Cerebral venous sinus thrombosis in a patient treated with ribociclib for metastatic breast cancer. Case reports and literature review.

Breast cancer is the most diagnosed cancer among women worldwide. Cyclin dependent kinase 4/6 inhibitors (ribociclib, palbociclib, and abemaciclib) modulate endocrine resistance and are widely used treatment for patients with advanced-stage hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer. Reports of both venous and arterial thromboembolic events, as a complication of cyclin dependent kinase 4/6 inhibitors, are increasingly recognized, but none involved cerebral venous sinus. We herein report on a 44-year-old female patient who initially presented with an early-stage breast cancer treated with surgery, chemotherapy, radiation therapy and finished 5 years of tamoxifen uneventfully. Eight years after her initial diagnosis, she relapsed with a solitary brain lesion which was resected and treated with radiation therapy, and was then started on aromatase inhibitors. Few months later, she progressed with biopsy-proven cervical and mediastinal lymph node metastasis. She was then switched to fulvestrant and ribociclib; both were well-tolerated. However, few weeks later she presented with increasing headache and mild dizziness. Imaging studies showed right lateral sinus acute non-occlusive thrombosis with no parenchymal changes. Patient was anticoagulated with low molecular weight heparin and follow-up visits showed stable disease with no bleeding.

Wurster Technology-Assisted Step-by-Step Engineering of Multi-layered Pellets (Sprinkles): Microscopy, Micro-CT, and e-Tongue-Based Analysis.

The advancement in the formulation and characterization techniques have paved the path for development of new as well as modification of existing dosage forms. The present work explores the role of micro-computed tomography (micro-CT) as advanced characterization technique for multi-layered-coated pellets to ascertain the quality of coated pellets. The work further explored in-house e-tongue technique for understanding palatability of formulation in early stages of development thus by reducing clinical taste evaluation time. The developed multi-layered-coated pellets were characterized using microscopy (optical and electron microscopy). The obtained results demonstrated formation of spherical-shaped pellets with uniform coating. The uniform coating was further confirmed by results obtained from scanning electron microscopy (SEM) and cross-sectional SEM analysis, which showed visible difference in pellet surface before and after multi-layered coating. The micro-CT results confirmed the visible demarcation of layers (drug and polymer, i.e., hydroxypropyl methylcellulose (HPMC) and eudragit (EPO)) along with uniform thickness of various layering. The dissolution study of developed pellets suggested the role of layering EPO on drug release from pellets. The e-tongue analysis proved to be an excellent tool for early prediction of taste masking of drug via multi-layered pellets and can serve as potential platform for taste masking with high specificity. The overall results suggest the suitability of developed multi-layered platform as efficient dosage form (sprinkle) in pediatric/geriatric product development.

Development of immediate release 3D-printed dosage forms for a poorly water-soluble drug by fused deposition modeling: Study of morphology, solid state and dissolution.

3D-printing technologies such as Fused Deposition Modeling (FDM) bring a unique opportunity for personalized and flexible near-patient production of pharmaceuticals, potentially improving safety and efficacy for some medications. However, FDM-printed tablets often exhibit tendency for slow dissolution due to polymer erosion-based dissolution mechanisms. Development of immediate release (IR) 3D-printed dosage with poorly water-soluble compounds is even more challenging but necessary to ensure wide applicability of the technology within pharmaceutical development portfolios. In this work, process and morphology were considered to achieve IR of BCS class IV compound lumefantrine as model active pharmaceutical ingredient (API) using basic butylated methacrylate copolymer (Eudragit EPO) as matrix former, as well as hydrophilic plasticizer xylitol and pore former maltodextrin. Grid-designed tablets with size acceptable for children from 6 years old and varying programmed infill density were successfully 3D-printed with 5% lumefantrine while higher drug load led to increased brittleness which is incompatible with 3D-printing. Lumefantrine assay was 92 to 97.5% of theoretical content depending on drug load and process parameters. 3D-printed tablets with 65% infill density met rapid release criteria, while 80% and 100% showed slower dissolution. Structural characteristics of 3D-printed tablets with non-continuous surface such as accessible porosity and specific surface area by weight and by volume were quantified by a non-destructive automated µCT-based methodology and were found to correlate with dissolution rate. Increase in accessible porosity, total surface area, specific surface area and decrease in relative density were statistically significant critical factors for modification of lumefantrine dissolution rate. Crystallinity in manufactured tablets and filaments was explored by highly sensitive Raman mapping technique. Lumefantrine was present in the fully amorphous state in the tablets exhibiting adequate stability for on-site manufacturing. The study demonstrates feasibility of immediate release FDM-3D-printed tablets with BCS class IV API and illustrates the correlation of FDM design parameters with morphological and dissolution characteristics of manufactured tablets.

Tracking immune-related cell responses to drug delivery microparticles in 3D dense collagen matrix.

Beyond the therapeutic purpose, the impact of drug delivery microparticles on the local tissue and inflammatory responses remains to be further elucidated specifically for reactions mediated by the host immune cells. Such immediate and prolonged reactions may adversely influence the release efficacy and intended therapeutic pathway. The lack of suitable in vitro platforms limits our ability to gain insight into the nature of immune responses at a single cell level. In order to establish an in vitro 3D system mimicking the connective host tissue counterpart, we utilized reproducible, compressed, rat-tail collagen polymerized matrices. THP1 cells (human acute monocytic leukaemia cells) differentiated into macrophage-like cells were chosen as cell model and their functionality was retained in the dense rat-tail collagen matrix. Placebo microparticles were later combined in the immune cell seeded system during collagen polymerization and secreted pro-inflammatory factors: TNFα and IL-8 were used as immune response readout (ELISA). Our data showed an elevated TNFα and IL-8 secretion by macrophage THP1 cells indicating that Placebo microparticles trigger certain immune cell responses under 3D in vivo like conditions. Furthermore, we have shown that the system is sensitive to measure the differences in THP1 macrophage pro-inflammatory responses to Active Pharmaceutical Ingredient (API) microparticles with different API release kinetics. We have successfully developed a tissue-like, advanced, in vitro system enabling selective "readouts" of specific responses of immune-related cells. Such system may provide the basis of an advanced toolbox enabling systemic evaluation and prediction of in vivo microparticle reactions on human immune-related cells.

Addition of nanoscaled bioinspired surface features: A revolution for bone related implants and scaffolds?

Our expanding ability to handle the "literally invisible" building blocks of our world has started to provoke a seismic shift on the technology, environment and health sectors of our society. During the last two decades, it has become increasingly evident that the "nano-sized" subunits composing many materials­living, natural and synthetic­are becoming more and more accessible for predefined manipulations at the nanosize scale. The use of equally nanoscale sized or functionalised tools may, therefore, grant us unprecedented prospects to achieve many therapeutic aims. In the past decade it became clear that nano-scale surface topography significantly influences cell behaviour and may, potentially, be utilised as a powerful tool to enhance the bioactivity and/ or integration of implanted devices. In this review, we briefly outline the state of the art and some of the current approaches and concepts for the future utilisation of nanotechnology to create biomimetic implantable medical devices and scaffolds for in vivo and in vitro tissue engineering,with a focus on bone. Based on current knowledge it must be concluded that not the materials and surfaces themselves but the systematic biological evaluation of these new material concepts represent the bottleneck for new biomedical product development based on nanotechnological principles.

Evaluation of early stage human bone marrow stromal proliferation, cell migration and osteogenic differentiation on µ-MIM structured stainless steel surfaces.

It is well established that surface topography greatly affect cell-surface interactions. In a recent study we showed that microstructured stainless steel surfaces characterized by the presence of defined hexagonally arranged hemisphere-like structures significantly affected cell architecture (shape and focal adhesion size) of primary human bone mesenchymal stromal cells. This study aimed at further investigating the influence these microstructures (microcline protruding hemispheres) on critical aspects of cell behaviour namely; proliferation, migration and osteogenic differentiation. As with previously reported data, we used primary human bone mesenchymal stromal cells to investigate such effects at an early stage in vitro. Cells of different patients were utilised for cell migration studies. Our data showed that an increase in cell proliferation was exhibited as a function of surface topography (hemispheres). Cell migration velocity also varied as a function of surface topography on patient specific basis and seems to relate to the differentiated state of the seeded cell population (as demonstrated by bALP positivity). Osteogenic differentiation, however, did not exhibit significant variations (both up and down-regulation) as a function of both surface topography and time in culture.

In vitro bioactivity of micro metal injection moulded stainless steel with defined surface features.

Micrometre- and nanometre-scale surface structuring with ordered topography features may dramatically enhance orthopaedic implant integration. In this study we utilised a previously optimised micron metal injection moulding (µ-MIM) process to produce medical grade stainless steel surfaces bearing micrometre scale, protruding, hemispheres of controlled dimensions and spatial distribution. Additionally, the structured surfaces were characterised by the presence of submicrometre surface roughness resulting from metal grain boundary formation. Following cytocompatibility (cytotoxicity) evaluation using 3T3 mouse fibroblast cell line, the effect on primary human cell functionality was assessed focusing on cell attachment, shape and cytoskeleton conformation. In this respect, and by day 7 in culture, significant increase in focal adhesion size was associated with the microstructured surfaces compared to the planar control. The morphological conformation of the seeded cells, as revealed by fluorescence cytoskeleton labelling, also appeared to be guided in the vertical dimension between the hemisphere bodies. Quantitative evaluation of this guidance took place using live cytoplasm fluorescence labelling and image morphometry analysis utilising both, compactness and elongation shape descriptors. Significant increase in cell compactness was associated with the hemisphere arrays indicating collective increase in focused cell attachment to the hemisphere bodies across the entire cell population. Micrometre-scale hemisphere array patterns have therefore influenced cell attachment and conformation. Such influence may potentially aid in enhancing key cellular events such as, for example, neo-osteogenesis on implanted orthopaedic surfaces.

Novel in vitro co-culture methodology to investigate heterotypic cell-cell interactions.

Cell-cell interactions are of crucial importance for the formation of tissues, homeostasis and regeneration processes as well as reactions on foreign bodies including implants. So far, however, the importance of heterotypic cell-cell interactions in the in vitro evaluation of implant surfaces has been largely neglected. This work aims to develop an in vitro methodology that enables the in-depth investigation of heterotypic cell-cell interactions in a mixed co-culture system, and to validate it with a primary adult human bone-derived osteoblast cells (HBCs) - abdominal fibroblasts (HAFs) system. The methodology proposed combines a simple live labelling step, semiautomated fluorescence image acquisition and analysis to characterize the interactions between different cell types (cell population dynamics) in co-culture in terms of cell proliferation and cell spatial distribution of each cell type. In this co-culture system, direct cell-cell contacts between the two cell types were permitted while the determination of cell-type specific responses could still be elucidated. We could show that HAF proliferation was reduced in a way negatively correlated with the seeding HBC/HAF ratio, i.e., a high proportion of HBC in the co-culture had an inhibitory effect on HAF proliferation. In all cultures segregation was found after 4 and 7 days of co-culture. HBCs were segregated at low ratios while HAFs were segregated at high ratios. Cell-cell distances depended on the total cell number in the co-culture but the dependence was different for each cell type.

Iron-phosphate glass fiber scaffolds for the hard-soft interface regeneration: the effect of fiber diameter and flow culture condition on cell survival and differentiation.

This work investigated the further development of a well-characterized, contiguous, glass fiber system for regeneration of the hard-soft tissue interface. We evaluated the effect of fiber diameter on human osteoblasts and fibroblasts attachment and viability using ternary glass fibers of the composition 0.48 CaO-0.02 Na(2)O-0.50 P(2)O(5). Fiber diameter significantly influenced cell attachment and survival, with fibers drawn at 800 revolutions per minute found to be optimal. Using a known composition of iron-phosphate glass fibers (composition 0.46 CaO-0.01 Na(2)O-0.03 Fe(2)O(3)-0.50 P(2)O(5)), scaffolds were produced. These scaffolds were incorporated within an open laminar flow culture system to provide nutrients, oxygen, and waste perfusion throughout the culture. The design of the chamber ensured that laminar flow was present, and changes in the differentiation of both osteoblast and fibroblast seeded scaffolds were assessed using quantitative polymerase chain reaction. Our data show that osteoblast and fibroblast differentiation is unaffected or enhanced by laminar flow when compared with static culture conditions. This system can therefore be adapted to construct larger, more complex, three-dimensional iron-phosphate fiber scaffolds for tissue engineering.

Effect of cell density on osteoblastic differentiation and matrix degradation of biomimetic dense collagen scaffolds.

Plastic compression of hyperhydrated collagen gels produces tissue-like scaffolds of enhanced biomechanical properties. By increasing collagen density, these scaffolds could be developed into highly Biomimetic cell-seeded templates. When utilizing three-dimensional (3-D) scaffold systems for tissue repair, and indeed when investigating the cytocompatibility of two-dimensional (2-D) surfaces, the cell seeding density is often overlooked. In this study, we investigated this potentially critical parameter using MG-63 cells seeded in the dense collagen scaffolds. This is conducted within the overall scope of developing these scaffolds for bone repair. Cell proliferation, osteoblastic differentiation, and matrix remodelling capacity in relation to various seeding densities, ranging from 10(5) to 10(8) cells/ml compressed collagen, were evaluated in vitro. This was performed using the AlamarBlue assay, quantitative polymerase chain reaction (qPCR), and tensile mechanical analysis respectively. Variations in cell seeding density significantly influenced cell proliferation where lower initial seeding density resulted in higher proliferation rates as a function of time in culture. Gene transcription levels for alkaline phosphatase (ALPL), runt-related transcription factor 2 (RUNX2), and osteonectin (SPARC) were also found to be dependent on the cell density. While ALPL transcription was down-regulated with culturing time for all seeding densities, there was an increase in RUNX2 and SPARC transcription, particularly for scaffolds with cell densities in the range 10(6)-10(7) cells/ml collagen. Furthermore, this range of seeding density affected cell capacity in conducting collagenous matrix degradation as established by analyzing matrix metalloproteinase 1 (MMP1) transcription and scaffold mechanical properties. This study has shown that the seeded cell population in the three-dimensional dense collagen scaffolds clearly affected the degree of osteoblastic cell proliferation, differentiation, and some aspects of matrix remodelling activity. The seeding density played a major role in influencing the corresponding cell differentiation and cell-matrix interaction.

In vitro bioactivity and gene expression by cells cultured on titanium dioxide doped phosphate-based glasses.

In our previous study, glasses with 50 P(2)O(5)-(20-15) Na(2)O-30 CaO-(0-5 mol%) TiO(2) have been prepared by the conventional melt-quenching process. MG63 cell proliferation, gene expression, in vivo biocompatibility, and bioactivity of these glasses is the concern of this study. The results showed that addition of TiO(2) in small amounts up to 5 mol% enhanced the biocompatibility of these glasses. The cell metabolic activity was conspicuous, on 3 and 5 mol% TiO(2) compositions in particular, with no significant difference from Thermanox control over a period of 21 days. The findings from the gene expression study showed that, at day 1 and on 5 mol% TiO(2) glass, core binding protein factor alpha 1 (Cbfa1) and alkaline phosphatase (ALP) showed significantly lower transcription level; however, collagen type I alpha subunit I (COLIAI) and Osteonectin (Sparc) showed no significant differences compared to the control. At day 7, all these genes transcription levels were not significantly different form the control, but at day 14, they were significantly higher than the control. Moreover, there were no significant differences detected in these genes on both 3 and 5 mol% TiO(2) glasses up to 7 days. At day 14; however, 5 mol% TiO(2) glasses showed significantly higher level than 3 mol% TiO(2) composition. This was also correlated by the presence of new bone tissue at the bone-particles interface for 5 mol% TiO(2) composition after 5 weeks of implantation in rat calvarium. Regardless of this favourable cell response and gene up-regulation, these glasses showed no evidence of apatite layer formation after 14 days incubation in SBF.

Effect of multiple unconfined compression on cellular dense collagen scaffolds for bone tissue engineering.

Plastic compression of hydrated collagen gels rapidly produces biomimetic scaffolds of improved mechanical properties. These scaffolds can potentially be utilised as cell seeded systems for bone tissue engineering. This work investigated the influence of multiple unconfined compression on the biocompatibility and mechanical properties of such systems. Single and double compressed dense collagen matrices were produced and characterised for protein dry weight, morphology and mechanical strength. Compression related maintenance of the seeded HOS TE85 cell line viability in relation to the extent of compression was evaluated up to 10 days in culture using the TUNEL assay. Fluorescence Live/Dead assay was conducted to examine overall cell survival and morphology. Cell induced structural changes in the dense collagenous scaffolds were assessed by routine histology. The mechanical properties of the cellular scaffolds were also evaluated as a function of time in culture. It is clear that a single plastic compression step produced dense collagenous scaffolds capable of maintaining considerable cell viability and function as signs of matrix remodeling, and maintenance of mechanical properties were evident. Such scaffolds should therefore be further developed as systems for bone tissue regeneration.

Soluble phosphate glasses: in vitro studies using human cells of hard and soft tissue origin.

This report describes the short-term response of two typical cellular components of a hard/soft tissue interface such as the periodontal ligament/mandible and patellar tendon/tibia. Tissue engineering of such interfaces requires a contiguous scaffold system with at least two cell types associated with it. Human oral osteoblasts, oral fibroblasts and hand flexor tendon fibroblasts were seeded on phosphate-based glasses of different dissolution rates. Quantitative and morphological assessment of cell adhesion and proliferation for all cell types was assessed, after first elucidating an experimental composition range using MG63 cells. In addition, immunolabelling of bone-specific non-collagenous proteins bone sialoprotein, osteonectin and osteopontin was performed to determine osteoblast phenotype. Fibroblast phenotype was established by immunolabelling for prolyl-4-hydroxylase, an enzyme vital for collagen biosynthesis. Results indicated that both cell types maintained their respective phenotypes over time in culture on glass discs of generic composition (CaO)x-(Na2O)(0.5-x)-(P2O5)0.5, remained attached and proliferated dependent on glass composition and cell type. Glasses containing at least 46 mol% CaO, produced no adverse cell reaction suggesting that these compositions that support both osteoblasts and fibroblasts would be ideal as a scaffold material for engineering the hard/soft tissue interface.