Eraldo Sanna Passino

Sheep embryonic stem-like cells transplanted in full-thickness cartilage defects.

Articular cartilage regeneration is limited. Embryonic stem (ES) cell lines provide a source of totipotent cells for regenerating cartilage. Anatomical, biomechanical, physiological and immunological similarities between humans and sheep make this animal an optimal experimental model. This study examines the repair process of articular cartilage in sheep after transplantation of ES‐like cells isolated from inner cell masses (ICMs) derived from in vitro‐produced (IVP) vitrified embryos. Thirty‐five ES‐like colonies from 40 IVP embryos, positive for stage‐specific embryonic antigens (SSEAs), were pooled in groups of two or three, embedded in fibrin glue and transplanted into osteochondral defects in the medial femoral condyles of 14 ewes. Empty defect (ED) and cell‐free glue (G) in the controlateral stifle joint served as controls. The Y gene sequence was used to detect ES‐like cells in the repair tissue by in situ hybridization (ISH). Two ewes were euthanized at 1 month post‐operatively, three each at 2 and 6 months and four at 12 months. Repairing tissue was examined by biomechanical, macroscopic, histological, immunohistochemical (collagen type II) and ISH assays. Scores of all treatments showed no statistical significant differences among treatment groups at a given time period, although ES‐like grafts showed a tendency toward a better healing process. ISH was positive in all ES‐like specimens. This study demonstrates that ES‐like cells transplanted into cartilage defects stimulate the repair process to promote better organization and tissue bulk. However, the small number of cells applied and the short interval between surgery and euthanasia might have negatively affected the results. Copyright

Magnetic resonance imaging of the brain and skull of sheep with cerebral coenurosis

OBJECTIVE To determine MRI characteristics of the skulls and brains of sheep with chronic cerebral coenurosis (CC) caused by naturally acquired Taenia multiceps infection. ANIMALS 33 sheep with CC and 10 healthy control sheep. PROCEDURES Sheep underwent MRI of the head. Volumes of the cranial cavity and rostral and caudal fossas of the cranial cavity were determined. For CC-affected sheep, the number, location, and volume of T multiceps cysts were determined and the percentage volumes of cysts in the cranial cavity and rostral and caudal fossas of the cranial cavity were calculated. Focal and diffuse abnormalities of cranial bones in CC-affected sheep were identified. Brain edema and hemorrhage and signs of increased cranial pressure (ICP) in MRI images were determined. RESULTS Volumes of the cranial cavity and rostral and caudal fossas of the cranial cavity were significantly larger for CC-affected sheep versus healthy control sheep. Total volumes of cysts ranged from 4.40% to 46.93% in cranial cavities of sheep, 4.12% to 51.53% in rostral fossas of cranial cavities of sheep, and 15.24% to 68.30% in caudal fossas of cranial cavities of sheep. Moderate to severe diffuse cranial bone abnormalities and signs of increased ICP in MRI images were detected in 21 and 24 sheep, respectively, and were positively correlated with cyst volumes. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that cranial cavity volume and morphological abnormalities can be detected in sheep with CC. These changes may reflect abnormalities in ossification of the cranial bones secondary to chronically increased ICP caused by development of T multiceps cysts.

The use of embryonic cells in the treatment of osteochondral defects of the knee: an ovine in vivo study.

PURPOSE the aim of this study was to determine whether local delivery of embryonic stem-like (ESL) cells into osteochondral defects in the femoral condyles of sheep would enhance regeneration of hyaline articular cartilage. METHODS male ESL cells embedded in fibrin glue were engrafted into osteochondral defects in the medial condyles (ESL-M) of the left femur in 22 ewes. An identical defect was created in the medial condyle of the contralateral stifle joint and left untreated as a control (empty defect, ED). The ewes were divided into 5 groups. Four sheep each were euthanized at 1, 2, 6, and 12 months from surgery, and 6 ewes were euthanized 24 months post-implantation. To study the effect of varying loads on the long-term regeneration process, an identical defect was also created and ESL cell engraftment performed in the lateral condyle (ESL-L) of the left stifle joint of the animals in the 12- and 24-month groups. The evaluation of regenerated tissue was performed by biomechanical, macroscopic, histological, immunohistochemical (collagen type II) and fluorescent in situ hybridization (FISH) assays. RESULTS no significant differences were found between treated and control sites in the biomechanical assays at any time point. ESL cell grafts showed significantly greater macroscopic evidence of regeneration as compared to controls at 24 months after surgery; significantly better histological evidence of repair in ESL-M samples versus controls was found throughout the considered period. At 24 months from surgery there was significantly improved integration of graft edges with the host tissue in the ESL-M as compared to the ESL-L samples, demonstrating that load bearing positively affects the long-term regeneration process. CONCLUSIONS ESL cells enhanced the regeneration of hyaline cartilage. FISH confirmed that the regenerative tissue originated from ESL cells. CLINICAL RELEVANCE ESL cells are able to self-renew for prolonged periods without differentiation and, most importantly, to differentiate into a large variety of tissues.

Physical reparative treatment in reptiles

BackgroundThe tissue growth necessary to achieve a complete or partial restitution ad integrum as a result of injury to soft tissue and/or hard times in reptiles is variable and often needs long time in relation to the species, to the habitat and to their intrinsic physiological characteristics. The purpose of this work was to see if the tissue optimization (TO) treatment with radio electric asymmetric conveyer (REAC) provided good results in these animals and whether its use translates into reduced time of tissue repair. This paper describes preliminary results with in promoting the tissue repair in reptiles.Cases presentationA 5 year old male Testudo graeca (Leo) and Trachemys scripta scripta (Mir) and a 15 year old female Testudo hermanni (Juta) were evaluated because of soft tissue injuries. A female 25 year old Trachemys scripta elegans (Ice), a female 2.5 year old Trachemys scripta scripta (Penelope) as well as a 50 year old male Testudo graeca (Margherito) were evaluated because of wounds of the carapace. Following debridement and traditional therapies, Leo, Penelope and Margherito were exposed to the radio electric asymmetric conveyer (REAC) device, with a specific treatment protocol, named tissue optimization-basic (TO-B). Also Ice and Mir were subjected to REAC treatment after wounds debridement. Juta was treated only with REAC treatment.Complete wound healing was evident after 17 days for Leo, 7 days for Penelope, 27 days for Mir, 78 days for Ice and after 14 days for Margherito. Juta showed a considerable tissue activation in 2 days and complete wound healing in 5 days.ConclusionOur findings suggest that REAC TO-B treatment may provide advantages over other traditional methods after complete wound healing in Leo, and also suitable healing in the other patients. Then REAC device with its specific treatment TO-B protocol, which induces tissue repair without causing severe stress to the patient, could be a potential therapy for tissue damage healing in reptiles. Further studies still need to be conducted to support our observations.

Clinical evolution of cerebral coenurosis from invasive to chronic infection in sheep and a goat

This survey describes the evolution in vivo of Coenurus cerebralis in small ruminants. At presentation, neurological signs and cerebrospinal fluid (CSF) features were suggestive of multifocal or diffuse inflammatory reaction. Magnetic resonance imaging (MRI) captured the transition between the invasive and quiescent phase of the infection, revealing the concurrent presence of meningitis and small cysts. During the quiescent phase, in all animals, neurological symptoms disappeared, and cerebrospinal fluid was unremarkable while cysts grew progressively. Subsequently, the onset of neurological symptoms coincided with MRI signs of diffuse or localized increase of intracranial pressure, as confirmed by direct intracranial pressure measuring. All the animals had an excellent post-surgical recovery. This is the first report describing the evolution of coenurosis in vivo. Sequential imaging allowed describing interesting such as the death of some coenuri and different parasite growth rate in the same host.

The effect of radio electric asymmetric conveyer treatment on sperm parameters of subfertile stallions: A pilot study

The Radio Electric Asymmetric Conveyer (REAC) has been mostly applied to treat symptoms related to psychological stress. In the study, we demonstrated the effect of REAC-Veterinary Neuro Psycho Physical Optimization (VNPPO) treatment protocol on sperm parameters of subfertile (n=11) and fertile (n=4) stallions. Subfertile stallions showed a reduced sperm concentration, progressive motility and normal morphology compared to fertile stallions. An increase in progressive sperm motility and quality of sperm morphology was found in subfertile stallions after the REAC-VNPPO treatment. The positive effect of the REAC-VNPPO treatment was visible in a reduced number of reacted or absent acrosomes, nuclei with marginated chromatin and presence of cytoplasmic residues. Thus, we suggest that the REAC-VNPPO treatment for stallions with idiopathic subfertility may enhance the reproductive performance of stallions.

Development of a Device-Assisted Nerve-Regeneration Procedure in Disruptive Lesions of the Brachial Plexus

Background We describe the development of a new surgical procedure to be used in the treatment of disruptive brachial plexus (BP) lesions. It is centered on an artificial device designed to assist nerve regeneration by providing a confined and protected environment. Nerve fibers can repair inside the device, while the adverse massive scar‐tissue formation is limited to the outside of the device. Methods Steps in the development of the procedure were (1) definition of the rationale, (2) design of the device, (3) choice of an in vivo translational model, (4)refinement of the surgical procedure, and (5) performance of an in vivo pilot study as a proof of concept. An interdisciplinary team from several laboratories was involved in this work over a period of 6 years. Results Results showed the absence of significant scar tissue in the regenerate and the presence of myelinated fibers aligned proximodistally between the stumps. This surgical approach can be seen not only as a definitive treatment but also as an early examination and stabilization before some different surgery will be later performed. It may also be used as additional protection for traditional surgery like end‐to‐end coaptation. Conclusions We conclude that the availability of a suitable device‐assisted early treatment, even if not to be considered definitive, could help in addressing the BP lesions at an earlier stage and this may improve the final outcome. Our evidence justifies further experimentation on this approach.

Additional file 2: Figure S2. of REAC technology as optimizer of stallion spermatozoa liquid storage

Effect of REAC treatment on the kinetic parameters of stallion spermatozoa during 72 h. of storage at 4°C. Panel A and B show the mean and 95,0 Bonferroni intervals per group and per stallion, respectively. A total of 16 ejaculates collected from 8 stallions of different breeds (1: Thoroughbred; 2, 3, 4, 6: Arabian; 5, 7, 8: Warmblood) were used. Panel B: a,b,c,d Different letters indicate a statistical difference among the ejaculates collected from the 8 stallions (General Linear Model): p>0.001. PM: percentage of progressive motile spermatozoa; Rapid: percentage of rapid spermatozoa; VAP: average path velocity (mm/s; the average velocity of the smoothed cell path); VCL: curvilinear velocity (mm/s; the average velocity measured over the actual point to point track followed by the cell); VSL: straight-line velocity (mm/s; the average velocity measured in a straight line from the beginning to the end of the track); LIN: linearity index (%; the average value of the ratio VSL/VCL); STR: straightness index (%; the average value of the ratio VSL/VAP); ALH: amplitude of lateral head displacement (mm; the mean width of the head oscillation as the sperm swim); BCF: beat cross-frequency (Hz; the frequency of sperm head crossing the average path in either direction); WOB: wobble (VAP/VCL à 100, %; a measure of the oscillation of the actual trajectory about its spatial average path). (TIF 541 kb)

Second Generation Needling Techniques for the Treatment of Chondral Defects in Animal Model

Purpose  To compare the macroscopic, histological, and immunohistochemical characteristics of the repair tissue of chondral defects treated with microfracture and nanofracture in an ovine model. Methods  Full-thickness chondral lesions were created in the medial femoral condyle of both knees in four adult sheep and were treated with microfracture on one side and with nanofracture on the contralateral side. Chondral repair was assessed after 12 months by macroscopic, histological, and immunohistochemical analyses. Results  Histological cartilage repair significantly improved in the samples treated with nanofracture for cellular morphological characteristics and cartilage architecture. The immunohistochemical analysis showed a significantly higher immunoreactivity to type II collagen in the defects treated with nanofracture. Conclusion  Nanofracture provided better repair tissue than microfracture, with a more satisfactory cartilage architecture renovation and tissue having greater type II collagen content. Clinical Relevance  Mesenchymal stem cell stimulation is the most frequently used primary cartilage repair procedure. Nanofracture represents a novel technique to stimulate bone marrow that results into a successful repair of chondral defects.

REAC regenerative treatment efficacy in experimental chondral lesions: a pilot study on ovine animal model

Radioelectric asymmetric conveyor (REAC) technology is a platform designed to optimize cell polarity. Cell polarity is a universal biological phenomenon that is implicated in cell differentiation, proliferation, morphogenesis, aging, and rejuvenation. In this work, we investigate a timing and administration protocol for tissue optimization regenerative treatment type C, in order to treat aging-related chondral damage or injuries and gain insights into regenerative processes of articular cartilage in humans. The chondral lesion produced in this study in an animal model (6 knee joints of 4 adult sheep) was 6 mm in diameter and about 2 mm deep. These lesions, which did not involve subchondral bone, tend to increase in size and depth and are not completely repaired with normal hyaline articular cartilage since adult articular cartilage is avascular and has a very slow turnover at the cellular and molecular level. Moreover, the hydration of articular cartilage is reduced with aging and with decreased mitotic activity, synthesis, and population size of chondrocytes. Six months posttreatment, lesions appeared filled, though not completely, with newly generated tissue of the light opalescent color of healthy articular cartilage, which otherwise covered the underlying subchondral bone. The newly formed tissue surface appeared to be quite regular. Nearly complete regeneration of subchondral bone occurred, with little vascularization and ossification nuclei almost absent. The results of this study confirm previous data obtained in vitro on the regenerative effects of REAC technology on human normal and osteoarthritic chondrocytes exposed to IL-1β. The present findings indicate that REAC tissue optimization-regenerative treatment type C is a promising therapeutic tool among the other REAC regenerative treatment protocols for the treatment of cartilage lesions.