Degradation-Dependent Stress Relaxing Semi-Interpenetrating Networks of Hydroxyethyl Cellulose in Gelatin-PEG Hydrogel with Good Mechanical Stability and Reversibility.

The mechanical milieu of the extracellular matrix (ECM) plays a key role in modulating the cellular responses. The native ECM exhibits viscoelasticity with stress relaxation behavior. Here, we reported the preparation of degradation-mediated stress relaxing semi-interpenetrating (semi-IPN) polymeric networks of hydroxyethyl cellulose in the crosslinked gelatin-polyethylene glycol (PEG) architecture, leveraging a newly developed synthesis protocol which successively includes one-pot gelation under physiological conditions, freeze-drying and a post-curing process. Fourier transform infrared (FTIR) confirmed the formation of the semi-IPN blend mixture. A surface morphology analysis revealed an open pore porous structure with a compact skin on the surface. The hydrogel showed a high water-absorption ability (720.00 ± 32.0%) indicating the ability of retaining a hydrophilic nature even after covalent crosslinking with functionalized PEG. Detailed mechanical properties such as tensile, compressive, cyclic compression and stress relaxation tests were conducted at different intervals over 28 days of hydrolytic degradation. Overall, the collective mechanical properties of the hydrogel resembled the mechanics of cartilage tissue. The rate of stress relaxation gradually increased with an increasing swelling ratio. Hydrolytic degradation led to a marked increase in the percentage dissipation energy and stress relaxation response, indicating the degradation-dependent viscoelasticity of the hydrogel. Strikingly, the hydrogel maintained the structural stability even after degrading two-thirds of its initial mass after a month-long hydrolytic degradation. This study demonstrates that this semi-IPN G-PEG-HEC hydrogel possesses bright prospects as a potential scaffolding material in tissue engineering.

Additive Manufacturing for Personalized Skull Base Reconstruction in Endoscopic Transclival Surgery: A Proof-of-Concept Study.

BACKGROUND: Endoscopic transnasal transclival intradural surgery is limited by a high postoperative cerebrospinal fluid leak rate. The aim of this study was to investigate the role of three-dimensional printing to create a personalized, rigid scaffold for clival reconstruction. METHODS: Two different types of clivectomy were performed in 5 specimens with the aid of neuronavigation, and 11 clival reconstructions were simulated. They were repaired with polylactide, three-dimensional-printed scaffolds that were manually designed in a computer-aided environment based either on the real or on the predicted defect. Scaffolds were printed with a fused filament fabrication technique and different offsets. They were positioned and fixed either following the gasket seal technique or with screws. Postdissection radiological evaluation of scaffold position was performed in all cases. In 3 specimens, the cerebrospinal fluid leak pressure point was measured immediately after reconstruction. RESULTS: The production process took approximately 30 hours. The designed scaffolds were satisfactory when no offset was added. Wings were added during the design to allow for screw positioning, but broke in 30% of cases. Radiological assessment documented maximal accuracy of scaffold positioning when the scaffold was created on the real defect; accuracy was satisfactory when the predicted clivectomy was performed under neuronavigation guidance. The cerebrospinal fluid leak pressure point was significantly higher when the scaffold was fixed with screws compared with the gasket technique. CONCLUSIONS: In this preclinical setting, additive manufacturing allows the creation of customized scaffolds that are effective in reconstructing even large and geometrically complex clival defects.

Size-Induced Constraint Effects on Crack Initiation and Propagation Parameters in Ductile Polymers.

Fracture mechanics are of high interest for the engineering design and structural integrity assessment of polymeric materials; however, regarding highly ductile polymers, many open questions still remain in terms of fully understanding deformation and fracture behaviors. For example, the influence of the constraint and specimen size on the fracture behavior of polymeric materials is still not clear. In this study, a polymeric material with an elastic plastic deformation behavior (ABS, acrylonitrile butadiene styrene) is investigated with regard to the influence of constraint and specimen size. Different single-edge notched bending (SENB) specimen sizes with constant geometrical ratios were tested. The material key curve was used to investigate differences in the constraint, where changes for small and large specimen sizes were found. Based on a size-independent crack resistance curve (J-R curve), two apparent initiation parameters (J0.2 and Jbl) were determined, namely, the initiation parameter Jini (based on the crack propagation kinetics curve) and the initiation parameter JI,lim (based on an ESIS TC 4 draft protocol). It was found that J0.2 and Jbl could be used as crack initiation parameters whereby Jini and JI,lim are indicative of the onset of stable crack growth.

Transclival approaches for intradural pathologies: historical overview and present scenario.

Recently, endoscopic transsphenoidal transclival approaches have been developed and their role is widely accepted for extradural pathologies. Their application to intradural pathologies is still debated, but is undoubtedly increasing. In the past five decades, different authors have reported various extracranial, anterior transclival approaches for intradural pathologies. The aim of this review is to provide a historical overview of transclival approaches applied to intradural pathologies. PubMed was searched in October 2018 using the terms transcliv*, cliv* intradural, transsphenoidal transcliv*, transoral transcliv*, transcervical transcliv*, transsphenoidal brainstem, and transoral brainstem. Exclusion criteria included not reporting reconstruction technique, anatomical studies, reviews without new data, and transcranial approaches. Ninety-one studies were included in the systematic review. Since 1966, transcervical, transoral, transsphenoidal microsurgical, and, recently, endoscopic routes have been used as a corridor for transclival approaches to treat intradural pathologies. Each approach presents a curve that follows Scott's parabola, with evident phases of enthusiasm that quickly faded, possibly due to high post-operative CSF leak rates and other complications. It is evident that the introduction of the endoscope has led to a significant increase in reports of transclival approaches for intradural pathologies. Various reconstruction techniques and materials have been used, although rates of CSF leak remain relatively high. Transclival approaches for intradural pathologies have a long history. We are now in a new era of interest, but achieving effective dural and skull base reconstruction must still be definitively addressed, possibly with the use of newly available technologies.

Graphene Layers Functionalized with A Janus Pyrrole-Based Compound in Natural Rubber Nanocomposites with Improved Ultimate and Fracture Properties.

The ultimate properties and resistance to fracture of nanocomposites based on poly(1,4-cis-isoprene) from Hevea Brasiliensis (natural rubber, NR) and a high surface area nanosized graphite (HSAG) were improved by using HSAG functionalized with 2-(2,5-dimethyl-1H-pyrrol-1-yl)propane-1,3-diol (serinol pyrrole) (HSAG-SP). The functionalization reaction occurred through a domino process, by simply mixing HSAG and serinol pyrrole and heating at 180 °C. The polarity of HSAG-SP allowed its dispersion in NR latex and the isolation of NR/HSAG-SP masterbatches via coagulation. Nanocomposites, based either on pristine HSAG or on HSAG-SP, were prepared through traditional melt blending and cured with a sulphur-based system. The samples containing HSAG-SP revealed ultimate dispersion of the graphitic filler with smaller aggregates and higher amounts of few layers stacks and isolated layers, as revealed by transmission electron microscopy. With HSAG-SP, better stress and elongation at break and higher fracture resistance were obtained. Indeed, in the case of HSAG-SP-based composites, fracture occurred at larger deformation and with higher values of load and, at the highest filler content (24 phr), deviation of fracture propagation was observed. These results have been obtained with a moderate functionalization of the graphene layers (about 5%) and normal lab facilities. This work reveals a simple and scalable way to prepare tougher NR-based nanocomposites and indicates that the dispersion of a graphitic material in a rubber matrix can be improved without using an extra-amount of mechanical energy, just by modifying the chemical nature of the graphitic material through a sustainable process, avoiding the traditional complex approach, which implies oxidation to graphite oxide and subsequent partial reduction.

Rational Design and Development of Anisotropic and Mechanically Strong Gelatin-Based Stress Relaxing Hydrogels for Osteogenic/Chondrogenic Differentiation.

Rational design and development of tailorable simple synthesis process remains a centerpiece of investigational efforts toward engineering advanced hydrogels. In this study, a green and scalable synthesis approach is developed to formulate a set of gelatin-based macroporous hybrid hydrogels. This approach consists of four sequential steps starting from liquid-phase pre-crosslinking/grafting, unidirectional freezing, freeze-drying, and finally post-curing process. The chemical crosslinking mainly involves between epoxy groups of functionalized polyethylene glycol and functional groups of gelatin both in liquid and solid state. Importantly, this approach allows to accommodate different polymers, chitosan or hydroxyethyl cellulose, under identical benign condition. Structural and mechanical anisotropy can be tuned by the selection of polymer constituents. Overall, all hydrogels show suitable structural stability, good swellability, high porosity and pore interconnectivity, and maintenance of mechanical integrity during 3-week-long hydrolytic degradation. Under compression, hydrogels exhibit robust mechanical properties with nonlinear elasticity and stress-relaxation behavior and show no sign of mechanical failure under repeated compression at 50% deformation. Biological experiment with human bone marrow mesenchymal stromal cells (hMSCs) reveals that hydrogels are biocompatible, and their physicomechanical properties are suitable to support cells growth, and osteogenic/chondrogenic differentiation, demonstrating their potential application for bone and cartilage regenerative medicine toward clinically relevant endpoints.

Chitosan-based scaffold counteracts hypertrophic and fibrotic markers in chondrogenic differentiated mesenchymal stromal cells.

Cartilage tissue engineering remains problematic because no systems are able to induce signals that contribute to native cartilage structure formation. Therefore, we tested the potentiality of gelatin-polyethylene glycol scaffolds containing three different concentrations of chitosan (CH; 0%, 8%, and 16%) on chondrogenic differentiation of human platelet lysate-expanded human bone marrow mesenchymal stromal cells (hBM-MSCs). Typical chondrogenic (SOX9, collagen type 2, and aggrecan), hypertrophic (collagen type 10), and fibrotic (collagen type 1) markers were evaluated at gene and protein level at Days 1, 28, and 48. We demonstrated that 16% CH scaffold had the highest percentage of relaxation with the fastest relaxation rate. In particular, 16% CH scaffold, combined with chondrogenic factor TGFß3, was more efficient in inducing hBM-MSCs chondrogenic differentiation compared with 0% or 8% scaffolds. Collagen type 2, SOX9, and aggrecan showed the same expression in all scaffolds, whereas collagen types 10 and 1 markers were efficiently down-modulated only in 16% CH. We demonstrated that using human platelet lysate chronically during hBM-MSCs chondrogenic differentiation, the chondrogenic, hypertrophic, and fibrotic markers were significantly decreased. Our data demonstrate that only a high concentration of CH, combined with TGFß3, creates an environment capable of guiding in vitro hBM-MSCs towards a phenotypically stable chondrogenesis.

3D gelatin-chitosan hybrid hydrogels combined with human platelet lysate highly support human mesenchymal stem cell proliferation and osteogenic differentiation.

Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin-chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin-chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin-chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%-90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin-chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin-chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells.

Dynamic freedom: substrate stress relaxation stimulates cell responses.

Tissue engineers have explored a set of materials cues that can allow control of cell viability and guide cell fate and functions. Although the effect of substrate stiffness on cell fate has been extensively studied and established, the role of substrate stress relaxation, the ability of a substrate to dissipate cell-induced forces, is only emerging. Recently, several studies have demonstrated that substrate stress relaxation is an important mechanical cue for cell spreading, proliferation and differentiation in vitro. In this mini-review, we highlight the influence of substrate stress relaxation on cell behavior and function as well as provide future perspectives. Firstly, we describe the methods used for characterizing the stress relaxation/creep responses of hydrogels along with the molecular origin of viscoelastic properties. Then, we highlight the most recent studies elucidating the stress relaxation effect on cellular behavior using physically cross-linked hydrogels. Finally, we report on an emerging alternative design of tunable viscoelastic hydrogels: chemically cross-linked (reversible linkages) adaptable hydrogels that have been used as stable 3D cell culture platforms for a few years in the era of hydrogel systems.

Cafeteria diet induce changes in blood flow that are more related with heat dissipation than energy accretion.

Background. A "cafeteria" diet is a self-selected high-fat diet, providing an excess of energy, which can induce obesity. Excess of lipids in the diet hampers glucose utilization eliciting insulin resistance, which, further limits amino acid oxidation for energy. Methods. Male Wistar rats were exposed for a month to "cafeteria" diet. Rats were cannulated and fluorescent microspheres were used to determine blood flow. Results. Exposure to the cafeteria diet did not change cardiac output, but there was a marked shift in organ irrigation. Skin blood flow decreased to compensate increases in lungs and heart. Blood flow through adipose tissue tended to increase in relation to controls, but was considerably increased in brown adipose tissue (on a weight basis). Discussion. The results suggest that the cafeteria diet-induced changes were related to heat transfer and disposal.

White adipose tissue urea cycle activity is not affected by one-month treatment with a hyperlipidic diet in female rats.

Under high-energy diets, amino acid N is difficult to dispose of, as a consequence of the availability of alternative substrates. We found, recently, that WAT contains a complete functional urea cycle, we analyzed the possible overall changes in the WAT urea cycle (and other-related amino acid metabolism gene expressions) in rats subjected to a cafeteria diet. Adult female Wistar rats were fed control or simplified cafeteria diets. Samples of WAT sites: mesenteric, periovaric, retroperitoneal and subcutaneous, were used for the estimation of all urea cycle enzyme activities and gene expressions. Other key amino acid metabolism gene expressions, and lactate dehydrogenase were also measured. Subcutaneous WAT showed a differentiated amino acid metabolism profile, since its cumulative (whole site) activity for most enzymes was higher than the activities of the other sites studied. After one month of eating an energy-rich cafeteria diet, and in spite of doubling the size of WAT, the transforming capacity of most amino acid metabolism enzymes remained practically unchanged in the tissue. This was not only due to limited changes in the overall enzyme activity, but also a consequence of a relative decrease in the expression of the corresponding genes. Overall, the results of this study support the consideration of WAT as an organ, disperse but under uniform control. The metabolic peculiarities between its different sites, and their ability to adapt to different energy availability conditions only add to the variable nature of adipose tissue. We have presented additional evidence of the significant role of WAT in amino acid metabolism.

Effects of sex and site on amino acid metabolism enzyme gene expression and activity in rat white adipose tissue.

Background and Objectives. White adipose tissue (WAT) shows marked sex- and diet-dependent differences. However, our metabolic knowledge of WAT, especially on amino acid metabolism, is considerably limited. In the present study, we compared the influence of sex on the amino acid metabolism profile of the four main WAT sites, focused on the paths related to ammonium handling and the urea cycle, as a way to estimate the extent of WAT implication on body amino-nitrogen metabolism. Experimental Design. Adult female and male rats were maintained, undisturbed, under standard conditions for one month. After killing them under isoflurane anesthesia. WAT sites were dissected and weighed. Subcutaneous, perigonadal, retroperitoneal and mesenteric WAT were analyzed for amino acid metabolism gene expression and enzyme activities. Results. There was a considerable stability of the urea cycle activities and expressions, irrespective of sex, and with only limited influence of site. Urea cycle was more resilient to change than other site-specialized metabolic pathways. The control of WAT urea cycle was probably related to the provision of arginine/citrulline, as deduced from the enzyme activity profiles. These data support a generalized role of WAT in overall amino-N handling. In contrast, sex markedly affected WAT ammonium-centered amino acid metabolism in a site-related way, with relatively higher emphasis in males' subcutaneous WAT. Conclusions. We found that WAT has an active amino acid metabolism. Its gene expressions were lower than those of glucose-lipid interactions, but the differences were quantitatively less important than usually reported. The effects of sex on urea cycle enzymes expression and activity were limited, in contrast with the wider variations observed in other metabolic pathways. The results agree with a centralized control of urea cycle operation affecting the adipose organ as a whole.

Influence of a hyperlipidic diet on the composition of the non-membrane lipid pool of red blood cells of male and female rats.

Background and objectives. Red blood cells (RBC) are continuously exposed to oxidative agents, affecting their membrane lipid function. However, the amount of lipid in RBCs is higher than the lipids of the cell membrane, and includes triacylglycerols, which are no membrane components. We assumed that the extra lipids originated from lipoproteins attached to the cell surface, and we intended to analyse whether the size and composition of this lipid pool were affected by sex or diet. Experimental design. Adult male and female Wistar rats were fed control or cafeteria diets. Packed blood cells and plasma lipids were extracted and analysed for fatty acids by methylation and GC-MS, taking care of not extracting membrane lipids. Results. The absence of ω3-PUFA in RBC extracts (but not in plasma) suggest that the lipids extracted were essentially those in the postulated lipid surface pool and not those in cell membrane. In cells' extracts, there was a marked depletion of PUFA (and, in general, of insaturation). Fatty acid patterns were similar for all groups studied, with limited effects of sex and no effects of diet in RBC (but not in plasma) fatty acids. Presence of trans fatty acids was small but higher in RBC lipids, and could not be justified by dietary sources. Conclusions. The presence of a small layer of lipid on the RBC surface may limit oxidative damage to the cell outer structures, and help explain its role in the transport of lipophilic compounds. However, there may be other, so far uncovered, additional functions for this lipid pool.

Evidences of basal lactate production in the main white adipose tissue sites of rats. Effects of sex and a cafeteria diet.

Female and male adult Wistar rats were fed standard chow or a simplified cafeteria diet for one month. Then, the rats were killed and the white adipose tissue (WAT) in four sites: perigonadal, retroperitoneal, mesenteric and subcutaneous (inguinal) were sampled and frozen. The complete WAT weight in each site was measured. Gene expression analysis of key lipid and glucose metabolism enzymes were analyzed, as well as tissue and plasma lactate and the activity of lactate dehydrogenase. Lactate gradients between WAT and plasma were estimated. The influence of sex and diet (and indirectly WAT mass) on lactate levels and their relationships with lactate dehydrogenase activity and gene expressions were also measured. A main conclusion is the high production of lactate by WAT, practically irrespective of site, diet or sex. Lactate production is a direct correlate of lactate dehydrogenase activity in the tissue. Furthermore, lactate dehydrogenase activity is again directly correlated with the expression of the genes Ldha and Ldhb for this enzyme. In sum, the ability to produce lactate by WAT is not directly dependent of WAT metabolic state. We postulate that, in WAT, a main function of the lactate dehydrogenase path may be that of converting excess available glucose to 3C fragments, as a way to limit tissue self-utilization as substrate, to help control glycaemia and/or providing short chain substrates for use as energy source elsewhere. More information must be gathered before a conclusive role of WAT in the control of glycaemia, and the full existence of a renewed glucose-lactate-fatty acid cycle is definitely established.

Altered nitrogen balance and decreased urea excretion in male rats fed cafeteria diet are related to arginine availability.

Hyperlipidic diets limit glucose oxidation and favor amino acid preservation, hampering the elimination of excess dietary nitrogen and the catabolic utilization of amino acids. We analyzed whether reduced urea excretion was a consequence of higher NO x ; (nitrite, nitrate, and other derivatives) availability caused by increased nitric oxide production in metabolic syndrome. Rats fed a cafeteria diet for 30 days had a higher intake and accumulation of amino acid nitrogen and lower urea excretion. There were no differences in plasma nitrate or nitrite. NO(x) and creatinine excretion accounted for only a small part of total nitrogen excretion. Rats fed a cafeteria diet had higher plasma levels of glutamine, serine, threonine, glycine, and ornithine when compared with controls, whereas arginine was lower. Liver carbamoyl-phosphate synthetase I activity was higher in cafeteria diet-fed rats, but arginase I was lower. The high carbamoyl-phosphate synthetase activity and ornithine levels suggest activation of the urea cycle in cafeteria diet-fed rats, but low arginine levels point to a block in the urea cycle between ornithine and arginine, thereby preventing the elimination of excess nitrogen as urea. The ultimate consequence of this paradoxical block in the urea cycle seems to be the limitation of arginine production and/or availability.

Cultured 3T3L1 adipocytes dispose of excess medium glucose as lactate under abundant oxygen availability.

White adipose tissue (WAT) produces lactate in significant amount from circulating glucose, especially in obesity;Under normoxia, 3T3L1 cells secrete large quantities of lactate to the medium, again at the expense of glucose and proportionally to its levels. Most of the glucose was converted to lactate with only part of it being used to synthesize fat. Cultured adipocytes were largely anaerobic, but this was not a Warburg-like process. It is speculated that the massive production of lactate, is a process of defense of the adipocyte, used to dispose of excess glucose. This way, the adipocyte exports glucose carbon (and reduces the problem of excess substrate availability) to the liver, but the process may be also a mechanism of short-term control of hyperglycemia. The in vivo data obtained from adipose tissue of male rats agree with this interpretation.