The dorsal portion of the bovine diaphragm as a useful tissue for producing a 3D muscle scaffold.

Three-dimensional (3D) organoids are an innovative approach to obtain an in vitro model for ex vivo studies to overcome the limitations of monolayer cell culture and reduce the use of animal models. An organoid of skeletal muscle requires the presence of the extracellular matrix to represent a functional muscle in vitro, which is why decellularized tissue is an optimal choice. Various muscles have been considered to produce a muscle organoid, most from rodents or small animals, and only recently some studies have been reported on the muscles of large animals. This work presents a muscular organoid produced from the bovine diaphragm, which has a peculiar multilayered structure with different fibre orientations depending on the considered area. This paper analyses the anatomical structure of the bovine diaphragm, selects the most appropriate portion, and presents a decellularization protocol for a multilayered muscle. In addition, a preliminary test of recellularization with primary bovine myocytes was presented with the future aim of obtaining a 3D muscle allogenic organoid, completely bovine-derived. The results demonstrate that the dorsal portion of bovine diaphragm presents a regular alternation of muscular and fibrous layers and that the complete decellularization does not affect the biocompatibility. These results provide a strong foundation for the potential application of this portion of tissue as a scaffold for in vitro studies of muscle organoids.

Mutable Collagenous Tissue Isolated from Echinoderms Leads to the Production of a Dermal Template That Is Biocompatible and Effective for Wound Healing in Rats.

The mutable collagenous tissue (MCT) of echinoderms possesses biological peculiarities that facilitate native collagen extraction and employment for biomedical applications such as regenerative purposes for the treatment of skin wounds. Strategies for skin regeneration have been developed and dermal substitutes have been used to cover the lesion to facilitate cell proliferation, although very little is known about the application of novel matrix obtained from marine collagen. From food waste we isolated eco-friendly collagen, naturally enriched with glycosaminoglycans, to produce an innovative marine-derived biomaterial assembled as a novel bi-layered skin substitute (Marine Collagen Dermal Template or MCDT). The present work carried out a preliminary experimental in vivo comparative analysis between the MCDT and Integra, one of the most widely used dermal templates for wound management, in a rat model of full-thickness skin wounds. Clinical, histological, and molecular evaluations showed that the MCDT might be a valuable tool in promoting and supporting skin wound healing: it is biocompatible, as no adverse reactions were observed, along with stimulating angiogenesis and the deposition of mature collagen. Therefore, the two dermal templates used in this study displayed similar biocompatibility and outcome with focus on full-thickness skin wounds, although a peculiar cellular behavior involving the angiogenesis process was observed for the MCDT.

From Food Waste to Innovative Biomaterial: Sea Urchin-Derived Collagen for Applications in Skin Regenerative Medicine.

Collagen-based skin-like scaffolds (CBSS) are promising alternatives to skin grafts to repair wounds and injuries. In this work, we propose that the common marine invertebrate sea urchin represents a promising and eco-friendly source of native collagen to develop innovative CBSS for skin injury treatment. Sea urchin food waste after gonad removal was here used to extract fibrillar glycosaminoglycan (GAG)-rich collagen to produce bilayer (2D + 3D) CBSS. Microstructure, mechanical stability, permeability to water and proteins, ability to exclude bacteria and act as scaffolding for fibroblasts were evaluated. Our data show that the thin and dense 2D collagen membrane strongly reduces water evaporation (less than 5% of water passes through the membrane after 7 days) and protein diffusion (less than 2% of BSA passes after 7 days), and acts as a barrier against bacterial infiltration (more than 99% of the different tested bacterial species is retained by the 2D collagen membrane up to 48 h), thus functionally mimicking the epidermal layer. The thick sponge-like 3D collagen scaffold, structurally and functionally resembling the dermal layer, is mechanically stable in wet conditions, biocompatible in vitro (seeded fibroblasts are viable and proliferate), and efficiently acts as a scaffold for fibroblast infiltration. Thus, thanks to their chemical and biological properties, CBSS derived from sea urchins might represent a promising, eco-friendly, and economically sustainable biomaterial for tissue regenerative medicine.

Covalently bound DNA on naked iron oxide nanoparticles: Intelligent colloidal nano-vector for cell transfection.

BACKGROUND: Conversely to common coated iron oxide nanoparticles, novel naked surface active maghemite nanoparticles (SAMNs) can covalently bind DNA. Plasmid (pDNA) harboring the coding gene for GFP was directly chemisorbed onto SAMNs, leading to a novel DNA nanovector (SAMN@pDNA). The spontaneous internalization of SAMN@pDNA into cells was compared with an extensively studied fluorescent SAMN derivative (SAMN@RITC). Moreover, the transfection efficiency of SAMN@pDNA was evaluated and explained by computational model. METHODS: SAMN@pDNA was prepared and characterized by spectroscopic and computational methods, and molecular dynamic simulation. The size and hydrodynamic properties of SAMN@pDNA and SAMN@RITC were studied by electron transmission microscopy, light scattering and zeta-potential. The two nanomaterials were tested by confocal scanning microscopy on equine peripheral blood-derived mesenchymal stem cells (ePB-MSCs) and GFP expression by SAMN@pDNA was determined. RESULTS: Nanomaterials characterized by similar hydrodynamic properties were successfully internalized and stored into mesenchymal stem cells. Transfection by SAMN@pDNA occurred and GFP expression was higher than lipofectamine procedure, even in the absence of an external magnetic field. A computational model clarified that transfection efficiency can be ascribed to DNA availability inside cells. CONCLUSIONS: Direct covalent binding of DNA on naked magnetic nanoparticles led to an extremely robust gene delivery tool. Hydrodynamic and chemical-physical properties of SAMN@pDNA were responsible of the successful uptake by cells and of the efficiency of GFP gene transfection. GENERAL SIGNIFICANCE: SAMNs are characterized by colloidal stability, excellent cell uptake, persistence in the host cells, low toxicity and are proposed as novel intelligent DNA nanovectors for efficient cell transfection.

Wound-healing markers after autologous and allogeneic epithelial-like stem cell treatment.

BACKGROUND AIMS: Several cytokines and growth factors play an essential role in skin regeneration and epithelial-like stem cells (EpSCs) have beneficial effects on wound healing in horses. However, there are no reports available on the expression of these growth factors and cytokines after EpSC therapy. METHODS: Wounds of 6 cm(2) were induced in the gluteus region of 6 horses and treated with (i) autologous EpSCs, (ii) allogeneic EpSCs, (iii) vehicle treatment or (iv) untreated control. Real time polymerase chain reaction was performed on tissue biopsies taken 1 and 5 weeks after these treatments to evaluate mRNA expression of interferon (IFN)-γ, interleukin (IL)-6, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF)-1 and epidermal keratin (eKER). RESULTS: One week after treatments, mRNA levels of IL-6 (P = 0.012) and VEGF (P = 0.008) were higher in allogeneic EpSC-treated wounds compared with controls. Also, mRNA levels of IGF-1 were higher at 1 week in both autologous (P = 0.027) and allogeneic (P = 0.035) EpSC-treated wounds. At week 5, all EpSC- and vehicle-treated wounds demonstrated significantly higher IFN-γ, VEGF and eKER mRNA expression compared with controls and compared with their respective levels at week 1. CONCLUSIONS: Equine wounds treated with allogeneic EpSCs demonstrate a significant increase in mRNA expression of IL-6, VEGF and IGF-1 in the acute phase. In the longer term, an increase in IFN-γ, VEGF and eKER mRNA was detected in the wounds treated with allogenic EpSCs, autologous EpSCs or their vehicle.

Production, characterization and biocompatibility of marine collagen matrices from an alternative and sustainable source: the sea urchin Paracentrotus lividus.

Collagen has become a key-molecule in cell culture studies and in the tissue engineering field. Industrially, the principal sources of collagen are calf skin and bones which, however, could be associated to risks of serious disease transmission. In fact, collagen derived from alternative and riskless sources is required, and marine organisms are among the safest and recently exploited ones. Sea urchins possess a circular area of soft tissue surrounding the mouth, the peristomial membrane (PM), mainly composed by mammalian-like collagen. The PM of the edible sea urchin Paracentrotus lividus therefore represents a potential unexploited collagen source, easily obtainable as a food industry waste product. Our results demonstrate that it is possible to extract native collagen fibrils from the PM and produce suitable substrates for in vitro system. The obtained matrices appear as a homogeneous fibrillar network (mean fibril diameter 30-400 nm and mesh < 2 µm) and display remarkable mechanical properties in term of stiffness (146 ± 48 MPa) and viscosity (60.98 ± 52.07 GPa·s). In vitro tests with horse pbMSC show a good biocompatibility in terms of overall cell growth. The obtained results indicate that the sea urchin P. lividus can be a valuable low-cost collagen source for mechanically resistant biomedical devices.

Description of a double centrifugation tube method for concentrating canine platelets.

BACKGROUND: To evaluate the efficiency of platelet-rich plasma preparations by means of a double centrifugation tube method to obtain platelet-rich canine plasma at a concentration at least 4 times higher than the baseline value and a concentration of white blood cells not exceeding twice the reference range. A complete blood count was carried out for each sample and each concentrate. Whole blood samples were collected from 12 clinically healthy dogs (consenting blood donors). Blood was processed by a double centrifugation tube method to obtain platelet concentrates, which were then analyzed by a flow cytometry haematology system for haemogram. Platelet concentration and white blood cell count were determined in all samples. RESULTS: Platelet concentration at least 4 times higher than the baseline value and a white blood cell count not exceeding twice the reference range were obtained respectively in 10 cases out of 12 (83.3%) and 11 cases out of 12 (91.6%). CONCLUSIONS: This double centrifugation tube method is a relatively simple and inexpensive method for obtaining platelet-rich canine plasma, potentially available for therapeutic use to improve the healing process.

Assessment of the quality of the healing process in experimentally induced skin lesions treated with autologous platelet concentrate associated or unassociated with allogeneic mesenchymal stem cells: preliminary results in a large animal model.

Regenerative medicine for the treatment of skin lesions is an innovative and rapidly developing field that aims to promote wound healing and restore the skin to its original condition before injury. Over the years, different topical treatments have been evaluated to improve skin wound healing and, among them, mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) have shown promising results for this purpose. This study sought to evaluate the quality of the healing process in experimentally induced full-thickness skin lesions treated with PRP associated or unassociated with MSCs in a sheep second intention wound healing model. After having surgically created full-thickness wounds on the back of three sheep, the wound healing process was assessed by performing clinical evaluations, histopathological examinations, and molecular analysis. Treated wounds showed a reduction of inflammation and contraction along with an increased re-epithelialization rate and better maturation of the granulation tissue compared to untreated lesions. In particular, the combined treatment regulated the expression of collagen types I and III resulting in a proper resolution of the granulation tissue contrary to what was observed in untreated wounds; moreover, it led to a better maturation and organization of skin adnexa and collagen fibers in the repaired skin compared to untreated and PRP-treated wounds. Overall, both treatments improved the wound healing process compared to untreated wounds. Wounds treated with PRP and MSCs showed a healing progression that qualitatively resembles a restitutio ad integrum of the repaired skin, showing features typical of a mature healthy dermis.

Extracellular ATP signaling during differentiation of C2C12 skeletal muscle cells: role in proliferation.

Evidence shows that extracellular ATP signals influence myogenesis, regeneration and physiology of skeletal muscle. Present work was aimed at characterizing the extracellular ATP signaling system of skeletal muscle C2C12 cells during differentiation. We show that mechanical and electrical stimulation produces substantial release of ATP from differentiated myotubes, but not from proliferating myoblasts. Extracellular ATP-hydrolyzing activity is low in myoblasts and high in myotubes, consistent with the increased expression of extracellular enzymes during differentiation. Stimulation of cells with extracellular nucleotides produces substantial Ca(2+) transients, whose amplitude and shape changed during differentiation. Consistently, C2C12 cells express several P2X and P2Y receptors, whose level changes along with maturation stages. Supplementation with either ATP or UTP stimulates proliferation of C2C12 myoblasts, whereas excessive doses were cytotoxic. The data indicate that skeletal muscle development is accompanied by major functional changes in extracellular ATP signaling.

A Prototype Skin Substitute, Made of Recycled Marine Collagen, Improves the Skin Regeneration of Sheep.

Skin wound healing is a complex and dynamic process that aims to restore lesioned tissues. Collagen-based skin substitutes are a promising treatment to promote wound healing by mimicking the native skin structure. Recently, collagen from marine organisms has gained interest as a source for producing biomaterials for skin regenerative strategies. This preliminary study aimed to describe the application of a collagen-based skin-like scaffold (CBSS), manufactured with collagen extracted from sea urchin food waste, to treat experimental skin wounds in a large animal. The wound-healing process was assessed over different time points by the means of clinical, histopathological, and molecular analysis. The CBSS treatment improved wound re-epithelialization along with cell proliferation, gene expression of growth factors (VEGF-A), and development of skin adnexa throughout the healing process. Furthermore, it regulated the gene expression of collagen type I and III, thus enhancing the maturation of the granulation tissue into a mature dermis without any signs of scarring as observed in untreated wounds. The observed results (reduced inflammation, better re-epithelialization, proper development of mature dermis and skin adnexa) suggest that sea urchin-derived CBSS is a promising biomaterial for skin wound healing in a "blue biotechnologies" perspective for animals of Veterinary interest.

Could cold plasma act synergistically with allogeneic mesenchymal stem cells to improve wound skin regeneration in a large size animal model?

Skin wound healing may sometimes lead to open sores that persist for long periods and expensive hospitalization is needed. Among different kinds of therapeutic innovative approaches, mesenchymal stem cells (MSCs) and low-temperature atmospheric pressure cold plasma (ionized gas) have been recently tested to improve this regenerative process. To optimize wound healing the present study intended to combine, for the first time, these two novel approaches in a large size animal wound healing model with the aim of assessing the putative dual beneficial effects. Based on clinical, histopathological, and molecular results a synergistic action in a second intention healing wound in sheep has been observed. Experimental wounds treated with cold plasma and MSCs showed a slower but more effective healing compared to the single treatment, as observed in previous studies. The combined treatment improved the correct development of skin appendages and structural proteins of the dermis showing the potential of the dual combination as a safe and effective tool for skin regeneration in the veterinary clinical field.

Autologous Platelet-Rich Plasma Enhances the Healing of Large Cutaneous Wounds in Dogs.

Platelet-rich plasma (PRP) is known to play a crucial role in skin wound healing, in both Human and Veterinary Medicine. Remarkably, until now, no studies have reported PRP treatment in subacute full-thickness skin wounds of the dog. The aim of this study was to evaluate the effects of two consecutive applications of autologous PRP, with the second application after 15 days, in 6 dogs showing large subacute skin wounds. The percentage of contraction, re-epithelialization and healing in all treated patients indicated that no complications or side effects, associated with consecutive PRP treatments, occurred in any patient and all wounds achieved complete closure and re-epithelialization. Our results suggest a positive effect of repeated autologous topical PRP treatments in large cutaneous subacute wounds of different etiology. Therefore, this PRP treatment could represent a simple, cost-effective, and valid alternative to promote healing processes in subacute large wounds cases in dogs.

An Assay System to Evaluate Riboflavin/UV-A Corneal Phototherapy Efficacy in a Porcine Corneal Organ Culture Model.

The purpose of this study was to investigate the response of porcine corneal organ cultures to riboflavin/UV-A phototherapy in the injury healing of induced lesions. A porcine corneal organ culture model was established. Corneal alterations in the stroma were evaluated using an assay system, based on an automated image analysis method able to (i) localize the holes and gaps within the stroma and (ii) measure the brightness values in these patches. The analysis has been performed by dividing the corneal section in 24 regions of interest (ROIs) and integrating the data analysis with a "multi-aspect approach." Three group of corneas were analyzed: healthy, injured, and injured-and-treated. Our study revealed a significant effect of the riboflavin/UV-A phototherapy in the injury healing of porcine corneas after induced lesions. The injured corneas had significant differences of brightness values in comparison to treated (p < 0.00) and healthy (p < 0.001) corneas, whereas the treated and healthy corneas showed no significant difference (p = 0.995). Riboflavin/UV-A phototherapy shows a significant effect in restoring the brightness values of damaged corneas to the values of healthy corneas, suggesting treatment restores the injury healing of corneas after lesions. Our assay system may be compared to clinical diagnostic methods, such as optical coherence tomography (OCT) imaging, for in vivo damaged ocular structure investigations.

Myostatin shows a specific expression pattern in pig skeletal and extraocular muscles during pre- and post-natal growth.

Myogenesis is driven by an extraordinary array of cellular signals that follow a common expression pattern among different animal phyla. Myostatin (mstn) is a secreted growth factor that plays a pivotal role in skeletal muscle mass regulation. The aim of the present study was to investigate mstn expression in a large mammal (the pig) in order to ascertain whether distinct expression changes of this factor might be linked to the fiber-type composition of the muscle examined and/or to specific developmental stages. To assess the expression pattern of mstn in relation to myogenic proliferative (Pax7 and MyoD) and differentiative (myogenin) markers, we evaluated muscles with different myosin heavy-chain compositions sampled during pre- and post-natal development and on myogenic cells isolated from the same muscles. Skeletal muscles showed higher levels of mRNA for mstn and all other genes examined during fetal development than after birth. The wide distribution of mstn was also confirmed by immunohistochemistry experiments supporting evidence for cytoplasmic staining in early fetal periods as well as the localization in type 1 fibers at the end of the gestation period. Extraocular muscles, in contrast, did not exhibit decreasing mRNA levels for mstn or other genes even in adult samples and expressed higher levels of both mstn mRNA and protein compared with skeletal muscles. Experiments carried out on myogenic cells showed that mstn mRNA levels decreased when myoblasts entered the differentiation program and that cells isolated at early post-natal stages maintained a high level of Pax7 expression. Our results showed that mstn had a specific expression pattern whose variations depended on the muscle type examined, thus supporting the hypothesis that at birth, porcine myogenic cells continue to be influenced by hyperplastic/proliferative mechanisms.

Embryonic chick cocultures of neuronal and muscle cells.

OBJECTIVE: The aim of this report was to set up an effective experimental model of cocultures between cells from spinal cord explants and myotubes from adductor muscle. METHODS: We obtained neuronal cells from chick spinal cord explants at embryonic day 5 (ED5) by means of an enzymatic digestion. Small spinal cord fragments were added in cultured muscle cells committed to the differentiative program. Myoblasts were isolated from the chick adductor muscle at ED12. RESULTS AND DISCUSSION: The validation of the experimental model was confirmed by a remarkable spreading pattern of neuronal cells, labeled with a NF200 antibody, and high concentration of myotubes, marked by alpha-actinin antibody. The indication of neuronal contacts was highlighted by the alpha-bungarotoxin. This communication reports one of the few morphologic description of muscular and neuronal coculture preparation, performed on chick embryos. CONCLUSION: The experimental model presented in this work might be a useful tool to study the cascade of myogenic positive and negative signals activated by paracrine neuronal factors.

Revisiting the peculiar regional distribution of muscle fiber types in rat Sternomastoid Muscle.

The sternomastoid (SM) muscle in rodents is known to have a peculiar distribution of fiber types with a steep gradient from surface to deep region. We here further characterize this peculiar regional distribution by quantitative histochemical morphometrys. In Hematoxylin-Eosin (H-E) stained transverse cryosections harvested in the medial portion of the muscle we counted around 10.000 myofibers with a mean diameter of 51.3±12.6 (µm). Cryisections of the SM stained by SDH reaction clearly show two distinct regions, toward the deep surface of the muscle a 40% area that contains packed SDH-positive myofibers, while the remaining area of the SM toward the external surface presents a more checker-board appearance. On the other hand, in the deep region of SM type 1 (slow contracting) muscle fibers, caracterized by positive acidic ATPase pH 4.35 reaction, are only the 24.5% of the fibers in the deep area of SM muscles, being restricted to the deepest region. The 75.5% of the myofibers in the deep region are of the fast contracting types (either 48.4% 2A, SDH -positive fibers or 27.1% 2B, SDH-negative fibers, respectively). As expected the 2B muscle fibers, acidic ATPase pH 4.3-negative and SDH-negative, present the largest size, while Type 1 fibers, acidic ATPase pH 4.3-positive and SDH-positive, present the smallest size in rat SM muscle. Based on present and previous observations, comparison of change in absolute number and/or percentage of the fiber types in any experimental model of muscle atrophy/hypertrophy/plasticity/pathology /recovery in the rat SM, and possibly of all mammals, will ask for morphometry of the whole muscle cross-sections, muscle sampling by bioptic approches will provide only comparable data on the size of the different types of muscle fibers.

Muscle spindles of the rat sternomastoid muscle.

The sternomastoid (SM) muscle in rodents presents a peculiar distribution of fiber types with a steep gradient from the ventral, superficial, white portion to the dorsal, deep, red region, where muscle spindles are restricted. Cross section of the medial longitudinal third of the rat SM contains around 10,000 muscle fibers with a mean diameter of 51.28±12.62 (µm +/- SD). Transverse sections stained by Succinate Dehydrogenase (SDH) reaction clearly presents two distinct regions: the dorsal deep red portion encompassing a 40% cross section area contains a high percentage of packed SDH-positive muscle fibers, and the ventral superficial region which contains mainly SDH-negative muscle fibers. Indeed, the ventral superficial region of the rat SM muscle contains mainly fast 2B muscle fibers. These acidic ATPase pH 4.3-negative and SDH-negative 2B muscle fibers are the largest of the SM muscle, while the acidic ATPase pH 4.3-positive and SDH-positive Type 1 muscle fibers are the smallest. Here we show that in thin transverse cryosections only 2 or 3 muscle spindle are observed in the central part of the dorsal deep red portion of the SM muscle. Azan Mallory stained sections allow at the same time to count the spindles and to evaluate aging fibrosis of the skeletal muscle tissue. Though restricted in the muscle red region, SM spindles are embedded in perimysium, whose changes may influence their reflex activity. Our findings confirm that any comparisons of changes in number and percentage of muscle spindles and muscle fibers of the rat SM muscle will require morphometry of the whole muscle cross-section. Muscle biopsies of SM muscle from large mammals will only provide partial data on the size of the different types of muscle fibers biased by sampling. Nonetheless, histology of muscle tissue continue to provide practical and low-cost quantitative data to follow-up translational studies in rodents and beyond.

Glial cell line-derived neurotrophic factor expression in the retina of adult zebrafish (Danio rerio).

The glial cell line-derived neurotrophic factor (GDNF) is a well-known growth factor acting on many neuronal populations of central, peripheral and autonomous nervous system. This factor was also previously detected in the retina of developing rat and chicken while no data are available for the zebrafish. In this study transcripts of GDNF mRNA were observed in adult retina extracts by RT-PCR. The presence of the GDNF protein was confirmed by SDS-PAGE and Western blotting analysis in adult retina homogenates. In situ hybridization and immunohistochemical experiments demonstrated that GDNF mRNA and protein localized in the photoreceptors, in the outer nuclear layer, in the inner plexiform layer and in the ganglion cell layer. These results showed that the expression of GDNF is not probably restricted during development but it might be involved in the physiology of adult zebrafish retina.

A mini-review of TAT-MyoD fused proteins: state of the art and problems to solve.

The transcriptional activator TAT is a small peptide essential for viral replication and possesses the property of entering the cells from the extracellular milieu, acting as a membrane shuttle. In order to safely differentiate cells an innovative methodology, based on the fusion of transcription factors and the TAT sequence, is discussed in this short review. In several studies, it has been demonstrated that TAT protein can be observed in the cell nucleus after few hours from the inoculation although its way of action is not fully understood. However, further studies will be necessary to develop this methodology for clinical purposes.

Tat-MyoD fused proteins, together with C2c12 conditioned medium, are able to induce equine adult mesenchimal stem cells towards the myogenic fate.

The Tat protein is able to translocate through the plasma membrane and when it is fused with other peptides may acts as a protein transduction system. This ability appears particularly interesting to induce tissue-specific differentiation when the Tat protein is associated to transcription factors. In the present work, the potential of the complex Tat-MyoD in inducing equine peripheral blood mesenchymal stem cells (PB-MSCs) towards the myogenic fate, was evaluated. Results showed that the internalization process of Tat-MyoD happens only in serum free conditions and that the nuclear localization of the fused complex is observed after 15 hours of incubation. However, the supplement of Tat-MyoD only was not sufficient to induce myogenesis and, therefore, in order to achieve the myogenic differentiation of PB-MSCs, conditioned medium from C2C12 cells was added without direct contact. Real Time PCR and immunofluorescence methods evaluated the establishment of a myogenic program. Our results suggest that TAT- transduction of Tat-MyoD, when supported by conditioned medium, represents a useful methodology to induce myogenic differentiation.