Incorporation of resident macrophages in engineered tissues: multiple cell type response to microenvironment controlled macrophage-laden gelatin hydrogels

The success of tissue engineering strategy is strongly related to the inflammatory response, mainly through the activity of macrophages that are key cells in initial immune response to implants. For engineered tissues, the presence of resident macrophages can be beneficial for maintenance of homeostasis and healing. Thus, incorporation of macrophages in engineered tissues can facilitate the integration upon implantation. In this study, we developed an in-vitro model of interaction between encapsulated naive monocytes, macrophages induced with M1/M2 stimulation and incoming cells for immune assisted tissue engineering applications. To mimic the wound healing cascade, Naive THP-1 monocytes, endothelial cells, and fibroblasts were seeded on the gels as incoming cells. The interaction was first monitored in the absence of the gels. In order to mimic resident macrophages, THP-1 cells were encapsulated in the presence or absence of IL-4 to control their phenotype and then these hydrogels were seeded with incoming cells. Without encapsulation, activated macrophages induce apoptosis in endothelial cells. Once encapsulated no adverse effects were seen. Macrophage-laden hydrogels attracted more endothelial cells and fibroblasts compared to monocytes-laden hydrogels. The induction (M2 stimulation) of encapsulated macrophages did not change the overall number of attracted cells; but significantly affected their morphology. M1 stimulation by a defined media resulted in secretion of both pro and anti-inflammatory cytokines compared to M2 stimulation. We demonstrated that there is a distinct effect of encapsulated macrophages on the behavior of the incoming cells; this effect can be harnessed to establish a microenvironment more prone to regeneration upon implantation

Utilisation d’hydrogels fonctionnalisés pour une ré-épithélialisation rapide des implants hybrides en ingénierie tissulaire

As part of the development of an artificial larynx, in vivo experiments and clinical trials have revealed a defect in re-epithelialization of the endoluminal side of the prosthesis. This respiratory epithelium is absolutely necessary to obtain an implantable device fully integrated into the body but also for the functionality of such an implant. In this work we have developed patches of interpenetrated and reticulated hydrogels based on collagen and hyaluronic acid to ensure rapid epithelial regrowth. These optimized hydrogel patches have sufficient resistance to hydrolysis to limit their early degradation once implanted. They have been functionalized by growth and cell differentiation factors that are released gradually with an objectified result on cell proliferation. Encapsulation of immune cells and the use of cytokines in these gels also modulate the inflammatory response towards a healing process rather than rejection.Dans le cadre du développement d’un larynx artificiel, les expérimentations sur l’animal et les essais cliniques ont mis en évidence un défaut de ré-épithélialisation de la face endoluminale de la prothèse. Cet épithélium respiratoire est absolument nécessaire pour obtenir un dispositif implantable totalement intégré dans le corps mais également pour la fonctionnalité d’un tel implant. Dans ce travail nous avons développé de nouveaux films d’hydrogels de collagène et d’acide hyaluronique interpénétrés et réticulés pour assurer une repousse épithéliale rapide. Ces films d’hydrogels optimisés ont une résistance suffisante à l’hydrolyse pour limiter leur dégradation précoce une fois implantés. Ils ont été fonctionnalisés par des facteurs de croissance et de différenciation cellulaire libérés de façon progressive avec un résultat objectivé sur la prolifération cellulaire. L’encapsulation de cellules immunitaire et l’utilisation de cytokines dans ces gels permettent également de moduler la réponse inflammatoire vers un processus de cicatrisation plutôt que de rejet

Dysphagia 27 years after cervical disc arthroplasty

Introduction: Dysphagia is a frequent postoperative symptom after anterior cervical disc arthroplasty. However, onset of dysphagia and neck pain a long time after surgery should suggest a diagnosis of prosthesis dislocation. Care report: A 65-year-old man with a history of cervical disc arthroplasty 27 years previously consulted for rapidly progressive dysphagia with no other associated symptoms. Physical examination and CT scan confirmed the diagnosis of anterior dislocation of the prosthesis with no signs of perforation. Surgical extraction via a neck incision allowed resolution of the symptoms. Discussion: Prosthesis dislocation should be considered in a patient with a history of cervical disc arthroplasty presenting with dysphagia and neck pain. The clinical and radiological assessment confirmed the diagnosis and early surgical management allowed resolution of the symptoms and avoided complications such as pharyngo-oesophageal perforation

Hypopharyngeal reconstruction using a circular stapler

Distal anastomosis by tubed free flap is one of the main technical difficulties encountered during hypopharyngeal reconstruction. Although high flap survival probability can be achieved by experienced surgical teams, two complications are commonly observed at the flap-oesophagus junction: fistula and stenosis. Use of a circular stapler reduced the frequency of these complications by ensuring a perfectly circular and resistant suture line. Salivary stent placement is therefore unnecessary, allowing earlier resumption of feeding. The stapling procedure is simple, but a few technical skills are required, as the stapler is not specifically designed for this purpose. We describe the indications, surgical procedure and global results based on our series. We consider the forearm flap to be the gold standard for this reconstruction, but thicker flaps, such as pectoralis major flap, can also be used, but with poorer results in terms of healing and swallowing performance. Keywords: Anastomosis; Circular stapler; Flap; Hypopharynx

Postoperative Correlation of Radiological and Surgical Findings in Management of Ethmoid Sinus Adenocarcinoma

Aims:Prognosis of ethmoid sinus adenocarcinoma (ADK) is essentially determined by local tumor control. There is a high rate of recurrence of these tumors across the range of patient series. Development of an optimal follow-up protocol of such tumors is recommended.Patients and methods:A retrospective, monocentric study was carried out including all patients diagnosed with ADK who underwent surgery and were followed up at our center between 2012 and 2016 and who were monitored postoperatively using magnetic resonance imaging (MRI) and histopathological verification of suspicious areas identified via imaging. Time to postoperative MRI, time to recurrence and sites of recurrence were obtained for each patient.Objectives:Performance evaluation of MRI in early screening of recurrence or residualtumors postoperatively in the management of ADK and identification of the main sites prone to risk of recurrence in these tumors.Results:We included 24 cases of ADK, there were 33% cases of recurrence with a mean time to recurrence of 35 months postoperatively. Mean time to completion of the first MRI scan was 65 days postoperatively.Performance parameters of screening for recurrence or residual tumors on the first postoperative MRI were:Se 64%; Sp 78%; PPV 69%; NPV 74%.Conclusions: Efficacy of postoperative MRI screening appears to be limited and regular endoscopic monitoring associated with imaging is required. Sites prone to risk should be subject to particular consideration in primary surgical resection and management of recurrence.The benefit of imaging in the immediate postoperative period has yet to be assessed in terms of disease-free survival and disease control

Double thin film-based sandwich-cell carrier design for multicellular tissue engineering

Many organs are multicellular and each cell type requires a different microenvironment. Thus, there is a need for modular structures where the microenvironment of each cell type can be tuned separately. Herein, we describe enzymatically crosslinked gelatin based double layered film structures where each layer can be loaded with growth factors separately. As a model, we have developed a bi-layer system to produce a respiratory epithelium. This system constitutes an in vitro “epithelial patch” that can be adhered to the lumen of any implant. Crosslinking of the patches with transglutaminase resulted in 7days of stability at 37°C. The film layer was first used to release growth factors and it was shown that the release significantly improved the proliferation over 5days. A549 human lung epithelial cells were used and under the release of an epithelial growth supplement mix, there was a significant improvement on the epithelial proliferation (p<0.01). The designed substrate was successfully attached to titanium implants and we demonstrated the stability of the epithelial patch under in vitro conditions for 7days without deterioration. Under co-culture conditions for three days both cell types were alive. Such patches can be used to obtain fast epithelialization of large implant surfaces

A composite Gelatin/hyaluronic acid hydrogel as an ECM mimic for developing mesenchymal stem cell‐derived epithelial tissue patches

Here we report fabrication of Gelatin-based biocomposite films and their application in developing epithelial patches. The films were loaded with an epithelial cell growth factor cocktail and used as an extracellular matrix mimic for in vitro regeneration of organized respiratory epithelium using Calu-3 cell line and mesenchymal stem cells (MSCs). Our data show differentiation of Calu-3 cells on composite films as evidenced by tight junction protein expression and barrier formation. The films also supported attachment, migration, and proliferation of alveolar basal epithelial cell line A549. We also show the suitability of the composite films as a biomimetic scaffold and growth factor delivery platform for differentiation of human MSCs to epithelial cells. MSCs differentiation to the epithelial lineage was confirmed by staining for epithelial and stem cell specific markers. Our data show that the MSCs acquire the epithelial characteristics after 2 weeks with significant reduction in vimentin, increase in pan cytokeratin expression, and morphological changes. However, despite the expression of epithelial lineage markers, these cells did not form fully functional tight junctions as evidenced by low expression of junctional protein ZO1. Further optimisation of culture conditions and growth factor cocktail is required to enhance tight junction formation in MSCs-derived epithelial cells on the composite hydrogels. Nevertheless, our data clearly highlight the possibility of using MSCs in epithelial tissue engineering and the applicability of the composite hydrogels as transferrable extracellular matrix mimics and delivery platforms with potential applications in regenerative medicine and in vitro modelling of barrier tissues