A novel extrusion-based 3D bioprinting system for skeletal muscle tissue engineering.

Three-dimensional (3D) bioprinting is an emerging technology, which turned out to be an optimal tool for tissue engineering approaches. To date, different printing systems have been developed. Among them, the extrusion-based approach demonstrated to be the most suitable for skeletal muscle tissue engineering, due to its ability to produce and deposit printing fibers in a parallel pattern that well mimic the native skeletal muscle tissue architecture. In tissue bioengineering, a key role is played by biomaterials, which must possess the key requisite of 'printability'. Nevertheless, this feature is not often well correlated with cell requirements, such as motives for cellular adhesion and/or absorbability. To overcome this hurdle, several efforts have been made to obtain an effective bioink by combining two different biomaterials in order to reach a good printability besides a suitable biological activity. However, despite being efficient, this strategy reveals several outcomes limitations. We report here the development and characterization of a novel extrusion-based 3D bioprinting system, and its application for correction of volumetric muscle loss (VML) injury in a mouse model. The developed bioprinting system is based on the use of PEG-Fibrinogen, a unique biomaterial with excellent biocompatibility, well-suited for skeletal muscle tissue engineering. With this approach, we obtained highly organized 3D constructs, in which murine muscle progenitors were able to differentiate into muscle fibers arranged in aligned bundles and capable of spontaneously contracting when culturedin vitro. Furthermore, to evaluate the potential of the developed system in future regenerative medicine applications, bioprinted constructs laden with either murine or human muscle progenitors were transplanted to regenerate theTibialis Anteriormuscle of a VML murine model, one month after grafting.

Proline oxidase-adipose triglyceride lipase pathway restrains adipose cell death and tissue inflammation.

The nutrient-sensing lipolytic enzyme adipose triglyceride lipase (ATGL) has a key role in adipose tissue function, and alterations in its activity have been implicated in many age-related metabolic disorders. In adipose tissue reduced blood vessel density is related to hypoxia state, cell death and inflammation. Here we demonstrate that adipocytes of poorly vascularized enlarged visceral adipose tissue (i.e. adipose tissue of old mice) suffer from limited nutrient delivery. In particular, nutrient starvation elicits increased activity of mitochondrial proline oxidase/dehydrogenase (POX/PRODH) that is causal in triggering a ROS-dependent induction of ATGL. We demonstrate that ATGL promotes the expression of genes related to mitochondrial oxidative metabolism (peroxisome proliferator-activated receptor-α, peroxisome proliferator-activated receptor-γ coactivator-1α), thus setting a metabolic switch towards fat utilization that supplies energy to starved adipocytes and prevents cell death, as well as adipose tissue inflammation. Taken together, these results identify ATGL as a stress resistance mediator in adipocytes, restraining visceral adipose tissue dysfunction typical of age-related metabolic disorders.

Evaluation of different types of dry-cured ham by Italian and French consumers.

The aim was to analyse the distribution of preferences between two group of consumers (100 consumers each) from different countries (Italy and France), in order to establish which type of dry-cured ham is most acceptable and to compare results across countries using preference mapping. A preference test was carried out on four types of dry-cured hams: Parma ham (P), Italian non-branded ham (I) and hams derived from pigs fed a diet containing sunflower oil (2.5%) (S) or extruded linseed (5%) (L). The consumers were requested to evaluate each single descriptor using a hedonic scale and assigning a score between 1 (dislike extremely) and 9 (like extremely). The four different dry-cured hams were sensorially well differentiated by the consumers of different nationalities. Italian consumers preferred P and S hams while French consumers preferred S, I and P hams in terms of overall acceptability and acceptability of aroma and flavour. L ham was negatively discriminated, compared to the other hams, by both consumer groups. A good differentiation between the dry-cured hams was obtained using the internal preference map method: P was discriminated from the other hams.

Effect of visual marbling on sensory properties and quality traits of pork loin.

Pork loins (N=53) were selected from a commercial packing plant to determine the influence of subjective marbling score on sensory attributes and eating quality properties. The pork loins were obtained from commercially raised hybrid barrows (average carcass weight=67.7 kg), originating from nine cooperating herds, and fed similar diets throughout the finishing period. Carcass quality measurements, trained sensory panel analyses, fatty acid composition, thiobarbituric acid-reactive substance (TBARS) index, and cholesterol content were assessed and analyzed on the individual pork loins. With an increase in marbling level, there was a corresponding decrease in drip loss (P=0.049) and observed increases in pH (P=0.001), sensory tenderness (P=0.001), and sensory juiciness scores (P=0.017). The most notable results demonstrated that protein concentrations were reduced as marbling levels amplified (P=0.012). The increase in marbling score was observed to be a significant source of variation in polyunsaturated fatty acid (PUFA) concentrations. Linoleic and arachidonic acids decreased in both raw and cooked samples as marbling score increased. The data demonstrated that visual marbling score does have an influence on sensory properties and pork quality.

Omega-3 polyunsaturated fatty acid from extruded linseed influences the fatty acid composition and sensory characteristics of dry-cured ham from heavy pigs.

Twenty-four barrows (78.1 +/- 1.7 kg of initial BW) were fed a control diet (2.5% sunflower oil) or an experimental diet containing 5% whole extruded linseed. Both diets were supplemented with 170 mg of vitamin E/kg of feed. At slaughter (160 +/- 9.2 kg of BW), 6 hams were collected per group and delivered to Stazione Sperimentale per l'Industria delle Conserve Alimentari of Parma for seasoning. There was no effect (P > 0.05) of diet on the physicochemical characteristics of dry-cured ham. The linseed diet increased (P < 0.05) the content of n-3 PUFA in trimmed fat (green ham), semimembranosus muscle, subcutaneous adipose tissue, and the whole slice (dry-cured ham). Moreover, there was a decrease in the n-6:n-3 PUFA ratio from 12 to 3 in all of those tissues. In contrast, the greater alpha-linolenic acid content in linseed caused an increase in the iodine value of green hams to a value that is not accepted by the Parma Ham Consortium. A sensory consumer test indicated that linseed-enriched dry-cured ham had the least acceptance score for odor, taste, and overall acceptability descriptors. These data suggest that the use of extruded linseed for pig feed is an acceptable way to improve the nutritional quality of long-cured pork products but could be limited by negative effects on the sensory characteristics of dry-cured ham.

Genetic analysis of metamorphic and premetamorphic Xenopus ciliary marginal zone.

A major event affecting the eye during amphibian metamorphosis is an asymmetrical growth of the ventrotemporal portion of the retina compared with its dorsonasal counterpart. This event is due to an increased proliferation of the precursors of the ventral ciliary marginal zone (CMZ). Here, we analyze the expression patterns of several key homeobox genes implicated in eye development (Xrx1, Xvax2, Xsix3, Xpax6, Xchx10, Xotx2) to understand whether they are active at the time in which the metamorphic changes of the retina occur. We also analyze their expression patterns in the ventral and dorsal CMZ and compare them with bromodeoxyuridine incorporation in the CMZ. Our results suggest that the metamorphic CMZ maintains the functional subdivisions described during embryonic development. Moreover, we find that genes involved in proliferation and cell type determination of the embryonic retina are actively transcribed in the proliferating CMZ, thus indicating a potential regulatory role for these genes in the metamorphic retina.

Designated "no smoking" areas provide from partial to no protection from environmental tobacco smoke.

OBJECTIVE: To determine the efficacy of designated "no smoking" areas in the hospitality industry as a means of providing protection from environmental tobacco smoke (ETS), and whether certain design features assist in achieving this end. METHODOLOGY: In the greater metropolitan region of Sydney, a representative group of 17 social and gaming clubs, licensed to serve alcoholic beverages and in which, apart from designated areas, smoking occurs, agreed to participate. In each establishment, simultaneous single measurements of atmospheric nicotine, particulate matter (10 microm; PM10) and carbon dioxide (CO2) levels were measured in a general use area and in a designated "no smoking" area during times of normal operation, together with the levels in outdoor air (PM10 and CO2 only). Analyses were made of these data to assess the extent to which persons using the "no smoking" areas were protected from exposure to ETS. RESULTS: By comparison with levels in general use areas, nicotine and particulate matter levels were significantly less in the "no smoking" areas, but were still readily detectable at higher than ambient levels. For nicotine, mean (SD) levels were 100.5 (45.3) microg/m3 in the areas where smoking occurred and 41.3 (16.1) microg/m3 in the "no smoking" areas. Corresponding PM10 levels were 460 (196) microg/m3 and 210 (210) microg/m3, while outdoor levels were 61 (23) microg/m3. The reduction in pollutants achieved through a separate room being designated "no smoking" was only marginally better than the reduction achieved when a "no smoking" area was contiguous with a smoking area. CO2 levels were relatively uninformative. CONCLUSION: Provision of designated "no smoking" areas in licensed (gaming) clubs in New South Wales, Australia, provides, at best, partial protection from ETS-typically about a 50% reduction in exposure. The protection afforded is less than users might reasonably have understood and is not comparable with protection afforded by prohibiting smoking on the premises.

Nerve-independence of limb regeneration in larval Xenopus laevis is correlated to the level of fgf-2 mRNA expression in limb tissues.

In both larval and adult urodele amphibians, limb blastema formation requires the presence of an adequate nerve supply. In previous research, we demonstrated that the hindlimb of early Xenopus laevis larvae formed a regeneration blastema even when denervated, while the denervated limb of late larvae did not. We hypothesized that the nerve-independence was due to the autonomous synthesis of a mitogenic neurotrophic-like factor by undifferentiated limb bud cells. In this paper, we demonstrate that fgf-2 mRNA is present in larval limb tissues and that its level is correlated to the extent of mesenchymal cells populating the limb: in early limbs, fgf-2 mRNA is present at high levels all over the limb, while, in late limbs, the fgf-2 expression is low and detectable only in the distal autopodium. After denervation, fgf-2 mRNA synthesis increases in amputated early limbs but not in amputated late limbs. The implantation of anti-FGF-2 beads into amputated early limbs hardly lowers the mitotic activity of blastema cells. However, FGF-2 beads implanted into the blastema of late limbs prevent the denervation-induced inhibition of mitosis and oppose blastema regression. Our data indicate that FGF-2 is a good candidate for the endogenous mitogenic factor responsible for blastema formation and growth in amputated and denervated early limbs. However, in amputated late limbs, the very limited fgf-2 expression is not sufficient to promote blastema formation in the absence of nerves.

Nerve-independence of limb regeneration in larval Xenopus laevis is related to the presence of mitogenic factors in early limb tissues.

Early limbs of larval Xenopus laevis can form a regeneration blastema in the absence of nerves. The nerve-independence could be due to the synthesis of neurotrophic-like factors by the limb bud cells. To test this hypothesis, two series of experiments were performed. Series A: the right hindlimbs of stage 57 larvae (acc. to Nieuwkoop and Faber. 1956. Normal table of Xenopus laevis [Daudin]. Amsterdam: North-Holland Pub. Co.), which are nerve-dependent for regeneration, were amputated through the tarsalia. The regenerating limbs were submitted to: sham denervation; denervation; denervation and implantation of a fragment of an early limb, or a late limb, or a spinal cord. Series B: froglets were subjected to amputation of both forelimbs. The cone blastemas were transplanted into denervated hindlimbs of stage 57 larvae, together with a fragment of an early or a late limb. The results in series A showed that the implantation of early limb tissue into the denervated blastema maintained cell proliferation at levels similar to those observed after the implantation of a spinal cord fragment or in sham denervated blastemas. However, the implantation of late limb tissues were ineffective. The results of series B showed that the implantation of early limb tissue, but not of late limb tissue prevented the inhibition of cell proliferation and the regression of denervated limb blastemas of juveniles. These results indicate that the nerve-independence is related to the synthesis of diffusible mitogenic neurotrophic-like factors in early limb tissues, and that nerve-dependence is established when differentiated cells of late limb tissues stop producing these factors.

Lens regeneration in larval Xenopus laevis: experimental analysis of the decline in the regenerative capacity during development.

In Xenopus laevis, the capacity to regenerate a new lens from the outer cornea gradually decreases between stages 50 and 58, is almost negligible during the metamorphic climax, and disappears after metamorphosis. The factors responsible for lens transdifferentiation of the outer cornea are produced by the neural retina and are located in the vitreous chamber. This decrease in the regenerative capacity may be due to: (1) a reduction of the inductive power of the retina, (2) a reduction of lens-forming competence of the outer cornea, (3) an inhibition of the lens transdifferentiation process, (4) a combination of these causes. In order to test these hypotheses, fragments of outer cornea or of outer and inner corneas joined together were isolated from early larvae, late larvae and froglets, and implanted into the eye of host larvae during the premetamorphosis or the metamorphic climax. Results from implants of outer cornea into the vitreous chamber showed that the drop in lens regeneration capacity during the metamorphic climax is not due to a decrease in the inductive power of the retinal factor and that the gradual decrease in the regenerative capacity observed between stages 50 and 58 is not related to a substantial diminution in the capacity of outer cornea cells to transdifferentiate into lens fibers. Results from implants of outer and inner corneas joined together showed that in these implants the lens transdifferentiation of the outer cornea was partially inhibited. These findings indicate that the decrease in lens regeneration is mainly due to an inhibition of the lens transdifferentiation process of the outer cornea by the inner cornea. However, even implants of cornea (multilayered epithelium and substantia propria) excised from metamorphosed animals were able to form lens fibers, although to a lesser percentage than that obtained after implantation of fragments of larval outer and inner corneas. Thus, the lens-forming competence in the corneal epithelium is still present to a certain degree even when lens regeneration capacity is lost. Several observations suggest that in the lentectomized eye of late larvae and froglets the mechanical inhibition of lens transdifferentiation process exerted by the inner cornea (or the substantia propria), due to the rapid formation of a connective barrier against the spreading of the retinal factor toward the outer cornea, has a decisive role in maintaining the phenotypic stability of the outer cornea.

Infection control and contaminated waste disposal practices in Southern Sydney Area Health Service Dental Clinics.

The sterilization and contaminated waste disposal practices in all 14 dental clinics operated by the Southern Sydney Area Health Service were surveyed. All of the clinics used autoclaves for sterilization. All hand instruments, handpieces and triplex syringes were autoclaved between patients. Chemical disinfection solutions were used in 12 of the 14 dental clinics, mainly for surface decontamination. Five dental clinics had separate storage areas for contaminated waste which compiled with contaminated waste separation and disposal guidelines. The practice of recapping needles with fingers and some inadequate washing facilities are areas that require particular attention.

Morphogenesis and differentiation of grafted blastemas formed in vitro from amputated hindlimbs of larval Xenopus laevis.

The present study was designed to test the morphogenetic potency of limb blastemas formed in vitro from amputated limbs of larval Xenopus laevis. Hindlimbs of larvae at stage 55 (according to Nieuwkoop and Faber [1956] Normal Table of Xenopus laevis (Daudin)) were amputated through the tarsalia, excised at the base of the thigh and cultured in Leibovitz's L-15 supplemented with 2% FCS. After 8-10 days, 50% of the cultured limbs formed a conic blastema on the amputation surface. However, on the excision surface no blastema was present. Three different parts (blastema, blastema with the shank region and proximal part of the limb) of the cultured limbs were then grafted to the axial musculature or to the hindlimb of stage 57 host larvae. Results showed that the blastema formed in vitro were true autodifferentiating regeneration blastemas, since they were able to form well-differentiated autopodia not only when grafted with the shank region to a neutral territory (axial musculature) or to the limb territory, but also when transplanted alone to the two environments. The morphological complexity (no. of toes) of the autopodia differentiated from the grafted blastemas was superimposable to that observed in vivo. Moreover, as in vivo, the entire regeneration process was nerve-independent. In fact, the regeneration blastemas, formed in vitro in the complete absence of nerves, could grow and differentiate also when grafted to denervated host limbs. The grafted proximal parts of the cultured limbs never formed a regenerate.

Effect of ammodytin L from the venom of Vipera ammodytes on Xenopus laevis differentiated muscle fibres and regenerating limbs.

Ammodytin L is a non-catalytic, phospholipase-like snake venom toxin from Vipera ammodytes, which shows a cytotoxic activity on differentiated myotubes when tested in vitro. In the range of concentrations in which ammodytin L induced necrosis of myogenic cells in culture, other cell types (erythrocytes, platelets, fibroblasts) did not appear to be affected. To test the in vivo toxicity and the effective cytolytic specificity of ammodytin L we have followed the morphological changes in muscle tissue of Xenopus laevis limbs after intramuscular toxin injection. Only muscular cells were affected by ammodytin L, and the toxin did not induce any morphological change in other cell types. Further evidence of the muscle-specific action of the toxin was obtained from experiments carried out using the Xenopus kidney cell line B3.2 in culture. Ammodytin L was unable to affect parameters of cell viability such as lactate dehydrogenase leakage, [3H]thymidine incorporation, growth curves and morphological changes. Moreover, direct ammodytin L application to cultured regenerative limbs did not provoke alterations in undifferentiated myoblasts. These data suggest that ammodytin L, like other phospholipase-like toxins, exerts its toxicity by selectively damaging differentiated muscle fibres.

The relationship of innervation and differentiation to regenerative capacity in the reamputated hindlimb of larval Xenopus laevis.

Regenerated hindlimbs of larval Xenopus laevis were reamputated at critical larval stages and levels, viz when amputation of the control limb at the same larval stage and level is followed by reduced regeneration. Reamputations were performed at the level of (1) the original plane of amputation, (2) the early regenerate (cone/palette stage), (3) the late regenerate (digit stage). Reamputation increased both the percentage rate of regeneration and the morphological complexity of the regenerates in all experimental series. Cell counts in lateral motor columns and spinal ganglia innervating the hindlimb, together with histological observations and mitotic index and labelling index determinations in reamputated and control limbs showed that improved regeneration in the reamputated limb was related to an increase in undifferentiated and proliferating cells in the stump. We did not find any evidence suggesting that renewed regeneration in reamputated anuran limbs results from an increase in innervation, as has previously been hypothesized. We support our conclusions by demonstrating an improvement in regenerationen in the reamputated and denervated hindlimbs.

Lens formation from cornea implanted into amputated hindlimbs of Xenopus laevis larvae requires innervation or proliferating cell populations in the stump.

The capacity of amputated early and late limbs of larval Xenopus laevis to promote lens-forming transformations of corneal implants in the absence of a limb regeneration blastema has been tested by implanting outer cornea fragments from donor larvae at stage 48 (according to Nieuwkoop and Faber 1956), into limb stumps of larvae at stage 52 and 57. Blastema formation has been prevented either by covering the amputation surface with the skin or by reconnecting the amputated part to the limb stump. Results show that stage 52 non-regenerating limbs could promote lens formation from corneal implants not only when innervated but also when denervated. A similar result was observed in stage 57 limbs where blastema formation was prevented by reconnecting the amputated part to the stump. In this case, relevant tissue dedifferentiation was observed in the boundary region between the stump and the autografted part of the limb. However, stage 57 limbs, where blastema formation was prevented by covering the amputation surface with skin, could promote lens formation from the outer cornea only when innervated. In this case, no relevant dedifferentiation of the stump tissues was observed. These results indicate that blastema formation is not a prerequisite for lens-forming transformations of corneal fragments implanted into amputated hindlimbs of larval X. laevis and that lens formation can be promoted by factors delivered by the nerve fibres or produced by populations of undifferentiated or dedifferentiated limb cells.

Acquisition of nerve dependence for the formation of a regeneration blastema in amputated hindlimbs of larval Xenopus laevis: the role of limb innervation and that of limb differentiation.

In larval and adult urodeles and late-stage larval anurans, blastema formation after limb amputation requires an adequate nerve supply. Experimental evidence obtained from aneurogenic limbs indicates that, in urodeles, the acquisition of nerve dependence during embryonic development is due to the "addiction" of limb tissues to factors released by the ingrowing nerves rather than to limb differentiation. The aim of this work was to establish whether, in the toad Xenopus laevis, nerve-dependence for blastema formation after hindlimb amputation, which is acquired gradually during larval development and becomes complete at stage 57 is due to limb innervation or to limb differentiation. Two series of experiments were carried out. In the first series, limb differentiation was inhibited by treating the larvae with an anti-thyroid drug, and innervation was maintained for an interval much longer than that normally required for development from nerve-independent stages to stage 57. In the second series, the limb was caused to differentiate in the absence of nerves by maintaining the limbs denervated. Limb differentiation was often accelerated by treating early-stage larvae with thyroxine or by grafting early-stage limbs onto denervated limbs of late larvae, which, being near metamorphic climax, possessed high levels of circulating thyroid hormones. Results showed that in the first series of experiments the denervated limbs formed regeneration blastemas after amputation, but in the second series they did not. It was therefore concluded that the acquisition of nerve dependence for blastema formation in larval Xenopus laevis is not directly imposed by factors released by the nerve fibers, but is strongly related to differentiation of limb tissues.

The inhibition of cell proliferation by mitomycin C does not prevent transdifferentiation of outer cornea into lens in larval Xenopus laevis.

The aim of the present work is to evaluate the relationship between cell proliferation and transdifferentiation (TS) of the outer cornea into lens in larval Xenopus laevis. Data obtained from corneal fragments treated with Mitomycin C (MMC) (0.1 mg/ml, 50 min) and implanted into the vitreous chamber (MMC/v ch) were compared with those obtained from untreated corneal fragments implanted into the vitreous chamber (contr/v ch) or between outer and inner corneas (contr/o c). Results demonstrated that in contr/v ch implants, which transdifferentiated into lenses or lentoid bodies in 88% of cases, the mitotic index (MI) showed a sharp increase during the period of lens vesicle formation (3 days) and became very low when the formation of lens fibres was under way (7 days). In contro c implants, which did not undergo any lens forming transformations, the MI remained unchanged in comparison to time O. In MMC/v ch implants, the inhibition of the mitotic activity was 100% up to the third day after implantation. On the fifth and seventh days, scant mitotic activity was observed in some cases, but the MI was much lower than the MI of contr/o c implants. The MMC/v ch implants transdifferentiated into lentoid bodies in 26% of cases. The lentoid bodies were much smaller than those observed in control implants, but they reacted positively with the lens antibodies at the same time after implantation as controls. Even the complete inhibition of proliferation due to stronger MMC treatments (e.g. 0.15 mg/ml, 50 min) did not prevent lens TS.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in the decrease in regenerative capacity of various brain regions of Xenopus laevis are related to differences in the undifferentiated cell populations.

The extent of the undifferentiated cell population in normal and regenerating brains of larvae and metamorphosed individuals of Xenopus laevis has been analyzed by means of an immunocytochemical method and mitotic index determinations. Results show that the decrease in regenerative capacity of the brain during larval development and after metamorphosis is in relation with the gradual reduction of the population of undifferentiated cells and that the different regenerative capacities of the various brain districts are related to quantitative and qualitative differences in this cell population. While in the early larval stages the cell population formed of actively cycling cells is very large and widespread, in late larval stages and after metamorphosis these cells localize in some encephalic areas (matrix zones). This localization occurs later in the telencephalon than in the rhombencephalon and in mesencephalon. The less conspicuous decrement in the regenerative capacity of the telencephalon than of other encephalic districts of froglets, particularly the mesencephalon, is related to the presence of a larger number of actively cycling cells together with a rather large number of undifferentiated cells which are in a temporary quiescent state from which they may re-enter the actively cycling state in response to proliferation promoting factors.

Medullary and gangliar regeneration after unilateral removal of a segment of spinal cord of the trunk and corresponding ganglion in adult newts.

Regeneration of the spinal cord, segmental nerves and sensory ganglia takes place after tail amputation in the newt. Many histological and immunocytochemical observations provide evidence that the ependymal tube is the source not only of new neurons and glial cells in the spinal cord, but also of some cells that go on to participate in the formation of the spinal ganglia of the regenerating tail. In previous experiments involving the removal of the spinal ganglia of the trunk, no regeneration was observed and it is thought that the trunk region differs from the tail region with regard to the ability to regenerate sensory ganglia. However, in these experiments the spinal cord of the trunk was not damaged. In the present work involving adult newts (Triturus carnifex Laur.), unilateral ablation of a segment of the spinal cord of the trunk in addition to removal of a corresponding spinal ganglion was performed. In these experimental conditions, regeneration of a rudimentary spinal ganglion near the regenerated side of the spinal cord segment was observed in several cases. Histological observations carried out 2, 4, 6 and 13 months after the operation support the view that some cells migrating from the lateroventral part of the regenerating side of the spinal cord via the developing ventral root could participate in the formation of the rudimentary spinal ganglion.