Treatment with embryonic stem-like cells into osteochondral defects in sheep femoral condyles

Background Articular cartilage has poor intrinsic capacity for regeneration because of its avascularity and very slow cellular turnover. Defects deriving from trauma or joint disease tend to be repaired with fibrocartilage rather than hyaline cartilage. Consequent degenerative processes are related to the width and depth of the defect. Since mesenchymal stem cells (MSCs) deriving from patients affected by osteoarthritis have a lower proliferative and chondrogenic activity, the systemic or local delivery of heterologous cells may enhance regeneration or inhibit the progressive loss of joint tissue. Embryonic stem cells (ESCs) are very promising, since they can self-renew for prolonged periods without differentiation and can differentiate into tissues from all the 3 germ layers. To date only a few experiments have used ESCs for the study of the cartilage regeneration in animal models and most of them used laboratory animals. Sheep, due to their anatomical, physiological and immunological similarity to humans, represent a valid model for translational studies. This experiment aimed to evaluate if the local delivery of male sheep embryonic stem-like (ES-like) cells into osteochondral defects in the femoral condyles of adult sheep can enhance the regeneration of articular cartilage. Twenty-two ewes were divided into 5 groups (1, 2, 6, 12 and 24 months after surgery). Newly formed tissue was evaluated by macroscopic, histological, immunohistochemical (collagen type II) and fluorescent in situ hybridization (FISH) assays. Results Regenerated tissue was ultimately evaluated on 17 sheep. Samples engrafted with ES-like cells had significantly better histologic evidence of regeneration with respect to empty defects, used as controls, at all time periods. Conclusions Histological assessments demonstrated that the local delivery of ES-like cells into osteochondral defects in sheep femoral condyles enhances the regeneration of the articular hyaline cartilage, without signs of immune rejection or teratoma for 24 months after engraftment. Electronic supplementary material The online version of this article (doi:10.1186/s12917-014-0301-9) contains supplementary material, which is available to authorized users.


INTRODUCTION
Background 3 a. Include sufficient scientific background (including relevant references to previous work) to understand the motivation and context for the study, and explain the experimental approach and rationale. Articular cartilage has a poor intrinsic capacity for regeneration because of its avascularity and very slow turnover both at the cellular and molecular levels. As consequence, defects occurring as a result of trauma or joint disease tend to be repaired with fibrocartilage rather than hyaline cartilage. With time, degenerative processes frequently occur in the regenerated tissue [1,2,3], which may stabilize or progress in relation to 2 main factors: the width and depth of the defect. It has been demonstrated that sheep articular cartilage osteochondral defects 3 mm wide or less re-fill with normal hyaline cartilage, whereas wider defects are replaced by fibrocartilage which, eventually, degenerate in fibrous tissue [4]. This inferior repair tissue is not capable of withstanding the mechanical loads exerted on the tissue during locomotion and the result is eburnation of the subchondral bone [4,5]. In relation to the depth, superficial defects involving only the articular cartilage do not heal spontaneously [1,2,6], while osteochondral defects, penetrating the subchondral bone and thus, gaining access to the mesenchymal stem cells (MSCs) that reside in the bone marrow space, can give rise to regenerated cartilage [7,8,9].Surgical treatments used to stimulate cartilage regeneration, in most cases, result only in a delay in the onset of degenerative processes [10, 11, 12, 13, 14 ,15]. Thus, there is a search for alternative solutions and cells engraftment is among the most advanced new technologies in cartilage repair [16,17,18,19,20]. Considering that in certain degenerative diseases, autologous stem cells are depleted and have reduced proliferative capacity and chondrogenic ability [21,22], the delivery of heterologous cells may enhance repair or inhibit the progressive loss of joint tissue [21,22]. Among the several factors to be considered in the choice of the type of cells, there are the ease of harvest, the cell yield and purity and their proliferative and chondrogenic capacity [23]. Autologous Chondrocytes Implantation (ACI), the first technique used to repair focal cartilage defects [24,25,26], is associated with donor site morbidity, loss of chondrocyte phenotype upon ex vivo expansion and inferior fibrocartilage formation at the defect site [27,28,29]. Thus, new extracorporeal cell sources are sought, mainly stem cells. Among them, Mesenchymal Stem Cells (MSCs) [13,18,19,21,30], shows the advantages of their immunoevasivity [31] and immunosuppressive effect [32,33], but they have a limited capacity for self-renewal and proliferation, and differentiation potential impaired with increasing donor age [34]. On the contrary, Embryonic Stem Cells (ESCs) are able to self-renew for prolonged periods without differentiation and, most importantly, to differentiate into a large variety of tissues derived from all 3 germ layers [35,36,37,38].
b. Explain how and why the animal species and model being used can address the scientific objectives and, where appropriate, the study's relevance to human biology.
The histological appearance of the articular regenerated tissue is essential for the validation of therapeutic interventions [9] and it is likely to be predictive of its functionality and durability [39]. Since in human is possible to perform only small arthroscopic sampling to evaluate the histological aspect of the regenerated tissue, in deep histological studies can be achieved in vivo only in animal models. To date few studies concerning cartilage regeneration have been performed in animal models in vivo [40,41,42,43] and, until recently, most of the research on stem cells has been performed in small laboratory animals [13,44]. However, they do not represent an optimal model for achieving cartilage regeneration in human. On the contrary, the larger size and weight of adult sheep, which places greater weight-bearing loads on the healing site, as well as the structural, biochemical, physiological and immunological similarities to man and the ease and cheap cost of its management in respect to other species, make sheep an optimal experimental model for future clinical application in human [45,46].

Objectives 4
Clearly describe the primary and any secondary objectives of the study, or specific hypotheses being tested.
a. Confirm the origin of the stem tissue newly generated by sexing the cells with the PCR in the liquid phase and in in situ hybridization; b. Evaluate the histopathological point of view the reparative process verifying the deposition of type II collagen and comparing the implants with the controls at 1, 2, 6, 12 and 24 months.

Study design 6
For each experiment, give brief details of the study design including: a. The number of experimental and control groups. 4 groups of 4 Sarda sheep (1, 2, 6 and 12 month from surgical procedure) and 1 group of 6 Sarda sheep (24 month from surgical procedure) each were studied (total 22 ewes). ES-like cells were engrafted in the osteochondral defect created in the left medial femoral condyle (ES), while the identical defect created in the controlateral stifle joint was left untreated (Empty Defect: ED) and served as a control.
b. Any steps taken to minimise the effects of subjective bias when allocating animals to treatment (e.g. randomisation procedure) and when assessing results (e.g. if done, describe who was blinded and when).
All animals before the surgery have been adapted to the environmental conditions in the locations of hospitalization at least one month. In this time have been performed rutine analysis for parassites or other disease in progress. After the surgery, all animals were released in paddocks with the same environmental conditions and feeding.
c. The experimental unit (e.g. a single animal, group or cage of animals).
In the study, n refers to number of animals. From each condyle we got 2 half-lesion ( Figure  1. A and B) and from each half lesion we got 4 representative slides for histological staining and immunohistochemistry.  . A lubricated stomach tube of 1 cm of inner diameter was inserted into the rumen to prevent bloating. The animal was positioned in dorsal recumbence in a cradle for thoracic containment, leaving posterior limbs free. With the knee in the maximum flexure, a lateral para-patellar arthrotomy of both stifle joints was performed using a lateral approach and medial patellar dislocation, to expose the articular surface of the weight-bearing area of the medial femoral condyle. A cranio-lateral cutaneous incision, of about 8 cm in length. A full-thickness defect was performed using a punch for chondral biopsy of 6 mm of diameter to mark off the edges of the defect and to cut the cartilage until the calcified portion. Thus, all defects had the same diameter (6 mm) and depth (2 mm) and involved the subchondral bone ( Figure 2).During drilling, the area was infused with saline solution to cool the tissue and to avoid dehydration of articular cartilage due to the loss of synovial fluid during the surgical procedure. All animals received an intra-surgical antibiotic and antinflammatory therapy (amoxicillin 25 mg/kg im and ketoprofen 2 mg/kg im), continued for the following post-surgical 4 days, before of the re-positioning of the patella and the suture of the several layers. All surgical procedures were performed by the same operator, in the respect of the welfare laws. Antibiotic and antinflammatory therapy (amoxicillin 40 mg/kg/day im and ketoprofen 2 mg/kg/day im) was administered for 5 days. All animals were kept confined in paddocks for 10 days in groups of 6 animals each, and then were allowed to roam freely on pasture for the rest of the study.  a. Provide details of the animals used, including species, strain, sex, developmental stage (e.g. mean or median age plus age range) and weight (e.g. mean or median weight plus weight range). all animals were reared in semi-extensive paddocks (figure 4) Figure 4 semiextensive paddocks (DVM) b. Husbandry conditions (e.g. breeding programme, light/dark cycle, temperature, quality of water etc for fish, type of food, access to food and water, environmental enrichment).
The water was always available and the feed was mixed hay and concentrates c. Welfare-related assessments and interventions that were carried out prior to, during, or after the experiment. The sequence for the analysis of the groups was: group 1, 2, 6, 12 and 24 Experimental outcomes 12 Clearly define the primary and secondary experimental outcomes assessed (e.g. cell death, molecular markers, behavioural changes).

Statistical methods 13
a. Provide details of the statistical methods used for each analysis.
The Mixed Procedure of SAS 8.2 (SAS Institute Inc., Cary, NC, USA) was used to perform the analysis. b. Specify the unit of analysis for each dataset (e.g. single animal, group of animals, single neuron). c. Describe any methods used to assess whether the data met the assumptions of the statistical approach.
An analysis of variance was performed on the macroscopic and histological data from the total and some selected categories scores of both treatments throughout all considered periods. The model of the analysis contained the main effects of treatment and time from surgery and the interaction between them, together with the random effect of the ewe within the period.
For each experimental group, report relevant characteristics and health status of animals (e.g. weight, microbiological status, and drug or test naïve) prior to treatment or testing. (This information can often be tabulated).
The animals' health status was monitored throughout the experiments by a health surveillance programme according to Ethical Committee of the University of Sassari and by the Veterinary control officers of Animal Protection in experimental and clinical studies made in the University of Sassari (see point 5)).

Numbers analysed 15
a. Report the number of animals in each group included in each analysis. Report absolute numbers (e.g. 10/20, not 50%2). Outcomes and estimation 16

ES
Report the results for each analysis carried out, with a measure of precision (e.g. standard error or confidence interval). In this study ES showed a significantly better histological healing process as compared to ED in most of the examined categories. In our knowledge, this is the first time that ES-like cells are engrafted in sheep and that engraftments have been evaluated until 24 months from surgery. Up to now, they have been assessed at a maximum period of 18 months in sheep [4], 12 months in goat [21], 8 months in horse [18] and 6 months in laboratory animals [13,45,46,47]. The treatment of ES-like cells, engrafted into osteochondral defects in sheep knee condyles, enhances the regeneration of the articular hyaline cartilage, above all in the long term periods. b. Comment on the study limitations including any potential sources of bias, any limitations of the animal model, and the imprecision associated with the results2.
In small ruminants (sheep [4,20,44,49,50] and goat [5,7,21]), most of experiments euthanized animals at 6 months, probably employing the later time period used in laboratory animals. According to the authors: this is a too short period to establish a complete regeneration process in large animals, in agreement with Schneider-Wald [55], who affirms that the follow-up period for assessment of the effectiveness of cartilage regeneration is 12months. According to the authors: this is a too short period to establish a complete regeneration process in large animals, in agreement with Schneider-Wald [55], who affirms that the follow-up period for assessment of the effectiveness of cartilage regeneration is 12months. Moreover, in sheep, several authors [45,49,51,52,53] perform chondral defect, despite such kind of defect can't heal spontaneously [1,2,6]. Even, most of experiments performed in sheep employed surgical techniques without the use of cells [4,20,50,54]. For all these reasons, a comparison of results obtained in this experiment with other trials resulted very difficult to perform. c. Describe any implications of your experimental methods or findings for the replacement, refinement or reduction (the 3Rs) of the use of animals in research.

Generalisability/ translation 19
Comment on whether, and how, the findings of this study are likely to translate to other species or systems, including any relevance to human biology.
Sheep demonstrated to be a valid model for human translational research when compared with laboratory animals, due to the increased stifle size, less effective native cartilage repair and longer lifespan. Moreover, a recent study comparing the differences in geometry and mechanical properties of human, porcine, bovine and ovine articular cartilage, found that human cartilage had a significant largest equilibrium elastic modulus in respect to porcine and bovine cartilage, but not with ovine cartilage [50]. In addition, from the regulatory point of view, the ovine model is one of the suggested large animal models for pre-clinical studies [48].

Funding 20
List all funding sources (including grant number) and the role of the funder(s) in the study.