Enthesitis on Chip - A Model for Studying Acute and Chronic Inflammation of the Enthesis and its Pharmacological Treatment.

Enthesitis, the inflammation of the enthesis, which is the point of attachment of tendons and ligaments to bones, is a common musculoskeletal disease. The inflammation often originates from the fibrocartilage region of the enthesis as a consequence of mechanical overuse or -load and consequently tissue damage. During enthesitis, waves of inflammatory cytokines propagate in(to) the fibrocartilage, resulting in detrimental, heterotopic bone formation. Understanding of human enthesitis and its treatment options is limited, also because of lacking in vitro model systems that can closely mimic the pathophysiology of the enthesis and can be used to develop therapies. In this study, an enthes(it)is-on-chip model is developed. On opposite sides of a porous culture membrane separating the chip's two microfluidic compartments, human mesenchymal stromal cells are selectively differentiated into tenocytes and fibrochondrocytes. By introducing an inflammatory cytokine cocktail into the fibrochondrocyte compartment, key aspects of acute and chronic enthesitis, measured as increased expression of inflammatory markers, can be recapitulated. Upon inducing chronic inflammatory conditions, hydroxyapatite deposition, enhanced osteogenic marker expression and reduced secretion of tissue-related extracellular matrix components are observed. Adding the anti-inflammatory drug celecoxib to the fibrochondrocyte compartment mitigates the inflammatory state, demonstrating the potential of the enthesitis-on-chip model for drug testing.

Uni- and Multivariate Analyses of Cancer Risk in Cytologically Indeterminate Thyroid Nodules: A Single-Center Experience.

Every year in Italy, about 60,000 new cases of nodular thyroid pathology are diagnosed, of which almost 30% are cytologically indeterminate (TIR3A/3B). The risk of malignancy reported in the literature on thyroid nodules ranges from 5% to 15% for TIR3A and from 15% to 30% for TIR3B. It is suspected that these percentages are higher in practice. We performed univariate and multivariate analyses of clinical risk factors. The medical records of 291 patients who underwent surgery for cytologically indeterminate nodular thyroid disease were retrospectively reviewed. Clinical parameters and preoperative serum markers were then compared between the benign nodular thyroid disease and thyroid cancer groups. For each patient, clinical characteristics, comorbidities, neck ultrasonographic features, and histological reports were statistically analyzed using Chi-squared and Fisher's exact tests. A total of 134 malignant neoplasms were found (46%), divided into 55 cases (35%) in the TIR3A group and 79 cases (59%) in the TIR3B group. Statistical analysis was not significant in both populations for both sex and age (TIR3A p-value = 0.5097 and p-value = 0.1430, TIR3B p-value = 0.5191 p-value = 0.3384), while it was statistically significant in patients with TIR3A nodules associated with thyroiditis (p-value = 0.0009). In addition, the patients with TIR3A and 3B nodules were stratified by ultrasound risk for the prediction of malignancy and it was significant (p = 0.0004 and p < 0.0001). In light of these results, it emerges that surgical treatment of nodular thyroid pathology with indeterminate cytology TIR3A should always be considered, and surgery for TIR3B is mandatory.

Microfluidically Aligned Collagen to Maintain the Phenotype of Tenocytes In Vitro.

Tendon is a highly organized tissue that transmits forces between muscle and bone. The architecture of the extracellular matrix of tendon, predominantly from collagen type I, is important for maintaining tenocyte phenotype and function. Therefore, in repair and regeneration of damaged and diseased tendon tissue, it is crucial to restore the aligned arrangement of the collagen type I fibers of the original matrix. To this end, a novel, user-friendly microfluidic piggyback platform is developed allowing the controlled patterned formation and alignment of collagen fibers simply on the bottom of culture dishes. Rat tenocytes cultured on the micropatterns of aligned fibrous collagen exhibit a more elongated morphology. The cells also show an increased expression of tenogenic markers at the gene and protein level compared to tenocytes cultured on tissue culture plastic or non-fibrillar collagen coatings. Moreover, using imprinted polystyrene replicas of aligned collagen fibers, this work shows that the fibrillar structure of collagen per se affects the tenocyte morphology, whereas the biochemical nature of collagen plays a prominent role in the expression of tenogenic markers. Beyond the controlled provision of aligned collagen, the microfluidic platform can aid in developing more physiologically relevant in vitro models of tendon and its regeneration.

Histomorphometric analysis of osteocyte lacunae in human and pig: exploring its potential for species discrimination.

In recent years, several studies have focused on species discrimination of bone fragments by histological analysis. According to literature, the most consistent distinguishing features are Haversian canal and Haversian system areas. Nonetheless, there is a consistent overlap between human and non-human secondary osteon dimensions. One of the features that have never been analyzed for the purpose of species discrimination is the osteocyte lacuna, a small oblong cavity in which the osteocyte is locked in. The aim of this study is to verify whether there are significant quantitative differences between human and pig lacunae within secondary osteons with similar areas. Study sample comprises the midshaft of long bones (humerus, radius, ulna, femur, tibia, and fibula) of a medieval human adult and a juvenile pig. Sixty-eight secondary osteons with similar areas have been selected for each species and a total of 1224 osteocyte lacunae have been measured. For each osteon, the total number of lacunae was counted, and the following measurements were taken: minimum and maximum diameter, area, perimeter, and circularity of nine lacunae divided between inner, intermediate, and outer lacunae. Statistical analysis showed minimal differences between human and pig in the number of lacunae per osteons and in the minimum diameter (P > 0.05). On the contrary, a significant difference (P < 0.001) has been observed in the maximum diameter, perimeter, area, and circularity. Although there is the need for further research on different species and larger sample, these results highlighted the potential for the use of osteocyte lacunae as an additional parameter for species discrimination. Concerning the difference between the dimensions of osteocyte lacunae based on their position within the osteon (inner, intermediate, and outer lacunae), results showed that their size decreases from the cement line towards the Haversian canal both in human and pig.

Development of a synthetic tissue engineered three-dimensional printed bioceramic-based bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro.

Over the last decade there have been increasing efforts to develop three-dimensional (3D) scaffolds for bone tissue engineering from bioactive ceramics with 3D printing emerging as a promising technology. The overall objective of the present study was to generate a tissue engineered synthetic bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro, thereby mimicking the advantageous properties of autogenous bone grafts and facilitating usage for reconstructing segmental discontinuity defects in vivo. To this end, 3D scaffolds were developed from a silica-containing calcium alkali orthophosphate, using, first, a replica technique - the Schwartzwalder-Somers method - and, second, 3D printing, (i.e. rapid prototyping). The mechanical and physical scaffold properties and their potential to facilitate homogenous colonization by osteogenic cells and extracellular bone matrix formation throughout the porous scaffold architecture were examined. Osteoblastic cells were dynamically cultured for 7 days on both scaffold types with two different concentrations of 1.5 and 3 × 109 cells/l. The amount of cells and bone matrix formed and osteogenic marker expression were evaluated using hard tissue histology, immunohistochemical and histomorphometric analysis. 3D-printed scaffolds (RPS) exhibited more micropores, greater compressive strength and silica release. RPS seeded with 3 × 109 cells/l displayed greatest cell and extracellular matrix formation, mineralization and osteocalcin expression. In conclusion, RPS displayed superior mechanical and biological properties and facilitated generating a tissue engineered synthetic bone graft in vitro, which mimics the advantageous properties of autogenous bone grafts, by containing homogenously distributed terminally differentiated osteoblasts and mineralizing bone matrix and therefore is suitable for subsequent in vivo implantation for regenerating segmental discontinuity bone defects. Copyright © 2016 John Wiley & Sons, Ltd.

Feasibility, efficacy and safety of stent insertion as a palliative treatment for malignant strictures in the cervical segment of the esophagus and the hypopharynx.

BACKGROUND: 50% of esophageal cancers are inoperable at the time of diagnosis, and around 15% involve the cervical esophagus. The hypopharynx is often involved by these malignancies as well. Palliation of cervical esophageal malignancies through stent insertion is considered limited due to technical challenges, poor patient tolerance and high complication rate. The aim of this study is to review our experience with stent insertion in the cervical segment of the esophagus and to evaluate outcome differences between stent insertions involving or sparing the hypopharynx. METHODS: We retrospectively reviewed data on 69 consecutive patients that underwent stent insertion for malignant strictures in the cervical esophagus at our Department. Patients were divided according to involvement or sparing of the lower hypopharynx. Dysphagia severity was measured with the Mellow-Pinkas scale before the procedure and on monthly follow-ups. Any complication and its management were recorded. The main outcome parameters were as follows: dysphagia improvement, rate of successful dysphagia palliation (i.e., a reduction of the score to 0 or 1 after stent insertion) and complication rate. Multivariable analysis was carried out to assess the influence of patient- and procedure-related factors on the outcome of the procedure. RESULTS: Stent insertion was achieved in 100% patients. At 4 weeks, dysphagia score improved from a median of 3-0 (p < 0.001), and a successful palliation was achieved in 76.8% patients. The 30-day mortality rate was 14.5%. Successful palliation throughout the follow-up was achieved in 72.9% of the surviving patients. Complications occurred in 31.9% patients. Dilation before stent insertion was associated with a less efficient short-term dysphagia palliation (OR 6.77, 95% CI 1.46-31.29, p = 0.02). CONCLUSIONS: Stent insertion is a safe and effective palliative treatment for malignant cervical esophageal strictures. Results are consistent even in patients with hypopharyngeal lesions. Dilation should be avoided before stent insertion.