Placental Stem Cells from Domestic Animals: Translational Potential and Clinical Relevance.

The field of regenerative medicine is moving toward clinical practice in veterinary science. In this context, placenta-derived stem cells isolated from domestic animals have covered a dual role, acting both as therapies for patients and as a valuable cell source for translational models. The biological properties of placenta-derived cells, comparable among mammals, make them attractive candidates for therapeutic approaches. In particular, stemness features, low immunogenicity, immunomodulatory activity, multilineage plasticity, and their successful capacity for long-term engraftment in different host tissues after autotransplantation, allo-transplantation, or xenotransplantation have been demonstrated. Their beneficial regenerative effects in domestic animals have been proven using preclinical studies as well as clinical trials starting to define the mechanisms involved. This is, in particular, for amniotic-derived cells that have been thoroughly studied to date. The regenerative role arises from a mutual tissue-specific cell differentiation and from the paracrine secretion of bioactive molecules that ultimately drive crucial repair processes in host tissues (e.g., anti-inflammatory, antifibrotic, angiogenic, and neurogenic factors). The knowledge acquired so far on the mechanisms of placenta-derived stem cells in animal models represent the proof of concept of their successful use in some therapeutic treatments such as for musculoskeletal disorders. In the next future, legislation in veterinary regenerative medicine will be a key element in order to certify those placenta-derived cell-based protocols that have already demonstrated their safety and efficacy using rigorous approaches and to improve the degree of standardization of cell-based treatments among veterinary clinicians.

Ovine amniotic epithelial cells: in vitro characterization and transplantation into equine superficial digital flexor tendon spontaneous defects.

In vitro expanded and frosted ovine amniotic epithelial cells (oAECs) were evaluated for their phenotype, stemness and attitude to differentiate into tenocytes. Fifteen horses with acute tendon lesions were treated with one intralesional injection of oAECs. Tendon recovery under controlled training was monitored. In vitro expanded oAECs showed a constant proliferative ability, a conserved phenotype and stable expression profile of stemness markers. Differentiation into tenocytes was also regularly documented. US controls showed the infilling of the defect and early good alignment of the fibers and 12 horses resumed their previous activity. Histological and immunohistochemical examinations in an explanted tendon demonstrated the low immunogenicity of oAECs that were able to survive in the healing site. In addition, oAECs supported the regenerative process producing ovine collagen type I amongst the equine collagen fibers. Considering our results, oAECs can be proposed as a new approach for the treatment of spontaneous equine tendon injuries.

Characterization, GFP gene Nucleofection, and allotransplantation in injured tendons of ovine amniotic fluid-derived stem cells.

Amniotic fluid has drawn increasing attention in the recent past as a cost-effective and accessible source of fetal stem cells. Amniotic fluid-derived mesenchymal stem cells (AFMSCs) that display high proliferation rate, large spectrum of differentiation potential, and immunosuppressive features are considered optimal candidates for allogeneic repair of mesenchymal damaged tissues. In this study, ovine AFMSCs (oAFMSCs) isolated from 3-month-old sheep fetuses were characterized for their proliferation rate, specific surface antigen and pluripotency marker expression, genomic stability, and mesenchymal lineage differentiation during their in vitro expansion (12 passages) and after nucleofection. The high proliferation rate of oAFMSCs gradually decreased during the first six subculture passages while the expression of surface molecules (CD29, CD58, CD166) and of pluripotency-associated markers (OCT4, TERT, NANOG, SOX2), the in vitro osteogenic differentiation potential, and a normal karyotype were maintained. Afterwards, oAFMSCs were nucleofected with a selectable plasmid coding for green fluorescent protein (GFP) using two different programs, U23 and C17, previously optimized for human mesenchymal stem cells. Transfection efficiencies were ∼63% and ∼37%, while cell recoveries were ∼10% and ∼22%, respectively. Nucleofected oAFMSCs expressing the GFP transgene conserved their pluripotency marker profile and retained a normal karyotype and the osteogenic differentiation ability. Seven single clones with a GFP expression ranging from 80% to 97% were then isolated and expanded over 1 month, thus providing stably transfected cells with long-term therapeutic potential. The in vivo behavior of GFP-labeled oAFMSCs was tested on a previously validated preclinical model of experimentally induced Achille's tendon defect. The allotransplanted oAFMSCs were able to survive within the host tissue for 1 month enhancing the early phase of tendon healing as indicated by morphological and biomechanical results. Altogether these data suggest that genetically modified oAFMSCs might represent a valuable tool for in vivo preclinical studies in a highly valid translational model.

Platelet rich plasma in tendinopathies: how to explain the failure.

Tendinopathies are very common in athletes and in people practicing sport activities. The experimental evidence that growth factors (GFs), present in platelets, enhance the recruitment, proliferation and differentiation of cells involved in tissue regeneration, has prompted the use of platelet rich plasma (PRP) preparations in the treatment of these diseases. However, at present, a sound demonstration of the clinical efficacy of PRP is still lacking. Several theoretical and practical reasons can explain the failure of the treatment: a) animal experiments have been carried out on normal tendons submitted to surgical lesions, and it is questionable whether these models may best mimic human pathology; b) the pathway of chronic tendinopathies is very complex, involving, besides GFs, many other pathogenetic factors, which operate at different stages of the disease; c) several methods have been used to produce PRP, which can result in a large variation in GF content, and in kinetics of release. Therefore, further research is desirable. As a preliminary step, it is necessary to standardize PRP preparation, and to establish the modalities of its activation and administration. Secondly, prospective, randomized, double-blind studies are needed, selecting subjects with homogenous forms of tendinopathies: load-bearing and non-load-bearing tendons, midportion and insertional tendinopathies, with or without neovascularization. Finally, new strategies in PRP use should be exploited: among them, the association of PRP with autologous stem cells or the administration of selective GFs (fibroblast growth factor, vascular endothelial growth factor, or anti-angiogenic factors), which could be better options in specific situations.

Achilles tendon regeneration can be improved by amniotic epithelial cell allotransplantation.

Amniotic epithelial cells (AECs) are ideal seed cells for tissue regeneration, but no research has yet been reported on their tendon regeneration potential. This study investigated the efficiency of AEC allotransplantation for tendon healing, as well as the mechanism involved. To this aim ovine AECs, characterized by specific surface and stemness markers (CD14(-), CD31(-), CD45(-), CD49f, CD29, CD166, OCT4, SOX2, NANOG, TERT), were allotransplanted into experimentally induced tissue defects in sheep Achilles tendon. In situ tissue repair revealed that AEC-treated tendons had much better structural and mechanical recoveries than control ones during the early phase of healing. Immunohistochemical and biochemical analyses indicated that extracellular matrix remodeling was more rapid and that immature collagen fibers were completely replaced by mature ones in 28 days. Moreover, spatial-temporal analysis of cellularity, proliferation index, vascular area, and leukocyte infiltration revealed that AECs induced a specific centripetal healing process that first started in the tissue closer to the healthy portion of the tendons, where AECs rapidly migrated to then progress through the core of the lesion. This peculiar healing evolution could have been induced by the growth factor stimulatory influence (TGF-ß1 and VEGF) and/or by the host progenitor cells recruitment, but also as the consequence of a direct tenogenic AEC differentiation resulting in the regeneration of new tendon matrix. These findings demonstrate that AECs can support tendon regeneration, and their effects may be used to develop future strategies to treat tendon disease characterized by a poor clinical outcome in veterinary medicine.

Experimental study on allografts of amniotic epithelial cells in calcaneal tendon lesions of sheep.

An experimental protocol was designed to study the survival and behaviour of an allograft of amniotic epithelial cells (AECs) in an ovine model. The study was conducted on three healthy adult sheep. A core lesion was created in both calcaneal tendons under ultrasound (US) guidance by injecting 400 UI of Type 1A collagenase diluted in 0.6 ml saline. The AECs were obtained from a 60-80-day-old fetus and cultured under standard conditions. After 15 days of collagenase treatment, 2 x 10(6) AECs stained with a vital membrane fluorescent probe (PHK26) were injected under US guidance in 500 microl saline solution into the lesion of one limb. The contralateral untreated limb was used as a control. Animals were euthanatized 7 (1) and 30 (2) days later. Histological analyses performed on explanted tendons clearly demonstrate that AECs survived for at least 1 month inside the lesion without any adverse reactions. The damaged tissue of the treated tendons showed a high number of reparative cells in active proliferation that were accumulating collagen within the extracellular matrix. In addition, after 1 month, the neo-collagen began to be organized into parallel arrays of fibers oriented along the longitudinal axis of the tendon.

Novel nanostructured scaffold for osteochondral regeneration: pilot study in horses.

The present in vivo preliminary experiment is aimed at testing mechanical and biological behaviour of a new nano-structured composite multilayer biomimetic scaffold for the treatment of chondral and osteochondral defects. The three-dimensional biomimetic scaffold (Fin-Ceramica Faenza S.p.A., Faenza-Italy) was obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles, in two configurations, bi- and tri-layered, to reproduce, respectively, chondral and osteochondral anatomy. Chondral defects (lateral condyle) and deep osteochondral defects (medial condyle) were made in the distal epiphysis of the third metacarpal bone of both forelimbs of two adult horses and treated respectively with the chondral and osteochondral grafts. Both animals were euthanised six months follow up. The images obtained at the second look arthroscopy evaluation, performed two months after surgery, demonstrated good filling of the chondral and osteo-chondral defects without any inflammatory reaction around and inside the lesions. At the histological analysis the growth of trabecular bone in the osteochondral lesion was evident. Only in one case, the whole thickness of the osteochondral lesion was filled by fibrocartilaginous tissue. The formation of a tidemark line was evident at the interface with the newly formed bone. Newly formed fibrocartilaginous tissue was present in the area of the chondral defect. Initial alignment of the collagen fibres was recognisable with polarised light in both groups. The results of the present pilot study showed that this novel osteochondral and chondral scaffold may act as a suitable matrix to facilitate orderly regeneration of bone and hyaline-like cartilage.

Selective deafferentation of hand cutaneous territory is followed by changes in fibre type distribution of a forearm muscle in the horse.

Based on previous observations that capsaicin can selectively damage group III and IV afferents and induce muscle fibre transformation, we hypothesized that eliminating, by means of capsaicin, the group III and IV afferents of a peripheral territory it could lead to a fibre transformation in a muscle involved in the flexor reflexes of the same peripheral territory. Therefore, capsaicin was injected into the palmar nerves of the forelimb of the horse to investigate if eliminating group III and IV afferents from the hand of the horse a muscle fibre transition would occur in the flexor carpi radialis (FCR) muscle, which is involved in the flexor reflexes of the finger itself. 120 days after capsaicin injection, type I slow fibres increased and type IIA fast fibres decreased. We presume that the long lasting deafferentation of the ergo-nociceptive fibres causes a plastic remodelling in the central nervous system and indirectly influences the motoneuron excitability via short or long loop-pathways enhancing their tonic discharge.

Synovial fluid parameters in normal and osteochondritic hocks of horses with open physis.

An investigation was carried out on most common synovial fluid parameters of normal and osteochondritic hocks of horses less than 12 months old in order to confirm the presence of an inflammatory process. Furthermore, a spectroscopic study was performed on synovial fluid from both normal and diseased hocks. A depolymerization of hyaluronic acid was demonstrated in synovial fluid from diseases joints, similar to that reported in human rheumatoid arthritis. A one month rest seem to normalize all parameters considered and in one joint, a return to normal infrared spectrum was demonstrated.

Ischemia-reperfusion syndrome: an alternative experimental model.

OBJECTIVE: To design an alternative experimental model of ischemia-reperfusion syndrome. Our model mimics the clinical pattern of the syndrome and also assesses the efficacy of therapeutical protocols. EXPERIMENTAL DESIGN: Ischemia was induced under general anaesthesia in the posterior limbs of 10 sheep by occluding the aorta and vena cava by means of two-way balloon catheters. Ischemia was stopped after 4 hours and blood and histologic parameters determined in the first three hours of revascularization. The animals were divided into three groups: a group of 3 sheep in which a sham operation was performed; a control group (5) to assess the efficacy of induced ischemia; the third group (5) to determine the effect of antioxidant and membrane protective drugs to assess the reliability of the model to study the ischemia-reperfusion syndrome. RESULTS: At the end of ischemia, skin temperature was decreased (p < 0.04) both in control and treated groups, pH decreased significantly soon after reperfusion in the control group (p < 0.04). Reperfusion in control sheep, compared with treated animals, was followed by a significant increase in CPK blood levels (p < 0.009), related to marked muscle damage, in particular after reperfusion. Tissue damage detected at TEM was less severe in treated animals. CONCLUSIONS: This model is an effective experimental strategy and a means of assessing preventive treatment.

Prevention of reperfusion syndrome in acute muscular ischaemia with free radical scavengers and membrane-protecting compounds: an experimental study.

The prevention of oxidant-induced damage following reperfusion was experimentally evaluated. Two pharmacological regimens containing different combinations of antioxidant factors and membrane-stabilizing compounds, such as alpha-tocopherol (vitamin E), methionine, dexamethasone, mannitol and cysteine, were administered. The reduced/oxidized glutathione (GSH/GSSG) ratio in muscle was used to evaluate oxidative stress. Ischaemia was induced by occluding the aorta and the inferior vena cava with an irrigation-occlusion catheter. After 4 h of ischaemia, five sheep were reperfused without any treatment (control group) and five treated with an endoaortic bolus administered at declamping (treatment 1). In five other sheep, treatment started during ischaemia (treatment 2). Ischaemia and, in particular, reperfusion significantly reduced the muscle GSH content, compared with the basal value in the control group; thus the GSH/GSSG ratio decreased significantly in the control group from 10.5(2.2) (mean(s.e.) basal value) to 0.687(0.3) at reperfusion (P < 0.009). Both treatments 1 and 2 significantly prevented a reduction in GSH content induced by reperfusion following ischaemia; the GSH/GSSG ratio (10.5(2.2) basal value) increased to 19.67(4.6) with reperfusion in the treatment group 1, mainly because of a lower decrease of GSH and a lower level of GSSG while it did not change in treatment group 2 (10.7(5.0)). Levels of creatine phosphokinase did not change in the treated groups, although they increased significantly in the control group (P < 0.006). Although oxidative stress is not the only cause of damage in revascularization, this study confirms the protective ability of treatment with free radical scavengers and membrane-stabilizing compounds.

Local haemofiltration with free radical scavenger treatment during revascularisation of severe muscular ischaemia induced in sheep limbs.

Many treatments have been proposed for the prevention of the revascularisation syndrome following embolectomy or thrombectomy in patients with severe ischaemia. These include the administration of diuretics, bicarbonate, buffer solutions, free radical scavengers, washing out the venous blood from the ischaemic leg, or systemic dialysis. The aim of our study was to investigate the effect of combining haemofiltration with a treatment using compound oxy-radical scavengers in order to prevent or to reduce the appearance of the revascularisation syndrome. The study was performed on 13 sheep. Eight animals underwent 4 h of aortic and vena cava occlusion using irrigation-occlusion catheters, followed by normal reperfusion (control group). Five sheep underwent the same period of ischaemia, followed by 1 h of local haemofiltration and re-oxygenation and 2 h of normal revascularisation. The priming solution for the ECC circuit consisted of 500 ml of 20% mannitol and 500 ml of 18/1000 HCO3- contained: superoxide dismutase (150,000 I.U.), methylprednisolone, 1 g, and heparin, 10,000 I.U. After the 3rd h of ischaemia, 2.1 g of acetate alpha-tocopherol (30 mg kg-1) were injected i.m. The treatment produced good protection against oxidative stress, shown by an increase in the glutathione ratio (GSH/GSSG), and reduced muscular damage, confirmed by a moderate increase in creatine phosphokinase (CPK) levels (significantly higher in the control group). Diuresis was significantly higher in the treated group, and the acid-basic and potassium balance returned to normal more rapidly. Our data suggest that this combined treatment could be effective in the prevention of the ischaemia-reperfusion syndrome.