s canal (SC). The TM/SC are highly specialised tissues that regulate AqH outflow; however, their exact mechanisms of AqH outflow control are still not fully understood. Emulating physiologically relevant 3D TM/S in vitro models poses challenges to accurately mimic the complex biophysical and biochemical cues that take place in healthy and glaucomatous TM/SC in vivo. With development of such models still in its infancy, there is a clear need for more well-defined approaches that will accurately contrast the two central regions that become dysfunctional in POAG; the juxtacanalicular tissue (JCT) region of the TM and inner wall endothelia of the Schlemm’s canal (eSC). This review will discuss the unique biological and biomechanical characteristics that are thought to influence AqH outflow and POAG progression. Further consideration into fundamental biomaterial attributes for the formation of a biomimetic POAG/AqH outflow model will also be explored for future success in pre-clinical drug discovery and disease translation.

Abstract The purpose of this study was to determine the effect of adiposity on the architecture and composition of hip OA subchondral bone, and to examine the pathological role of adipokines. Femoral heads were collected from normal-weight or over-weight/obese patients with hip OA. Structural parameters of subchondral bone were determined by MicroCT and type I collagen α1/α2 ratio was determined by SDS PAGE and by qRT-PCR in ex-vivo bone explants. The serum concentration of adipokines was determined by Luminex. The effect of resistin on primary OA osteoblasts was determined by analysis of Wnt pathway signal transduction, bone nodule formation, and osteoblast metabolic activity. Subchondral bone from over-weight/obese hip OA patients exhibited reduced trabecular thickness, increased bone surface/bone volume ratio, and an increase in the Type I collagen α1/α2, compared to normal-weight hip OA patients. The serum concentration of resistin was higher in overweight/obese OA patients, compared to normal-weight OA patients. Stimulation of normal-weight bone explant with recombinant resistin increased the Type I collagen α1/α2 ratio. Stimulation of primary OA osteoblasts with recombinant resistin increased Wnt signalling activation, osteoblast metabolic activity, and bone nodule formation. Increased adiposity in hip OA patients is associated with altered subchondral bone architecture and type I collagen composition.

Abstract Background Calcium sulphate (CS) has been used in bone grafting since the 1800s. It has not replaced autograft as the gold standard, however, since its dissolution occurs rapidly in bodily fluids, meaning that the material cannot support long-term bone growth. Here, the polymer poly (ether ether ketone) (PEEK) was used to slow dissolution in in vitro physiological environments and augment the mechanical properties of the material. Methods PEEK/CS specimens were fabricated by combining powders of PEEK and CS with water, resulting in a hardening paste. To enhance physical interactions between phases, cylindrical specimens were heat-treated to melt and fuse the PEEK. Following analysis of physical and chemical interactions by SEM and FT-IR respectively, dynamic ageing in PBS and compression testing was undertaken to measure how the PEEK influenced the mechanical properties of the final parts. Changes in structure and chemistry were determined using helium pycnometry, SEM and analysis of powder XRD patterns. Results Powders of PEEK and CS hemihydrate (CSH) (CaSO4.0.5H2O) were combined with PEEK at 0 wt%, 2.5 wt%, 20 wt%, 40 wt% and 80 wt% and at a P:L ratio of 0.85 g/mL. The subsequently hardened structures were heat-treated, which initiated the melting of PEEK and dehydration of CSD (CaSO4.2H2O) to the CS anhydrite (CSA) (CaSO4) phase, which changed colour and apparent volume. FT-IR and SEM analysis revealed heat treatment of PEEK/CS specimens facilitated both physical and chemical interactions between phases. Over a period of 21 days of ageing in PBS, the hydration of CS was determined by XRD and improved specimen longevity at all levels of PEEK wt% loading was measured compared with the control. Importantly, increasing PEEK wt% loading resulted in a marked increase in the mechanical properties of PEEK/CS specimens in terms of both compressive strength and modulus. Conclusions Reinforcement of CS with PEEK significantly enhanced in vitro dissolution resistance, in addition to enhancing mechanical properties. This composite therefore has significant future potential as a bone graft replacement.

Osteoblast-derived extracellular vesicles (EV) are a collection of secreted (sEVs) and matrix-bound nanoparticles that function as foci for mineral nucleation and accumulation. Due to the fact sEVs can be isolated directly from the culture medium of mineralizing osteoblasts, there is growing interest their application regenerative medicine. However, at present therapeutic advancements are hindered by a lack of understanding of their precise temporal contribution to matrix mineralization. This study advances current knowledge by temporally aligning sEV profile and protein content with mineralization status. sEVs were isolated from mineralizing primary osteoblasts over a period of 1, 2, and 3 weeks. Bimodal particle distributions were observed (weeks 1 and 3: 44 and 164 nm; week 2: 59 and 220 nm), indicating a heterogeneous population with dimensions characteristic of exosome- (44 and 59 nm) and microvesicle-like (164 and 220 nm) particles. Proteomic characterization by liquid chromatography tandem-mass spectrometry (LC-MS/MS) revealed a declining correlation in EV-localized proteins as mineralization advanced, with Pearson correlation-coefficients of 0.79 (week 1 vs. 2), 0.6 (2 vs. 3) and 0.46 (1 vs. 3), respectively. Principal component analysis (PCA) further highlighted a time-dependent divergence in protein content as mineralization advanced. The most significant variations were observed at week 3, with a significant (p < 0.05) decline in particle concentration, visual evidence of EV rupture and enhanced mineralization. A total of 116 vesicle-localized proteins were significantly upregulated at week 3 (56% non-specifically, 19% relative to week 1, 25% relative to week 2). Gene ontology enrichment analysis of these proteins highlighted overrepresentation of genes associated with matrix organization. Of note, increased presence of phospholipid-binding and calcium channeling annexin proteins (A2, A5, and A6) indicative of progressive variations in the nucleational capacity of vesicles, as well as interaction with the surrounding ECM. We demonstrate sEV-mediated mineralization is dynamic process with variations in vesicle morphology and protein content having a potential influence on developmental changes matrix organization. These findings have implications for the selection and application of EVs for regenerative applications.

One of the main challenges in additive manufacturing (AM) of medical implants for the treatment of bone tissue defects is to optimise the mechanical and biological performance. The use of post-processing can be a necessity to improve the physical properties of customised AM processed implants. In this study, Ti-6Al-4V coupons were manufactured using selective laser melting (SLM) in two build orientations (vertical and horizontal) and subsequently post-processed using combinations of hot isostatic pressing (HIP), sandblasting (SB), polishing (PL) and chemical etching (CE). The effect of the different post-manufacturing strategies on the tensile and fatigue performance of the SLMed parts was investigated and rationalised by observing the surface topography. Vertically built samples showed higher yield strength (YS) and ultimate tensile strength (UTS) than the horizontal samples, increasing from 760.9 ± 22.3 MPa and 961.3 ± 50.2 MPa in the horizontal condition to 820.09 ± 16.5 MPa and 1006.7 ± 6.3 MPa in the vertical condition, respectively. After the HIP treatment, the ductility was substantially improved in both orientations; by 2.1 and 2.9 folds in the vertical and horizontal orientations, respectively. The vertically built samples demonstrated a superior ductility of 22% following HIP and polishing. Furthermore, chemical etching was found to be the most effective surface post-processing treatment to improve the fatigue performance after HIP, achieving the highest run-out strength of 450 MPa. Most importantly, chemical etching after HIP enhanced the cellular affinity of the surface, in addition to its good fatigue performance, making it a promising post-processing approach for bone implants where tissue integration is needed.

Human articular chondrocytes lose their native phenotype when expanded in traditional monolayer cultures. As a consequence, hydrogel encapsulation has been investigated as a means to maintain the natural phenotype. Alginate has been widely used for cartilage engineering as it has been shown to enable the recovery of a native collagen type II expressing chondrocyte phenotype. This study has evaluated whether the capacity of the materials to maintain/revert the phenotype is due to the composition of the material or the physical entrapment provided by the gel. To achieve this, an alginate “fluid gel” (a shear-thinning structured gel system) was produced of identical chemistry to a traditionally gelled alginate structure. Both were seeded with passaged primary human articular chondrocytes. Chondrocytes in quiescent alginate showed the recovery of the native phenotype and a spherical morphology. Chondrocytes in alginate fluid gel were unable to maintain the recovered phenotype despite having a spherical morphology and were shown to have a lower level of entrapment than those in quiescent alginate. These findings indicate that geometric entrapment is essential for the maintenance of a recovered chondrocyte phenotype in alginate.

Background There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models. Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year). Methods Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including RT-qPCR, immunofluorescence staining and XRF, are described in detail. Results Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture. Conclusions Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.

Background There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models. Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year). Methods Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including RT-qPCR, immunofluorescence staining and XRF, are described in detail. Results Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture. Conclusions Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.

Background: There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models. Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year). Methods: Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including qPCR, immunofluorescence staining and XRF, are described in detail. Results: Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture. Conclusions: Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.