Alessandro Spadari

Isolation, growth and differentiation of equine mesenchymal stem cells: effect of donor, source, amount of tissue and supplementation with basic fibroblast growth factor

Mesenchymal stem cells (MSC) are increasingly used as therapeutical aid for the orthopaedic injuries in the horse. MSC populate different tissues but the most commonly used for clinical purposes are isolated from bone marrow or adipose tissue. The first objective of this study was to investigate if the donor animal, the tissue of origin and the technique of isolation could influence the number of MSC available for transplantation after a short-term expansion. The second aim was to devise a culture system capable of increasing MSC lifespan and we tested the effect of basic fibroblast growth factor (bFGF). Results indicate that MSC can be efficiently isolated from both sources and supplementation of bFGF enhances proliferation rate maintaining differentiation potential. In addition, this study shows that collection, expansion and storage of frozen MSC can be performed for later therapeutic use.

Sympathetic innervation of the ileocecal junction in horses.

The distribution and chemical phenotypes of sympathetic and dorsal root ganglion (DRG) neurons innervating the equine ileocecal junction (ICJ) were studied by combining retrograde tracing and immunohistochemistry. Immunoreactivity (IR) for tyrosine hydroxylase (TH), dopamine beta‐hydroxylase (DBH), neuronal nitric oxide synthase (nNOS), calcitonin gene‐related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY) was investigated. Sympathetic neurons projecting to the ICJ were distributed within the celiac (CG), cranial mesenteric (CranMG), and caudal mesenteric (CaudMG) ganglia, as well as in the last ganglia of the thoracic sympathetic chain and in the splanchnic ganglia. In the CG and CranMG 91 ± 8% and 93 ± 12% of the neurons innervating the ICJ expressed TH‐ and DBH‐IR, respectively. In the CaudMG 90 ± 15% and 94 ± 5% of ICJ innervating neurons were TH‐ and DBH‐IR, respectively. Sympathetic (TH‐IR) fibers innervated the myenteric and submucosal ganglia, ileal blood vessels, and the muscle layers. They were more concentrated at the ICJ level and were also seen encircling myenteric plexus (MP) and submucosal plexus (SMP) descending neurons that were retrogradely labeled from the ICJ. Among the few retrogradely labeled DRG neurons, nNOS‐, CGRP‐, and SP‐IR nerve cells were observed. Dense networks of CGRP‐, nNOS‐, and SP‐IR varicosities were seen around retrogradely labeled prevertebral ganglia neurons. The CGRP‐IR fibers are probably the endings of neurons projecting from the intestine to the prevertebral ganglia. These findings indicate that this crucial region of the intestinal tract is strongly influenced by the sympathetic system and that sensory information of visceral origin influences the sympathetic control of the ICJ. J. Comp. Neurol. 518:4046–4066, 2010.

CAD-CAM-generated hydroxyapatite scaffold to replace the mandibular condyle in sheep: preliminary results.

In this study, rapid CAD-CAM prototyping of pure hydroxyapatite to replace temporomandibular joint condyles was tested in sheep. Three adult animals were implanted with CAD-CAM-designed porous hydroxyapatite scaffolds as condyle substitutes. The desired scaffold shape was achieved by subtractive automated milling machining (block reduction). Custom-made surgical guides were created by direct metal laser sintering and were used to export the virtual planning of the bone cut lines into the surgical environment. Using the same technique, fixation plates were created and applied to the scaffold pre-operatively to firmly secure the condyles to the bone and to assure primary stability of the hydroxyapatite scaffolds during masticatory function. Four months post-surgery, the sheep were sacrificed. The hydroxyapatite scaffolds were explanted, and histological specimens were prepared. Different histological tissues penetrating the scaffold macropores, the sequence of bone remodeling, new apposition of bone and/or cartilage as a consequence of the different functional anatomic role, and osseointegration at the interface between the scaffold and bone were documented. This animal model was found to be appropriate for testing CAD-CAM customization and the biomechanical properties of porous, pure hydroxyapatite scaffolds used as joint prostheses.

Intrinsic innervation of the horse ileum

This paper describes the morphology and distribution of the enteric nervous system (ENS) cells and fibres immunoreactive for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene-related peptide (CGRP), NF200kDa (NF200), and S100 protein. The percentages of subclasses of enteric neurons in the total neuronal population were investigated by the use of anti-PGP 9.5 or anti-NSE antibodies. ChAT-IR myenteric plexus (MP) and submucosal plexus (SMP) neurons were 66+/-7% and 74+/-15%, respectively, whereas those cells expressing nNOS-IR were 38+/-7% and 5+/-1%, respectively. MP and SMP neurons expressing both phenotypes were also present. SP-IR was expressed by 14+/-13% of MP and 66+/-8% of SMP neurons whereas CGRP-IR was observed only in the SMP (43+/-6%). NF200-IR was expressed by 61+/-15% and 91+/-10% of the MP and SMP neurons, respectively. The majority of the CGRP-IR SMP neurons expressed also SP-IR. Almost all SP-IR neurons in both the plexuses were cholinergic. The present study quantifies the main neuronal subpopulations of the ENS of the horse ileum; these data might be utilized to understand the neuronal modifications which occur in several gastrointestinal tract disorders.

Mesenchymal stem cells and platelet gel improve bone deposition within CAD-CAM custom-made ceramic HA scaffolds for condyle substitution.

Purpose. This study evaluated the efficacy of a regenerative approach using mesenchymal stem cells (MSCs) and CAD-CAM customized pure and porous hydroxyapatite (HA) scaffolds to replace the temporomandibular joint (TMJ) condyle. Methods. Pure HA scaffolds with a 70% total porosity volume were prototyped using CAD-CAM technology to replace the two temporomandibular condyles (left and right) of the same animal. MSCs were derived from the aspirated iliac crest bone marrow, and platelets were obtained from the venous blood of the sheep. Custom-made surgical guides were created by direct metal laser sintering and were used to export the virtual planning of the bone cut lines into the surgical environment. Sheep were sacrificed 4 months postoperatively. The HA scaffolds were explanted, histological specimens were prepared, and histomorphometric analysis was performed. Results. Analysis of the porosity reduction for apposition of newly formed bone showed a statistically significant difference in bone formation between condyles loaded with MSC and condyles without (P < 0.05). The bone ingrowth (BI) relative values of split-mouth comparison (right versus left side) showed a significant difference between condyles with and without MSCs (P < 0.05). Analysis of the test and control sides in the same animal using a split-mouth study design was performed; the condyle with MSCs showed greater bone formation. Conclusion. The split-mouth design confirmed an increment of bone regeneration into the HA scaffold of up to 797% upon application of MSCs.

Clinical findings, rhinoscopy and histological evaluation of 54 dogs with chronic nasal disease

Nasal diseases are very common in dogs and rhinoscopy is often required for a definitive diagnosis. Rhinoscopy, while superficial in nature, can guide the clinician to the final diagnosis. In this study, rhinoscopy was performed on 54 dogs with symptoms of chronic nasopharyngeal disease. The endoscopic diagnosis of neoplasia or chronic nasal inflammation was validated with histological examination of pathological samples, in order to evaluate the degree of concordance between endoscopic findings and histological diagnosis. The agreement between endoscopy and histology was tested by application of Cohens kappa coefficient. We conclude that correlation between endoscopic results and histological diagnosis, expressed by a Cohens kappa coefficient of 0.73, is only possible with a constant cooperation between the clinician and the pathologist.

Cytological, immunohistochemical and mutational analysis of a gastric gastrointestinal stromal tumour in a cat.

Gastrointestinal stromal tumours (GISTs) represent a distinctive group of primary mesenchymal tumours of the gastrointestinal tract identified immunohistochemically by expression of CD117. A 10-year-old neutered female domestic shorthair cat with a history of recurrent vomiting was examined. The presence of a gastric mass was recognized and a laparotomy was performed. Cytological examination was consistent with a low-grade malignant mesenchymal tumour and histopathological investigation suggested myogenic differentiation of tumour cells. The diagnosis of GIST was confirmed by immunohistochemical expression of CD117. Sequence analysis of the KIT gene identified a deletion in exon 11. The same mutation is found often in human GISTs.

Application of the 'extraction bag' in laparoscopic treatment of unilateral and bilateral abdominal cryptorchidism in dogs.

Laparoscopy is a mini-invasive surgical technique, particularly indicated in the treatment of unilateral and bilateral abdominal cryptorchidism in dogs (Bianco et al., 1997; Petrizzi et al., 1998; Pena et al., 1998; Gallagher et al., 1992). Following testicular removal via laparoscopy, we found it advantageous to use an ‘extraction bag’, a technique that had been previously described by Gallagher (Gallagher et al., 1992). By placing the isolated testicle inside this device, asepsis of the operating field is maintained without limiting intra-abdominal surgical manoeuvres. In the case of bilateral cryptorchidism, as the first testicle is isolated and introduced into the extraction bag, it is possible to continue with the contralateral testicle with final extraction of the two testicles through the same surgical incision.

Serum amyloid A, haptoglobin, and ferritin in horses with colic: Association with common clinicopathological variables and short-term outcome

Equine colic may be associated with an acute phase response (APR). Measurement of acute phase proteins (APPs) allows the detection of an APR and may help clinicians in monitoring the disease; however, the role of APPs in colic is unclear. This study aimed to evaluate the clinical usefulness of serum amyloid A (SAA), haptoglobin and ferritin in combination with an extended clinicopathological profile in equine colic. The medical records of 54 horses were retrospectively selected. Horses were grouped based on outcome (survivors vs. non-survivors), diagnosis (ischaemic/strangulating vs. non-ischaemic/non-strangulating), and treatment (medical treatment vs. surgery). Laboratory data were compared, and a logistic regression analysis was performed for outcome prediction upon admission. A high percentage of horses had abnormal SAA (29/54), haptoglobin (20/54), and ferritin (31/54) concentrations. In particular, haptoglobin was below the reference interval in 13/54 horses. Non-survivors had significantly decreased haptoglobin and increased ferritin concentrations compared with survivors. The ischaemic/strangulating group had significantly increased creatinine and ferritin and decreased haptoglobin concentrations compared with the non-ischaemic/non-strangulating group. Creatinine was the only significant predictor of mortality in the regression analysis. In conclusion, APPs including SAA, haptoglobin, and ferritin combined with clinicopathological variables may help clinicians to understand the pathogenesis of APR and underline potential complications of equine colic. The reduction in haptoglobin concentration may suggest haemolysis or muscle fibre damage; ferritin may indicate alteration in iron metabolism and tissue damage. Further prospective studies are needed to assess diagnostic and prognostic values of APPs in colic horses.

Effects of intraarticular treatment with stanozolol on synovial membrane and cartilage in an ovine model of osteoarthritis

Aim of the study was to verify the clinical and morphological effects of intra-articular stanozolol or placebo treatment, lasting 3 and 9 months, in sheep in which a femoro-tibial osteo-arthritis (OA) were surgically induced (medial bilateral meniscectomy). Twenty healthy sheep divided into four groups and two control animals group, after surgical medial bilateral meniscectomy, were weekly injected in femoral-tibial joint (FTJ) with stanozolol or placebo. Lameness evaluation was performed and synovial fluid was collected from all sheep at each treatment time. Necropsies were performed after 3 or 9 month as described in experimental design. Gross pathologies were described and specimen tissues collected from femoro-tibial articular joints were processed for routine histological examination. The gross anatomy of the FTJ was well-preserved in stanozolol-treated sheep; this also applied to the histological features of articular cartilage. Joint aseptic inflammation and fibrosis were observed in placebo-treated sheep, associated with a different degree of severity of condylar and tibial plate cartilage degeneration. Stanozolol intra-articular treatment reduces osteophytes formation and subchondral bone reaction and promotes articular cartilage regeneration.