Characterization of the Protein Corona of Three Chairside Hemoderivatives on Melt Electrowritten Polycaprolactone Scaffolds.

In tissue engineering, the relationship between a biomaterial surface and the host's immune response during wound healing is crucial for tissue regeneration. Despite hemoderivative functionalization of biomaterials becoming a common tissue-engineering strategy for enhanced regeneration, the characteristics of the protein-biomaterial interface have not been fully elucidated. This study characterized the interface formed by the adsorbed proteins from various hemoderivatives with pristine and calcium phosphate (CaP)-coated polycaprolactone (PCL) melt electrowritten scaffolds. PCL scaffolds were fabricated by using melt electrospinning writing (MEW). Three hemoderivatives (pure platelet-rich plasma (P-PRP), leucocyte platelet-rich plasma (L-PRP) and injectable platelet-rich fibrin (i-PRF)) and total blood PLASMA (control) were prepared from ovine blood. Hemoderivatives were characterized via SEM/EDX, cross-linking assay, weight loss, pH and protein quantification. The interface between PCL/CaP and hemoderivative was examined via FTIR, XPS and electrophoresis. i-PRF/PCL-CaP (1653 cm-1), PLASMA/PCL-CaP (1652 cm-1) and i-PRF/PCL (1651 cm-1) demonstrated a strong signal at the Amide I region. PLASMA and i-PRF presented similar N1s spectra, with most of the nitrogen involved in N-C=O bonds (≈400 eV). i-PRF resulted in higher adsorption of low molecular weight (LMW) proteins at 60 min, while PLASMA exhibited the lowest adsorption. L-PRP and P-PRP had a similar pattern of protein adsorption. The characteristics of biomaterial interfaces can be customized, thus creating a specific hemoderivative-defined layer on the PCL surface. i-PRF demonstrated a predominant adsorption of LMW proteins. Further investigation of hemoderivative functionalized biomaterials is required to identify the differential protein corona composition, and the resultant immune response and regenerative capacity.

Resorbable additively manufactured scaffold imparts dimensional stability to extraskeletally regenerated bone.

Dimensionally stable vertical bone regeneration outside of the existing bony envelope is a major challenge in the field of orofacial surgery. In this study, we demonstrate that a highly porous, resorbable scaffold fabricated using additive manufacturing techniques enables reproducible extra-skeletal bone formation and prevents bone resorption. An additively manufactured medical grade polycaprolactone (mPCL) biphasic scaffold mimicking the architecture of the jaw bone, consisting of a 3D-printed outer shell overlying an inner highly porous melt electrowritten scaffold, was assessed for its ability to support dimensionally stable bone regeneration in an extraskeletal ovine calvarial model. To investigate bone formation capacity (stage 1), 7 different constructs placed under a protective dome were assessed 8 weeks post-implantation: Empty control, Biphasic scaffold with hydrogel (PCL-Gel), PCL-Gel with 75 or 150 µg of BMP-2 (PCL-BMP-75 and PCL-BMP-150), hydrogel only (Gel), Gel containing 75 or 150 µg of BMP-2 (Gel-BMP-75 and Gel-BMP-150). To assess dimensional stability (stage 2), in a separate cohort, 5 animals were similarly implanted with 2 samples of each of the Gel-BMP-150 and PCL-BMP-150 groups, and after 8 weeks of healing, the protective domes were removed and titanium implants were placed in the regenerated bone and allowed to heal for a further 8 weeks. Bone formation and osseointegration were assessed using micro-computed tomography, histology and histomorphometry. In stage 1, enhanced bone formation was found in the BMP-2 containing groups, especially the PCL-BMP constructs whereby regeneration of full bone height was achieved in a reproducible manner. There was no significant bone volume increase with the higher dose of BMP-2. In the dimensional stability assessment (stage 2), after the rtemoval of the protective dome, the biphasic scaffold prevented bone resorption whereas in the absence of the scaffold, the bone previously formed in the hydrogel underwent extensive resorption. This was attributed to the space maintenance properties and dimensional stability of the biphasic scaffold. Titanium implants osseointegrated into the newly formed bone within the biphasic scaffolds. In conclusion, additively manufactured biphasic scaffolds functionalized with BMP-2 facilitated dimensionally stable bone regeneration that supported dental implant osseointegration.

An overview of decellularisation techniques of native tissues and tissue engineered products for bone, ligament and tendon regeneration.

Decellularised tissues and organs have been successfully used in a variety of tissue engineering/regenerative medicine applications. Because of the complexity of each tissue (size, porosity, extracellular matrix (ECM) composition etc.), there is no standardised protocol and the decellularisation methods vary widely, thus leading to heterogeneous outcomes. Physical, chemical, and enzymatic methods have been developed and optimised for each specific application and this review describes the most common strategies utilised to achieve decellularisation of soft and hard tissues. While removal of the DNA is the primary goal of decellularisation, it is generally achieved at the expense of ECM preservation due to the harsh chemical or enzymatic processing conditions. As denaturation of the native ECM has been associated with undesired host responses, decellularisation conditions aimed at effectively achieving simultaneous DNA removal and minimal ECM damage will be highlighted. Additionally, the utilisation of decellularised matrices in regenerative medicine is explored, as are the most recent strategies implemented to circumvent challenges in this field. In summary, this review focusses on the latest advancements and future perspectives in the utilisation of natural ECM for the decoration of synthetic porous scaffolds.

Periodontal Tissue Engineering with a Multiphasic Construct and Cell Sheets.

This study reports on scaffold-based periodontal tissue engineering in a large preclinical animal model. A biphasic scaffold consisting of bone and periodontal ligament compartments manufactured by melt and solution electrospinning, respectively, was used for the delivery of in vitro matured cell sheets from 3 sources: gingival cells (GCs), bone marrow-derived mesenchymal stromal cells (Bm-MSCs), and periodontal ligament cells (PDLCs). The construct featured a 3-dimensional fibrous bone compartment with macroscopic pore size, while the periodontal compartment consisted of a flexible porous membrane for cell sheet delivery. The regenerative performance of the constructs was radiographically and histologically assessed in surgically created periodontal defects in sheep following 5 and 10 wk of healing. Histologic observation demonstrated that the constructs maintained their shape and volume throughout the entirety of the in vivo study and were well integrated with the surrounding tissue. There was also excellent tissue integration between the bone and periodontal ligament compartments as well as the tooth root interface, enabling the attachment of periodontal ligament fibers into newly formed cementum and bone. Bone coverage along the root surface increased between weeks 5 and 10 in the Bm-MSC and PDLC groups. At week 10, the micro-computed tomography results showed that the PDLC group had greater bone fill as compared with the empty scaffold, while the GC group had less bone than the 3 other groups (control, Bm-MSC, and PDLC). Periodontal regeneration, as measured by histologically verified new bone and cementum formation with obliquely inserted periodontal ligament fibers, increased between 5 and 10 wk for the empty, Bm-MSC, and PDLC groups, while the GC group was inferior to the Bm-MSC and PDLC groups at 10 wk. This study demonstrates that periodontal regeneration can be achieved via the utilization of a multiphasic construct, with Bm-MSCs and PDLCs obtaining superior results as compared with GC-derived cell sheets.

Three-Dimensional Bioprinting for Regenerative Dentistry and Craniofacial Tissue Engineering.

Craniofacial tissues are organized with complex 3-dimensional (3D) architectures. Mimicking such 3D complexity and the multicellular interactions naturally occurring in craniofacial structures represents one of the greatest challenges in regenerative dentistry. Three-dimensional bioprinting of tissues and biological structures has been proposed as a promising alternative to address some of these key challenges. It enables precise manufacture of various biomaterials with complex 3D architectures, while being compatible with multiple cell sources and being customizable to patient-specific needs. This review describes different 3D bioprinting methods and summarizes how different classes of biomaterials (polymer hydrogels, ceramics, composites, and cell aggregates) may be used for 3D biomanufacturing of scaffolds, as well as craniofacial tissue analogs. While the fabrication of scaffolds upon which cells attach, migrate, and proliferate is already in use, printing of all the components that form a tissue (living cells and matrix materials together) to produce tissue constructs is still in its early stages. In summary, this review seeks to highlight some of the key advantages of 3D bioprinting technology for the regeneration of craniofacial structures. Additionally, it stimulates progress on the development of strategies that will promote the translation of craniofacial tissue engineering from the laboratory bench to the chair side.

Decellularized periodontal ligament cell sheets with recellularization potential.

The periodontal ligament is the key tissue facilitating periodontal regeneration. This study aimed to fabricate decellularized human periodontal ligament cell sheets for subsequent periodontal tissue engineering applications. The decellularization protocol involved the transfer of intact human periodontal ligament cell sheets onto melt electrospun polycaprolactone membranes and subsequent bi-directional perfusion with NH4OH/Triton X-100 and DNase solutions. The protocol was shown to remove 92% of DNA content. The structural integrity of the decellularized cell sheets was confirmed by a collagen quantification assay, immunostaining of human collagen type I and fibronectin, and scanning electron microscopy. ELISA was used to demonstrate the presence of residual basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) in the decellularized cell sheet constructs. The decellularized cell sheets were shown to have the ability to support recellularization by allogenic human periodontal ligament cells. This study describes the fabrication of decellularized periodontal ligament cell sheets that retain an intact extracellular matrix and resident growth factors and can support repopulation by allogenic cells. The decellularized hPDL cell sheet concept has the potential to be utilized in future "off-the-shelf" periodontal tissue engineering strategies.

Multiphasic scaffolds for periodontal tissue engineering.

For a successful clinical outcome, periodontal regeneration requires the coordinated response of multiple soft and hard tissues (periodontal ligament, gingiva, cementum, and bone) during the wound-healing process. Tissue-engineered constructs for regeneration of the periodontium must be of a complex 3-dimensional shape and adequate size and demonstrate biomechanical stability over time. A critical requirement is the ability to promote the formation of functional periodontal attachment between regenerated alveolar bone, and newly formed cementum on the root surface. This review outlines the current advances in multiphasic scaffold fabrication and how these scaffolds can be combined with cell- and growth factor-based approaches to form tissue-engineered constructs capable of recapitulating the complex temporal and spatial wound-healing events that will lead to predictable periodontal regeneration. This can be achieved through a variety of approaches, with promising strategies characterized by the use of scaffolds that can deliver and stabilize cells capable of cementogenesis onto the root surface, provide biomechanical cues that encourage perpendicular alignment of periodontal fibers to the root surface, and provide osteogenic cues and appropriate space to facilitate bone regeneration. Progress on the development of multiphasic constructs for periodontal tissue engineering is in the early stages of development, and these constructs need to be tested in large animal models and, ultimately, human clinical trials.

Biomimetic tubular nanofiber mesh and platelet rich plasma-mediated delivery of BMP-7 for large bone defect regeneration.

There is a growing need for successful bone tissue engineering strategies and advanced biomaterials that mimic the structure and function of native tissues carry great promise. Successful bone repair approaches may include an osteoconductive scaffold, osteoinductive growth factors, cells with an osteogenic potential and capacity for graft vascularisation. To increase osteoinductivity of biomaterials, the local combination and delivery of growth factors has been developed. In the present study we investigated the osteogenic effects of calcium phosphate (CaP)-coated nanofiber mesh tube-mediated delivery of BMP-7 from a PRP matrix for the regeneration of critical sized segmental bone defects in a small animal model. Bilateral full-thickness diaphyseal segmental defects were created in twelve male Lewis rats and nanofiber mesh tubes were placed around the defect. Defects received either treatment with a CaP-coated nanofiber mesh tube (n = 6), an un-coated nanofiber mesh tube (n=6) a CaP-coated nanofiber mesh tube with PRP (n=6) or a CaP-coated nanofiber mesh tube in combination with 5 µg BMP-7 and PRP (n = 6). After 12 weeks, bone volume and biomechanical properties were evaluated using radiography, microCT, biomechanical testing and histology. The results demonstrated significantly higher biomechanical properties and bone volume for the BMP group compared to the control groups. These results were supported by the histological evaluations, where BMP group showed the highest rate of bone regeneration within the defect. In conclusion, BMP-7 delivery via PRP enhanced functional bone defect regeneration, and together these data support the use of BMP-7 in the treatment of critical sized defects.

In vitro biocompatibility of different polyester membranes.

Nowadays, synthetic biodegradable polymers, such as aliphatic polyesters, are largely used in tissue engineering. They provide several advantages compared to natural materials which use is limited by immunocompatibility, graft availability, etc. In this work, poly(L-lactic) acid (PLLA), poly(DL-lactic) acid (PDLA), poly-epsilon-caprolactone (PCL), poly(L-lactic)-co-caprolactone (molar ratio 70/30) (PLCL) were selected because of their common use in tissue engineering. The membranes were elaborated by solvent casting. Membrane morphology was investigated by atomic force microscopy. The membranes were seeded with human fibroblasts from cell line CRL 2703 in order to evaluate the biocompatibility by the Alamar blue test. The roughness of the membranes ranged from 4 nm for PDLA to 120 nm and they presented very smooth surface except for PCL which beside a macroscopic structure due to its hydrophobicity. Human fibroblasts proliferated over 28 days on the membranes proving the non-in vitro toxicity of the materials and of the processing method. A further step will be the fabrication of three-dimensional scaffold for tissue engineering and the treatment of the scaffolds to augment cell adhesion.

[Endotracheal tube for laryngeal CO2 laser microsurgery. 208 cases]. / Sonde d'intubation pour la microchirurgie laryngée par laser CO2. 208 observations.

To suppress the risk inherent in laryngeal microsurgery performed with a CO2 laser beam, the authors suggest to use a reinforced silicone endotracheal tube, the cuff of which is protected by a silicone plus aluminium powder shield. The resistance of silicone to fire is augmented during laser shots by a nitrogen flow on the upper side of the shield at the rates of 2 l/min in patients breathing air and 30 l/min in patients give oxygen.

[Technics and indications of general anesthesia for tonsillectomy and adenoidectomy. Apropos of an experience using anesthetic perfusion and cold loop technic]. / Techniques et indications de l'anesthésie générale pour amygdalectomie et adénoïdectomie. A propos d'une expérience d'utilisation d'un narcotique en perfusion et de la technique de l'anse froide

The authors present the results of an enquiry carried out in University and regional hospitals in France and recall the interest of general anaesthesia with tracheal intubation during tonsillectomy. This makes possible, even in young children, the use of the technique of cold loop dissection. Its main advantage is in the absolute control of the airways which permits the operator to carry out careful hemostasis. Propanidide used in perfusion, allows these patients to wake up rapidly and the awakening is of good quality; this is essential in this type of operation.