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The aim of this article was to study clinical and radiographic outcomes following treatment of bone healing disorders with a novel osteoinductive system that utilizes poly (ethyl acrylate), fibronectin and an ultra-low concentration of recombinant human bone morphogenetic protein-2. A case series of nine dogs and two cats were treated, and clinical records and radiographs were reviewed. Radiographs were scored by two blinded observers using the modified Radiographic Union Score for Tibial Fractures. Long-term follow-up was obtained using the Canine Orthopaedic Index and Feline Musculoskeletal Pain Index. Follow-up data were available for 11 treatments (10 cases). Complications: three minor, three major, one catastrophic (non-union requiring amputation). Lameness median 320 (range: 42–1,082) days postoperatively: ‘sound’ (three cases), ‘subtle’ (two), ‘mild’ (three), ‘moderate’ (one), and ‘non-weightbearing’ (one). The attending clinician judged 9 of 11 treatments achieved radiographic union; modified Radiographic Union Score for Tibial Fractures observers 1 and 2 agreed with the clinician in 8/9 and 5/9 treatments respectively. Long-term Canine Orthopaedic Index scores for five dogs median 650 (range: 544–1,724) days postoperatively: 15/64 (median) for four dogs with acceptable outcomes, 30/64 in one dog with a poor outcome. Feline Musculoskeletal Pain Index scores for two cats 433 and 751 days postoperatively: 48/60 and 60/60. Eight of 10 cases were sound or showed subtle or mild lameness in the short- or long-term, and radiographic union occurred in the majority of treatments.

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells that are of considerable clinical potential in transplantation and anti-inflammatory therapies due to their capacity for tissue repair and immunomodulation. However, MSCs rapidly differentiate once in culture, making their large-scale expansion for use in immunomodulatory therapies challenging. Although the differentiation mechanisms of MSCs have been extensively investigated using materials, little is known about how materials can influence paracrine activities of MSCs. Here, we show that nanotopography can control the immunomodulatory capacity of MSCs through decreased intracellular tension and increasing oxidative glycolysis. We use nanotopography to identify bioactive metabolites that modulate intracellular tension, growth and immunomodulatory phenotype of MSCs in standard culture and during larger scale cell manufacture. Our findings demonstrate an effective route to support large-scale expansion of functional MSCs for therapeutic purposes.

Peer reviewed: True, Funder: University of Glasgow; Id: http://dx.doi.org/10.13039/501100000853, Pancreatic cancer is a deadly and highly metastatic disease, although how metastatic lesions establish is not fully understood. A key feature of pancreatic tumours is extensive fibrosis and deposition of extracellular matrix (ECM). While pancreatic cancer cells are programmed by stimuli derived from a stiff ECM, metastasis requires loss of attachment and adaptation to a softer microenvironment at distant sites. Growing evidence suggests that stiff ECM influences pancreatic cancer cell behaviour. Here, we argue that this influence is reversible and that pancreatic cancer cells can be reprogrammed upon sensing soft substrates. Using engineered polyacrylamide hydrogels with tuneable mechanical properties, we show that collagen VI is specifically upregulated in pancreatic cancer cells on soft substrates, due to a lack of integrin engagement. Furthermore, the expression of collagen VI is inversely correlated with mechanosensing and activity of YAP (also known as YAP1), which might be due to a direct or indirect effect on transcription of genes encoding collagen VI. Collagen VI supports migration in vitro and metastasis formation in vivo. Metastatic nodules formed by pancreatic cancer cells lacking Col6a1 display stromal cell-derived collagen VI deposition, suggesting that collagen VI derived from either cancer cells or the stroma is an essential component of the metastatic niche. This article has an associated First Person interview with Vasileios Papalazarou, joint first author of the paper.

Inflammation is essential for responding to infections and subsequent tissue healing. However, chronic unresolved inflammation can become a serious health problem, as exemplified in the joints during Rheumatoid Arthritis (RA). Why does inflammation persist in RA? The answer could lie with synovial fibroblasts, non-haemopoietic cells that can adopt a pathogenic phenotype that fuels disease progression for years. Critically, targeting local fibroblasts could stop joint inflammation without suppressing systemic immunity. Nevertheless, basic research findings have not been translated to new drugs, perhaps because non-physiological data can be inadvertently generated in 2D cultures. Thus, developing better in vitro platforms is an urgent need in biomedical research. In this work, we sought to understand how distinct 3D environments affect fibroblast-mediated inflammation. Arthritic synovial fibroblasts were expanded and cultured in 2D, 3D rigid scaffolds and engineered hydrogels. The results reveal that SFs are plastic and adopt inflammatory or remission-like phenotypes in response to their surroundings. This work identifies new directions to develop better models for drug testing, and even signposts candidate mechanisms by which to rewire destructive SFs.

[EN] Nanotopography presents an effective physical approach for biomaterial cell manipulation mediated through material-extracellular matrix interactions. The extracellular matrix that exists in the cellular microenvironment is crucial for guiding cell behaviours, such as determination of integrin ligation and interaction with growth factors. These interactions with the extracellular matrix regulate downstream mechanotransductive pathways, such as rearrangements in the cytoskeleton and activation of signal cascades. Protein adsorption onto nanotopography strongly influences the conformation and distribution density of extracellular matrix and, therefore, subsequent cell responses. In this review, we first discuss the interactive mechanisms of protein physical adsorption on nanotopography. Secondly, we summarise advances in creating nanotopographical features to instruct desired cell behaviours. Lastly, we focus on the cellular mechanotransductive pathways initiated by nanotopography. This review provides an overview of the current state-of-the-art designs of nanotopography aiming to provide better biomedical materials for the future., We acknowledge support from the Leverhulme Trust through gran t RPG-2019-252 and the Engineering and Physical Sciences Research Council (EPSRC) grant EP/P001114/1.

Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via allogeneic stem cell transplantation (alloSCT), to engraft and repopulate the blood system. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs (ST-HSC) and progenitor populations at the expense of naïve LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs or pericytes). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1α expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1α expressing PerSCs, LT-HSC numbers were maintained with normal clonal ability and without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders.

Nanotopography is an effective method to regulate cells' behaviors to improve Ti orthopaedic implants' in vivo performance. However, the mechanism underlying cellular matrix-nanotopography interactions that allows the modulation of cell adhesion has remained elusive. In this study, we have developed novel nanotopographic features on Ti substrates and studied human osteoblast (HOb) adhesion on nanotopographies to reveal the interactive mechanism regulating cell adhesion and spreading. Through nanoflat, nanoconvex, and nanoconcave TiO

The tumor microenvironment plays an important role in cancer development and the use of 3D in vitro systems that decouple different elements of this microenvironment is critical for the study of cancer progression. In neuroblastoma (NB), vitronectin (VN), an extracellular matrix protein, has been linked to poor prognosis and appears as a promising therapeutic target. Here, we developed hydrogels that incorporate VN into 3D polyethylene glycol (PEG) hydrogel networks to recapitulate the native NB microenvironment. The stiffness of the VN/PEG hydrogels was modulated to be comparable to the in vivo values reported for NB tissue samples. We used SK-N-BE (2) NB cells to demonstrate that PEGylated VN promotes cell adhesion as the native protein does. Furthermore, the PEGylation of VN allows its crosslinking into the hydrogel network, providing VN retention within the hydrogels that support viable cells in 3D. Confocal imaging and ELISA assays indicate that cells secrete VN also in the hydrogels and continue to reorganize their 3D environment. Overall, the 3D VN-based PEG hydrogels recapitulate the complexity of the native tumor extracellular matrix, showing that VN-cell interaction plays a key role in NB aggressiveness, and that VN could potentially be targeted in preclinical drug studies performed on the presented hydrogels.

[EN] The architectural space to which the use of spherical geometry, gives rise has a prominent symbolic charge. Proof of this is that historically it has been used in the most notable architectural compositions. However, geometric, programmatic and implementation difficulties it raises in its realization. In particular to address the challenges raised by this bet compositional, investigating the extent to which causes the identification between form and function. We highlight its strong links with one-functional programs. It shows, singularly effective since ancient times, to places of worship, entertainment in covered enclosures, and more recently the scientific popularization of astronomy. You also find advantage which has resulted in the use of new materials for better formal identification., [ES] El espacio arquitectónico al que da lugar el uso de la geometría esférica, posee una destacada carga simbólica. Prueba de ello es que a lo largo de la historia ha sido utilizada en las más notables composiciones arquitectónicas. Sin embargo, en su materialización plantea dificultades geométricas, programáticas y de ejecución. En particular se abordan los retos suscitados por esta apuesta compositiva, indagando el alcance que provoca la identificación entre forma y función. Destacamos su fuerte vinculación a programas monofuncionales. Se muestra, singularmente eficaz desde tiempos antiguos, para lugares de culto, espectáculos en recintos cubiertos, y más recientemente, la divulgación científica de la astronomía. Asimismo, se constatan las ventajas que ha supuesto el uso de nuevos materiales para una mejor identificación formal.

Systemically circulating microbubbles are used as contrast agents to aid both drug targeting and delivery using ultrasound. Exploiting their acoustic behaviour in small diameter vessels is critical for both applications, but the highly controlled experiments required to support this are not possible in vivo and challenging in vitro. Experimental platforms with small diameter channels (below 200 microns) are not readily available nor able to represent vascular geometries, leaving the existence and extent of microbubble-microvessel interactions incompletely defined. In this work we present a 3D-printed microchannel platform using tissue-mimicking hydrogels featuring radii down to 75 microns. We demonstrate application to study microbubble behaviour via acoustic backscatter under controlled environments in physiologically-relevant conditions.

Pancreatic cancer is a deadly disease with high rates of metastasis, though how tumor cells establish metastatic lesions is not fully understood. A key feature of primary pancreatic tumors is extensive fibrosis due to deposition of extracellular matrix. While pancreatic cancer cells are programmed by stimuli derived from a stiff ECM, metastasis requires loss of attachment as well as adaptation to a softer microenvironment upon reaching distant sites. Growing evidence suggests that stiff ECM influences pancreatic cancer cell behaviour. Here we argue that this influence is reversible and that pancreatic cancer cells can be reprogrammed upon sensing of soft substrates. Through use of engineered polyacrylamide hydrogels with tuneable mechanical properties, we show that Collagen-VI is specifically upregulated on soft substrates, due to a lack of integrin engagement and low YAP1 activity. Collagen-VI supports migration in vitro and metastasis formation in vivo. Metastatic nodules formed by pancreatic cancer cells lacking Col6a1 expression, were characterised by stromal cell-derived collagen-VI deposition, suggesting that collagen-VI, either cancer or stroma derived, is an essential component of the metastatic niche.Summary StatementCollagen-VI is expressed by pancreatic tumors and metastases in a mechanosensitive way to promote niche colonisation.

The Federal Highway Administration (FHWA) mandates biannual bridge inspections to assess the condition of all bridges in the United States. These inspections are recorded in the National Bridge Inventory (NBI) and the respective state’s databases to manage, study, and analyze the data. As FHWA specifications become more complex, inspections require more training and field time. Recently, element-level inspections were added, assigning a condition state to each minor element in the bridge. To address this new requirement, a machine-aided bridge inspection method was developed using artificial intelligence (AI) to assist inspectors. The proposed method focuses on the condition state assessment of cracking in reinforced concrete bridge deck elements. The deep learning-based workflow integrated with image classification and semantic segmentation methods is utilized to extract information from images and evaluate the condition state of cracks according to FHWA specifications. The new workflow uses a deep neural network to extract information required by the bridge inspection manual, enabling the determination of the condition state of cracks in the deck. The results of experimentation demonstrate the effectiveness of this workflow for this application. The method also balances the costs and risks associated with increasing levels of AI involvement, enabling inspectors to better manage their resources. This AI-based method can be implemented by asset owners, such as Departments of Transportation, to better serve communities.

It is established that the mechanical properties of hydrogels control the fate of (stem) cells. However, despite its importance, a one-to-one correspondence between gels' stiffness and cell behaviour is still missing from literature. In this work, the viscoelastic properties of Poly(ethylene-glycol) (PEG)-based hydrogels - broadly used in 3D cell cultures and whose mechanical properties can be tuned to resemble those of different biological tissues - are investigated by means of rheological measurements performed at different length scales. When compared with literature values, the outcomes of this work reveal that conventional bulk rheology measurements may overestimate the stiffness of hydrogels by up to an order of magnitude. It is demonstrated that this apparent stiffening is caused by an induced 'tensional state' of the gel network, due to the application of a compressional normal force during measurements. Moreover, it is shown that the actual stiffness of the hydrogels is instead accurately determined by means of passive-video-particle-tracking (PVPT) microrheology measurements, which are inherently performed at cells length scales and in absence of any externally applied force. These results underpin a methodology for measuring the linear viscoelastic properties of hydrogels that are representative of the mechanical constraints felt by cells in 3D hydrogel cultures.

[EN] Embryonic stem cells (ESCs) possess remarkable abilities, as they can differentiate into all cell types (pluripotency) and be self-renewing, giving rise to two identical cells. These characteristics make ESCs a powerful research tool in fundamental embryogenesis as well as candidates for use in regenerative medicine. Significant efforts have been devoted to developing protocols to control ESC fate, including soluble and complex cocktails of growth factors and small molecules seeking to activate/inhibit key signaling pathways for the maintenance of pluripotency states or activate differentiation. Here we describe a novel method for the effective maintenance of mouse ESCs, avoiding the supplementation of complex inhibitory cocktails or cytokines, e.g., LIF. We show that the addition of zinc to ESC cultures leads to a stable pluripotent state that shares biochemical, transcriptional and karyotypic features with the classical LIF treatment. We demonstrate for the first time that ESCs maintained in long-term cultures with added zinc, are capable of sustaining a stable ESCs pluripotent phenotype, as well as differentiating efficiently upon external stimulation. We show that zinc promotes long-term ESC self-renewal (>30 days) via activation of ZIP7 and AKT signaling pathways. Furthermore, the combination of zinc with LIF results in a synergistic effect that enhances LIF effects, increases AKT and STAT3 activity, promotes the expression of pluripotency regulators and avoids the expression of differentiation markers., PR acknowledges support from the Spanish Ministry of Science, Innovation and Universities (RTI2018-096794), and Fondo Europeo de Desarrollo Regional (FEDER). CIBER-BBN was an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. MS-S acknowledges support from the UK Engineering and Physical Sciences Research Council (EPSRC - EP/P001114/1).

The tissue engineering applications of coaxial electrospinning are growing due to the potential increased functionality of the fibres compared to basic electrospinning. Previous studies of core and shell scaffolds have placed the active elements in the core, however, the surface response to a biomaterial affects the subsequent behaviour, thus here hydroxyapatite (HA) was added to the shell. Coaxial electrospun polycaprolactone (PCL)-polylactic acid (PLA)/HA (core-shell) scaffolds were produced in 2D sheets using a plate collector, or 3D tubes for bone tissue engineering using a rotating needle collector. The scaffolds include high hydroxyapatite content while retaining their structural and mechanical integrity. The effect of the collector type on fibre diameter, fibre alignment and mechanical properties have been evaluated, and the impact of HA incorporation on bioactivity, BMP-2 release, cell behaviour and mechanical properties for up to 12 weeks degradation were assessed. Fibre uniformity in coaxial electrospinning depends on the relative flow rate of the core and shell solutions. Using a rotating needle collector increased fibre alignment compared to a stationary collector, without affecting fibre diameter significantly, while HA content increased fibre non-uniformity. Coaxial PCL-PLA/HA fibres exhibited significantly higher bioactivity compared to PCL-PLA scaffolds due to the surface exposure of the HA particles. Apatite formation increased with increasing SBF immersion time. Coaxial tubular scaffolds with and without HA incorporation showed gradual reductions in their mechanical properties over 12 weeks in PBS or SBF but still retained their structural integrity. Coaxial scaffolds with and without HA exhibited gradual and sustained BMP-2 release and supported MSCs proliferation and differentiation with no significant difference between the two scaffolds types. These materials therefore show potential applications as bone tissue engineering scaffolds.

This paper presents a method for identification of cell forces. We propose both a local and non-local method. The former assumes rotation free field of surface tractions, while the latter introduces an intrinsic length scale parameter, such that cells can locally apply a moment on the material surface. This paper focuses attention on a numerical aspect of force identification without investigating, at this stage, the true nature of that forces.

The clinicopathologic and immunohistochemical features of three metastasizing fibrous histiocytomas of the skin are presented. The first patient had a 1.3-cm nodule in the right thigh, with right inguinal lymph node metastases 19 years later. The second patient, who had a 3-cm nodule excised from his left thigh and inguinal lymph node metastasis after 4 months, had a favorable outcome 14 years after local radiotherapy and chemotherapy. The third had a 2-cm nodule in his neck, which recurred 16 months later. Four months later, cervical lymph node metastases were found. The patient was alive and well 26 months after initial surgery. All three primary skin tumors involved the dermis and subcutis, appeared well-delineated but nonencapsulated, were associated with some degree of epidermal hyperplasia, and showed features of aneurysmal/atypical or cellular fibrous histiocytoma. The number of mitoses ranged from 6 to 11 per 10 high-power fields. Recurrences and metastases showed morphologic features similar to primary lesions. Tumor cells were positive, at least focally, for CD 68, Ki-M1p, and Factor XIIIa, and occasionally for smooth muscle actin. Desmin, CD 34, S-100 protein, and cytokeratin stainings were negative. Primary neoplasms, recurrences, and metastases showed a Mib-1 labeling index of 10% or less. Cellular, aneurysmal, and atypical (pseudosarcomatous) fibrous histiocytomas of the skin can metastasize, yet they often show a protracted clinical course. Risk factors for metastatic dissemination include large size, high cellularity, aneurysmal changes, marked cellular pleomorphism, high mitotic activity, tumor necrosis, and repeated local recurrences.

BACKGROUND. Malignant mixed mesodermal tumors (malignant mixed Mullerian tumors [MMMT]) occur rarely in extragenital sites. METHODS. The authors analyzed the clinical, pathologic, and immunohistochemical features of three cases of primary MMMT of the female peritoneum. RESULTS. The neoplasms occurred in 60-, 64- and 84-year-old women and arose from pelvic peritoneum. Two patients died with disseminated disease 8 and 24 months postoperatively. The third died of cardiac failure 12 months postoperatively with questionable metastatic disease. Microscopically, two tumors were of the heterologous type, containing foci of rhabdomyosarcomatous (case 1) and chondrosarcomatous (case 3) differentiation. Immunohistochemically, coexpression of keratin and vimentin was observed focally in both carcinomatous and sarcomatous components in all three neoplasms, whereas coexpression of low molecular weight cytokeratin, vimentin and actin was observed focally in case 2. Rhabdomyosarcomatous areas were positive with desmin and actin, and chondrosarcomatous areas for S-100 protein. Both epithelial and mesenchymal components were positive for alpha-1 antichymotrypsin in all cases. CONCLUSIONS. On the basis of the present cases and a review of 15 reports from the literature, primary MMMT of the female peritoneum proved to be a rare but highly malignant neoplasm occurring in elderly postmenopausal women. Of 15 patients with available follow-up, 12 died with disease, mostly within 1 year, regardless of the initial tumor stage, histology (homologous versus heterologous MMMT) or treatments attempted. The tumor developed within pelvic peritoneum in half the cases. Histogenetically, peritoneal MMMT are thought to represent "metaplastic" carcinomas originating from the secondary Mullerian system

INTRODUCTION During recent years, microgels have emerged as an effective type of drug delivery system (DDS), showing advantages such as tuneable size, increased surface area and injectability1. Collagen has been extensively studied as a scaffold for regenerative medicine and drug delivery due to its biocompatibility, non-immunogenicity and degradability2,3. The current study focuses on the use of microfluidic techniques for the automated generation of monodisperse type-I collagen (col-I) microgels crosslinked with PEG-4S, encapsulating hollow collagen spheres with glial-derived neurotrophic factor (GDNF) and bone morphogenetic protein 2 (BMP-2) for regenerative therapies in Parkinson’s Disease and bone repair, respectively. MATERIALS AND METHODS Col-1 (Collagen Solutions, UK) stock was characterized in a rotational viscometer and UV-Vis spectrophotometer to determine the fibrillogenesis conditions. Collagen was crosslinked with 4S-Star-PEG succinimide glutarate (Jenkem, USA) to form hydrogels. Crosslinking occurred after merging col-I with PEG-4S crosslinker in the nozzle of a double-chamber capillary within an oil flow. A coiled tube was placed at the outlet for microgels to gelify inside the device. Microgel size was controlled varying the nozzle diameter and flow speeds, and the mechanical properties modulated through pH and concentration of collagen/crosslinker. Microgels were characterized with TNBS assay and their cytotoxicity with LIVE-DEAD® assay (Neu-7 astrocytes, n=3). Hollow spheres were prepared by covalently attaching collagen to silica templates that are later dissolved with hydrofluoric acid. They were characterized morphologically and chemically, namely: scanning electron microscopy, dynamic light scattering and Fourier-Transform Infrared (FTIR). Hollow spheres were loaded with GDNF and BMP-2 by diffusion, seeded with different cell-types and encapsulated in the microgels within the microfluidic device. RESULTS AND DISCUSSION Microgels with different size and stiffness were successfully synthetized in a glass microfluidic device. The microgels are non-cytotoxic to cells (fig.1C) and foster cell growth at different crosslinker concentrations. In the synthesis of hollow spheres, no harmful by-products appeared (fig.1D), and their size ranged around 200 nm (fig.1E). Microfluidic-generated hydrogels encapsulating hollow spheres can therefore be used for sustained delivery of GDNF and BMP-2. CONCLUSIONS We demonstrate that microfluidics is an adequate technique for automatically generating monodisperse collagen microgels and provides a simple and useful tool for the posterior encapsulation of nanospheres and cells. The combination of microgels encapsulating hollow spheres is expected to provide a controlled and sustained delivery system of different therapeutic factors. AKNOWLEDGEMENTS The authors would like to thank European Union H2020 Programme (H2020-MSCA-ITN-2015) and Grant No. 676408 for providing financial support to this project. &nbsp