Elisabetta Giannessi

Isolation and expansion of equine umbilical cord-derived matrix cells (EUCMCs)

Stem cells from extra‐embryonic sources can be obtained by non‐invasive procedures. We have standardized a method for the expansion of equine umbilical cord‐derived matrix cells (EUCMCs) for potential therapy.

Different media and supplements modulate the clonogenic and expansion properties of rabbit bone marrow mesenchymal stem cells

Background -Rabbits provide an excellent model for many animal and human diseases, such as cardiovascular diseases, for the development of new vaccines in wound healing management and in the field of tissue engineering of tendon, cartilage, bone and skin.The study presented herein aims to investigate the biological properties of bone marrow rabbit MSCs cultured in different conditions, in order to provide a basis for their clinical applications in veterinary medicine.Findings -MSCs were isolated from 5 New Zealand rabbits. Fold increase, CFU number, doubling time, differentiation ability and immunophenotype were analyzed.With the plating density of 10 cells/cm2 the fold increase was significantly lower with DMEM-20%FCS and MSCs growth was significantly higher with αMEM-hEGF. The highest clonogenic ability was found at 100 cell/cm2 with MSCBM and at 10 cell/cm2 with M199. Both at 10 and 100 cells/cm2, in αMEM medium, the highest CFU increase was obtained by adding bFGF. Supplementing culture media with 10%FCS-10%HS determined a significant increase of CFU.Conclusion -Our data suggest that different progenitor cells with differential sensitivity to media, sera and growth factors exist and the choice of culture conditions has to be carefully considered for MSC management.

An autologously generated platelet-rich plasma suturable membrane may enhance peripheral nerve regeneration after neurorraphy in an acute injury model of sciatic nerve neurotmesis.

BACKGROUND The aim of this study was to investigate the ability of suturable platelet-rich plasma (PRP) membrane to promote peripheral nerve regeneration after neurotmesis and neurorraphy. METHODS A total of 36 rats were used: 32 animals underwent surgery and were split in two groups. An interim sacrifice was performed at 6 weeks postsurgery and final sacrifice at 12 weeks; four animals did not sustain nerve injury and served as control. Clinical, electromyographic (EMG), gross, and histological changes were assessed. The EMG signal was evaluated for its amplitude and frequency spectrum. Number of regenerating fibers, their diameter, and myelin thickness were histologically analyzed. RESULTS Both EMG parameters showed a significant (p < 0.05) effect of treatment at 6 and 12 weeks postsurgery. At 6 weeks, the fiber density was statistically different between treated and untreated animals with a higher observed density in treated nerves. No difference in fiber density was observed at 12 weeks postsurgery. The distribution of fiber diameters showed an effect at 12 weeks when only the sections of the nerves sutured with PRP showed fibers with diameters greater than 6 µm. DISCUSSION Our data show that the application of a PRP fibrin membrane around the neurorraphy improves the nerve regeneration process in a rat sciatic nerve model. The use of PRP as a suturable membrane could perform an action not only as a source of bioactive proteins but also as a nerve guide to hold the scar reaction and thus improve axonal regeneration.

Isolation and characterization of equine amnion mesenchymal stem cells

The amnion is a particular tissue whose cells show features of multipotent stem cells proposed for use in cellular therapy and regenerative medicine. From equine amnion collected after the foal birth we have isolated MSCs (mesenchymal stem cells), namely EAMSCs (equine amnion mesenchymal stem cells), from the mesoblastic layer. The cells were grown in α-MEM (α-modified minimum essential medium) and the effect of EGF (epidermal growth factor) supplementation was evaluated. To assess the growth kinetic of EAMSCs we have taken into account some parameters [PD (population doubling), fold increase and DT (doubling time)]. The differentiation in chondrogenic, adipogenic and osteogenic types of cells and their epitope expression by a cytofluorimetric study have been reported. EGF supplementation of the culture medium resulted in a significant increase in PD growth parameter and in the formation of bone nodules for the osteogenic differentiation. By immunohistochemistry the amnion tissue shows a positivity for the c-Kit (cluster tyrosine-protein kinase), CD105 and Oct-4 (octamer-binding transcription factor 4) antigens that confirmed the presence of MSCs with embryonic phenotype.

Tailored star poly (ε-caprolactone) wet-spun scaffolds for in vivo regeneration of long bone critical size defects:

One of the most challenging requirements of a successful bone tissue engineering approach is the development of scaffolds specifically tailored to individual tissue defects. Besides materials chemistry, well-defined scaffold’s structural features at the micro- and macro-levels are needed for optimal bone in-growth. In this study, polymeric fibrous scaffolds with a controlled internal network of pores and modelled on the anatomical shape and dimensions of a critical size bone defect in a rabbit’s radius model were developed by employing a computer-aided wet-spinning technique. The tailored scaffolds made of star poly(ε-caprolactone) or star poly(ε-caprolactone)–hydroxyapatite composite material were implanted into 20-mm segmental defects created in radial diaphysis of New Zealand white rabbits. Bone regeneration and tissue response were assessed by X-rays and histological analysis at 4, 8 and 12 weeks after surgery. No signs of macroscopic and microscopic inflammatory reactions were detected, and the developed scaffolds showed a good ability to support and promote the bone regeneration process. However, no significant differences in osteoconductivity were observed between star poly(ε-caprolactone) and star poly(ε-caprolactone)–hydroxyapatite scaffolds. Long-term study on implanted star poly(ε-caprolactone) scaffolds confirmed the presence of signs of bone regeneration and remodelling, particularly evident at 24 weeks.

Immunohistochemical distribution of the cannabinoid receptor 1 and fatty acid amide hydrolase in the dog claustrum

Cannabinoid receptor 1 (CB1R) and fatty acid amide hydrolase (FAAH) are part of the endocannabinoid system (ECB) which exerts a neuromodulatory activity on different brain functions and plays a key role in neurogenesis. Although many studies have reported FAAH and CB1R expression in the brain of different animal species, to the best of our knowledge they have never been described in the canine claustrum. Claustrum samples, obtained from necropsy of four neurologically normal dogs, were formalin fixed for paraffin embedding. Sections were either stained for morpho-histological analysis or immunostained for CB1R and FAAH. Analysis of adjacent sections incubated with the two antisera showed a complementary labeling pattern in the claustrum, with CB1R antibody staining fibers while anti-FAAH antibody stained cell bodies and the proximal portion of dendrites; this particular anatomical relationship suggests a retrograde endocannabinoid action via CB1R. CB1R and FAAH complementary immunostaining and their cellular localization reported here provide the first anatomical evidence for existence of the ECB in the dog claustrum.

A unified approach to model peripheral nerves across different animal species

Peripheral nerves are extremely complex biological structures. The knowledge of their response to stretch is crucial to better understand physiological and pathological states (e.g., due to overstretch). Since their mechanical response is deterministically related to the nature of the external stimuli, theoretical and computational tools were used to investigate their behaviour. In this work, a Yeoh-like polynomial strain energy function was used to reproduce the response of in vitro porcine nerve. Moreover, this approach was applied to different nervous structures coming from different animal species (rabbit, lobster, Aplysia) and tested for different amount of stretch (up to extreme ones). Starting from this theoretical background, in silico models of both porcine nerves and cerebro-abdominal connective of Aplysia were built to reproduce experimental data (R2 > 0.9). Finally, bi-dimensional in silico models were provided to reduce computational time of more than 90% with respect to the performances of fully three-dimensional models.

Preferential epithelial expression of type-1 cannabinoid receptor (CB1R) in the developing canine embryo

The use of cannabinoid receptor agonists is gaining a strong interest both in human and veterinary medicine. The potential use of cannabimimetic compounds in companion animals was reviewed in 2007 for their role in tissue inflammation and pain. A better knowledge of type-1 cannabinoid receptor (CB1R) expression on the target population may help in risk management in order to prevent unwanted side effects. We used 30-days old canine embryos to describe the distribution of CB1R by means of immunohistochemistry with a commercially available antibody.CB1R immunoreactivity was mainly epithelial and included most structures of central and peripheral nervous system, inner ear, olfactory epithelium and related structures, eye and thyroid. Further investigative research on the role of the endocannabinoid system in the developmental biology field is needed, however, we show that in the canine species we must consider pregnancy as risk condition for developmental abnormalities that may arise upon the use of CB1R receptor agonists.

Glucagon-like peptide 1 (GLP-1) in the gastrointestinal tract of the pheasant (Phasianus colchicus)

The distribution of Glucagon-like peptide 1 (GLP-1) was investigated in the gastrointestinal tract of the pheasant using immunohistochemistry. GLP-1 immunoreactive cells were common in the small intestine, in the proventriculus and in the pancreas. Immunostained cells were not seen in the crop, in the gizzard and in the large intestine. Double labelling demonstrated that GLP-1 and pituitary adenylate cyclase-activating polypeptide (PACAP) were occasionally co-localized only in the duodenal villi. In contrast to what was previously described in the chicken and ostrich, we noted GLP-1 positive cells in the duodenum. These data were consistent with the presence of proglucagon mRNA in the chicken duodenum. Our findings indicate that GLP-1 might have an inhibitory effect on gastric and crop emptying and on acid secretion also in the pheasant. Moreover, the results of the present research regarding the initial region of the small intestine suggest a further direct mechanism of the GLP-1 release during the early digestion phase and an enhancement of its incretin role.

Expanded polytetrafluoroethylene as tendon replacement: an experimental study in chickens.

We designed an experimental animal model (in chickens) to assess the potential applications, above all in hand surgery, of an expanded polytetrafluoroethylene (e-PTFE) graft as a replacement for a natural tendon. The results of macroscopic, histological, and functional observations made five weeks, and three, four, five, and six months after implantation showed that the e-PTFE seemed to be a good replacement for tendons, because it integrated well with surrounding tissue and permitted good functional recovery within a reasonable time period.We designed an experimental animal model (in chickens) to assess the potential applications, above all in hand surgery, of an expanded polytetrafluoroethylene (e-PTFE) graft as a replacement for a natural tendon. The results of macroscopic, histological, and functional observations made five weeks, and three, four, five, and six months after implantation showed that the e-PTFE seemed to be a good replacement for tendons, because it integrated well with surrounding tissue and permitted good functional recovery within a reasonable time period.