Gellan Gum-based luminal fillers for peripheral nerve regeneration: an in vivo study in the rat sciatic nerve repair model.

Peripheral nerve injuries (PNI) resulting in a gap to be bridged between the transected nerve ends are commonly reconstructed with autologous nerve tissue, but there is a need for valuable alternatives. This experimental work considers the innovative use of the biomaterial Gellan Gum (GG) as a luminal filler for nerve guidance channels made from chitosan with a 5% degree of acetylation. The engineered constructs should remodel the structural support given to regenerating axons by the so-called bands of Büngner. Four different GG formulations were produced by combining varying amounts of High-Acyl GG (HA-GG) and Methacrylated GG (MA-GG). The effective porosity of the freeze-dried networks was analysed by SEM and micro-CT 3D reconstructions, while the degradation and swelling abilities were characterized in vitro for up to 30 days. The metabolic activity and viability of immortalized Schwann cells seeded onto the freeze-dried networks were also evaluated. Finally, the developed hydrogel formulations were freeze-dried within the chitosan nerve guides and implanted in a 10 mm rat sciatic nerve defect. Functional and histomorphological analyses after 3, 6, and 12 weeks in vivo revealed that although it did not result in improved nerve regeneration, the NGC25:75 formulations could provide a basis for further development of GG scaffolds as luminal fillers for hollow nerve guidance channels.

Posterior talar process as a suitable cell source for treatment of cartilage and osteochondral defects of the talus.

Osteochondral defects of the ankle are common lesions affecting the talar cartilage and subchondral bone. Current treatments include cell-based therapies but are frequently associated with donor-site morbidity. Our objective is to characterize the posterior process of the talus (SP) and the os trigonum (OT) tissues and investigate their potential as a new source of viable cells for application in tissue engineering and regenerative medicine. SP and OT tissues obtained from six patients were characterized by micro-computed tomography and histological, histomorphometric and immunohistochemical analyses. Proliferation and viability of isolated cells were evaluated by MTS assay, DNA quantification and live/dead staining. The TUNEL assay was performed to evaluate cell death by apoptosis. Moreover, the production of extracellular matrix was evaluated by toluidine blue staining, whereas cells phenotype was investigated by flow cytometry. Characterization of ankle explants showed the presence of a cartilage tissue layer in both SP and OT tissues, which represented at least 20%, on average, of the explant. The presence of type II collagen was detected in the extracellular matrix. Isolated cells presented a round morphology typical of chondrocytes. In in vitro studies, cells were viable and proliferating for up to 21 days of culture. No signs of apoptosis were detected. Flow-cytometry analysis revealed that isolated cells maintained the expression of several chondrocytic markers during culture. The results indicated that the SP and OT tissues were a reliable source of viable chondrocytes, which could find promising applications in ACI/MACI strategies with minimal concerns regarding donor zone complications. Copyright © 2015 John Wiley & Sons, Ltd.

Peripheral Nerve Injury: Current Challenges, Conventional Treatment Approaches, and New Trends in Biomaterials-Based Regenerative Strategies.

Damage to peripheral nerves is a widely extended health problem, causing important socioeconomic costs worldwide. Indeed, peripheral nerve injuries (PNI) have been concerning the medical community for many decades. Nevertheless, despite the increase in knowledge in the injury physiopathology and the great research efforts being undertaken, the current standard grafting strategies used to repair PNI are not as efficient as desired. Although alternative engineered nerve grafts are already commercialized, their clinical performance is suboptimal. In this review, a general description of the circumstances and repercussions surrounding the PNI pathological state are presented, together with the treatment limitations and current challenges when addressing both short- and long-gap defects. In addition, potential therapeutic molecules are considered, while innovative regenerative strategies have been identified. Finally, the most relevant advances on the use of a wide range of biomaterials for the development of novel medical devices are also overviewed in depth, considering strategies making use of either empty or filled nerve conduits for guided tissue regeneration.

Custom-tailored tissue engineered polycaprolactone scaffolds for total disc replacement.

Degeneration of the intervertebral disc (IVD) represents a significant musculoskeletal disease burden. Tissue engineering has proposed several strategies comprising the use of biodegradable materials to prepare scaffolds that can present mechanical properties similar to those of native IVD tissues. However, this might be insufficient, since the patient's intervertebral space geometry must be replicated to allow for appropriate implant fixation and integration. Herein, we propose the use of reverse engineering and rapid prototyping techniques with the goal of preparing custom-tailored annulus fibrosus scaffolds; these techniques have previously been applied to rabbit models. The IVD reverse-engineered architecture was obtained by means of microcomputed tomography acquisition and three-dimensional modelling, resulting in a computer-aided design (CAD) that replicates the original rabbit IVD. Later, a fused deposition-modelling three-dimensional printer was used to produce the scaffolds with different geometries provided by the CAD, using polycaprolactone (PCL) with 100% infill density. The microstructure of the PCL scaffolds was investigated by scanning electron microscopy (SEM), which allowed us to observe an adequate fusion adhesion between the layers. The SEM images revealed that, up to the point of moderate resolution, the porosities manually designed in the CAD model were successfully replicated. The PCL scaffolds' three-dimensional architecture was also assessed by means of microcomputed tomography analysis. Compressive stiffness was determined using a mechanical testing system. Results showed higher values than those of human IVDs (5.9-6.7 kN mm(-1) versus 1.2 kN mm(-1), respectively). In vitro studies were performed to investigate the possible cytotoxicity of the polycaprolactone scaffolds' leachables. The results showed that the custom-tailored PCL scaffolds do not have any deleterious cytotoxic effect over annulus fibrosus cells or the mouse lung fibroblast's cell line. This study proposed a simple, rapid, and low-cost strategy to fabricate custom-tailored annulus fibrosus scaffolds. In the future, this strategy might be used in association with nucleus pulposus regeneration strategies to facilitate the development of tissue-engineered total disc replacement implants specific to each patient, with a goal of full IVD regeneration.

Biological evaluation of intervertebral disc cells in different formulations of gellan gum-based hydrogels.

Gellan gum (GG)-based hydrogels are advantageous in tissue engineering not only due to their ability to retain large quantities of water and provide a similar environment to that of natural extracellular matrix (ECM), but also because they can gelify in situ in seconds. Their mechanical properties can be fine-tuned to mimic natural tissues such as the nucleus pulposus (NP). This study produced different formulations of GG hydrogels by mixing varying amounts of methacrylated (GG-MA) and high-acyl gellan gums (HA-GG) for applications as acellular and cellular NP substitutes. The hydrogels were physicochemically characterized by dynamic mechanical analysis. Degradation and swelling abilities were assessed by soaking in a phosphate buffered saline solution for up to 170 h. Results showed that as HA-GG content increased, the modulus of the hydrogels decreased. Moreover, increases in HA-GG content induced greater weight loss in the GG-MA/HA-GG formulation compared to GG-MA hydrogel. Potential cytotoxicity of the hydrogel was assessed by culturing rabbit NP cells up to 7 days. An MTS assay was performed by seeding rabbit NP cells onto the surface of 3D hydrogel disc formulations. Viability of rabbit NP cells encapsulated within the different hydrogel formulations was also evaluated by Calcein-AM and ATP assays. Results showed that tunable GG-MA/HA-GG hydrogels were non-cytotoxic and supported viability of rabbit NP cells.

Optical projection tomography as a tool for 3D imaging of hydrogels.

An Optical Projection Tomography (OPT) system was developed and optimized to image 3D tissue engineered products based in hydrogels. We develop pre-reconstruction algorithms to get the best result from the reconstruction procedure, which include correction of the illumination and determination of sample center of rotation (CoR). Existing methods for CoR determination based on the detection of the maximum variance of reconstructed slices failed, so we develop a new CoR search method based in the detection of the variance sharpest local maximum. We show the capabilities of the system to give quantitative information of different types of hydrogels that may be useful in its characterization.

Biomechanical and cellular segmental characterization of human meniscus: building the basis for Tissue Engineering therapies.

OBJECTIVE: To overcome current limitations of Tissue Engineering (TE) strategies, deeper comprehension on meniscus biology is required. This study aims to combine biomechanical segmental analysis of fresh human meniscus tissues and its correlation with architectural and cellular characterization. METHOD: Morphologically intact menisci, from 44 live donors were studied after division into three radial segments. Dynamic mechanical analysis (DMA) was performed at physiological-like conditions. Micro-computed tomography (CT) analysis of freeze-dried samples assessed micro-structure. Flow cytometry, histology and histomorphometry were used for cellular study and quantification. RESULTS: Anterior segments present significantly higher damping properties. Mid body fresh medial meniscus presents higher values of E' compared to lateral. Cyclic loads influence the viscoelastic behavior of menisci. By increasing the frequency leads to an increase in stiffness. Conversely, with increasing frequencies, the capacity to dissipate energy and damping properties initially decrease and then rise again. Age and gender directly correlate with higher E' and tan δ. Micro-CT analysis revealed that mean porosity was 55.5 (21.2-89.8)% and 64.7 (47.7-81.8)% for freeze-dried lateral and medial meniscus, respectively. Predominant cells are positive for CD44, CD73, CD90 and CD105, and lack CD31, CD34 and CD45 (present in smaller populations). Histomorphometry revealed that cellularity decreases from vascular zone 1 to zone 3. Anterior segments of lateral and medial meniscus have inferior cellularity as compared to mid body and posterior ones. CONCLUSION: Menisci are not uniform structures. Anterior segments have lower cellularity and higher damping. Cyclic loads influence viscoelastic characteristics. Future TE therapies should consider segmental architecture, cellularity and biomechanics of fresh tissue.

Current concepts: tissue engineering and regenerative medicine applications in the ankle joint.

Tissue engineering and regenerative medicine (TERM) has caused a revolution in present and future trends of medicine and surgery. In different tissues, advanced TERM approaches bring new therapeutic possibilities in general population as well as in young patients and high-level athletes, improving restoration of biological functions and rehabilitation. The mainstream components required to obtain a functional regeneration of tissues may include biodegradable scaffolds, drugs or growth factors and different cell types (either autologous or heterologous) that can be cultured in bioreactor systems (in vitro) prior to implantation into the patient. Particularly in the ankle, which is subject to many different injuries (e.g. acute, chronic, traumatic and degenerative), there is still no definitive and feasible answer to 'conventional' methods. This review aims to provide current concepts of TERM applications to ankle injuries under preclinical and/or clinical research applied to skin, tendon, bone and cartilage problems. A particular attention has been given to biomaterial design and scaffold processing with potential use in osteochondral ankle lesions.

Hydrogels in acellular and cellular strategies for intervertebral disc regeneration.

Low back pain is an extremely common illness syndrome that causes patient suffering and disability and requires urgent solutions to improve the quality of life of these patients. Treatment options aimed to regenerate the intervertebral disc (IVD) are still under development. The cellular complexity of IVD, and consequently its fine regulatory system, makes it a challenge to the scientific community. Biomaterials-based therapies are the most interesting solutions to date, whereby tissue engineering and regenerative medicine (TE&RM) strategies are included. By using such strategies, i.e., combining biomaterials, cells, and biomolecules, the ultimate goal of reaching a complete integration between native and neo-tissue can be achieved. Hydrogels are promising materials for restoring IVD, mainly nucleus pulposus (NP). This study presents an overview of the use of hydrogels in acellular and cellular strategies for intervertebral disc regeneration. To better understand IVD and its functioning, this study will focus on several aspects: anatomy, pathophysiology, cellular and biomolecular performance, intrinsic healing processes, and current therapies. In addition, the application of hydrogels as NP substitutes will be addressed due to their similarities to NP mechanical properties and extracellular matrix. These hydrogels can be used in cellular strategies when combined with cells from different sources, or in acellular strategies by performing the functionalization of the hydrogels with biomolecules. In addition, a brief summary of therapies based on simple injection for primary biological repair will be examined. Finally, special emphasis will focus on reviewing original studies reporting on the use of autologous cells and biomolecules such as platelet-rich plasma and their potential clinical applications.

Gellan gum-based hydrogels for intervertebral disc tissue-engineering applications.

Intervertebral disc (IVD) degeneration is a challenging clinical problem that urgently demands viable nucleus pulposus (NP) implant materials. The best suited biomaterial for NP regeneration has yet to be identified, but it is believed that biodegradable hydrogel-based materials are promising candidates. In this work, we have developed ionic- and photo-crosslinked methacrylated gellan gum (GG-MA) hydrogels to be used in acellular and cellular tissue-engineering strategies for the regeneration of IVDs. The physicochemical properties of the developed hydrogels were investigated by Fourier-transform infrared spectroscopy, (1) H nuclear magnetic resonance and differential scanning calorimetry. The swelling ability and degradation rate of hydrogels were also analysed in phosphate-buffered saline solution at physiological pH for a period of 30 days. Additionally, the morphology and mechanical properties of the hydrogels were assessed under a scanning electron microscope and dynamic compression, respectively. An in vitro study was carried out to screen possible cytotoxicity of the gellan gum-based hydrogels by culturing rat lung fibroblasts (L929 cells) with hydrogel leachables up to 7 days. The results demonstrated that gellan gum was successfully methacrylated. We observed that the produced GG-MA hydrogels possess improved mechanical properties and lower water uptake ability and degradation rate as compared to gellan gum. This work also revealed that GG-MA hydrogels are non-cytotoxic in vitro, thus being promising biomaterials to be used in IVD tissue-engineering strategies.

Regulation of Nod1-mediated signaling pathways.

Nod1 is a member of the NLR/Nod/CATERPILLER family. It acts as a sensor for intracellular bacteria by recognizing specific glycopeptides derived from peptidoglycan. Nod1 activation mediates distinct cellular responses including activation of MAP kinases, IL-8 release, apoptosis and suppression of several estrogen-dependent responses in MCF-7 cells. Here we have extended these studies by identifying key regulatory steps in Nod1-dependent signaling pathways. We provide multiple lines of data showing that Nod1-dependent apoptosis is a caspase 8-mediated event and that apoptosis requires RIP2. In contrast, several lines of evidence show that Nod1-dependent JNK activation and IL-8 production did not require the presence of caspase 8 but required activation of TAK1 as well as RIP2. Thus, we have identified several key control points that lie downstream of Nod1. This work provides the basis for further studies of the biological significance and regulation of the Nod1 pathway.

Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. transfer from CD14 to TLR4 and MD-2.

The structural features of some proteins of the innate immune system involved in mediating responses to microbial pathogens are highly conserved throughout evolution. Examples include members of the Drosophila Toll (dToll) and the mammalian Toll-like receptor (TLR) protein families. Activation of Drosophila Toll is believed to occur via an endogenous peptide rather than through direct binding of microbial products to the Toll protein. In mammals there is a growing consensus that lipopolysaccharide (LPS) initiates its biological activities through a heteromeric receptor complex containing CD14, TLR4, and at least one other protein, MD-2. LPS binds directly to CD14 but whether LPS then binds to TLR4 and/or MD-2 is not known. We have used transient transfection to express human TLRs, MD-2, or CD14 alone or in different combinations in HEK 293 cells. Interactions between LPS and these proteins were studied using a chemically modified, radioiodinated LPS containing a covalently linked, UV light-activated cross-linking group ((125)I-ASD-Re595 LPS). Here we show that LPS is cross-linked specifically to TLR4 and MD-2 only when co-expressed with CD14. These data support the contention that LPS is in close proximity to the three known proteins of its membrane receptor complex. Thus, LPS binds directly to each of the members of the tripartite LPS receptor complex.