Ardanaz et al. BMC Veterinary Research (2016) 12:65 DOI 10.1186/s12917-016-0692-x RESEARCH ARTICLE Open Access Inflammatory response to the administration of mesenchymal stem cells in an equine experimental model: effect of autologous, and single and repeat doses of pooled allogeneic cells in healthy joints N. Ardanaz1, F. J. Vázquez1, A. Romero1, A. R. Remacha2, L. Barrachina1,2, A. Sanz2, B. Ranera2, A. Vitoria1, J. Albareda3, M. Prades4, P. Zaragoza2, I. Martín-Burriel2 and C. Rodellar2* Abstract Background: Mesenchymal stem cells (MSCs) transplantation has become a promising therapeutic choice for musculoskeletal injuries. Joint-related disorders are highly prevalent in horses. Therefore, these animals are considered as suitable models for testing MSC-based therapies for these diseases. The aim of this study was to investigate the clinical and inflammatory responses to intra-articular single and repeat dose administration of autologous or of pooled allogeneic MSCs in healthy equine healthy joints. Six horses were intra-articularly injected with a single autologous dose of bone marrow derived MSCs (BM-MSCs) and two separate doses of allogeneic BM-MSCs pooled from several donors. All contralateral joints were injected with Lactated Ringer’s Solution (LRS) as the control vehicle. Signs of synovitis and lameness were evaluated at days 0, 1, 2, 3, 5 and 10 after injection. Total protein (TP), white blood cell count (WBC) and neutrophil count (NC) in synovial fluid were also measured at the same time-points. Results: A mild synovial effusion without associated lameness was observed after all BM-MSCs injections. The second allogeneic injection caused the lowest signs of synovitis. Local temperature slightly increased after all BM-MSCs treatments compared to the controls. TP, WBC and NC in synovial fluids also increased during days 1 to 5 after all BM-MSCs injections. Both, clinical and synovial parameters were progressively normalized and by day 10 post-inoculation appeared indistinguishable from controls. Conclusions: Intra-articular administration of an allogeneic pool of BM-MSCs represents a safe therapeutic strategy to enhance MSCs availability. Importantly, the absence of hypersensitivity response to the second allogeneic BM-MSCs injection validates the use of repeat dose treatments to potentiate the therapeutic benefit of these cells. These results notably contribute to the development of stem cell based therapies for equine and human joint diseases. Keywords: Allogeneic, Horses, Joint, Mesenchymal stem cells, MSCs * Correspondence: rodellar@unizar.es 2 Laboratorio de Genética Bioquímica LAGENBIO, Universidad de Zaragoza, C/ Miguel Servet, 177, Zaragoza 50013, Spain Full list of author information is available at the end of the article © 2016 Ardanaz et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ardanaz et al. BMC Veterinary Research (2016) 12:65 Page 2 of 9 Background antigens CD90, CD105, CD73 and CD166 were positive, Osteoarthritis (OA) is an irreversible degenerative dis- whereas no mRNA was detected for haematopoietic ease characterized by articular cartilage loss and syn- markers CD45 and CD34. Specific dyes confirmed the ovial inflammation. Current OA therapeutic strategies ability of the cells to differentiate into osteogenic, adipo- are focused on reducing pain, physical disability and genic and chondrogenic lineages after induction with handicap, and try to limit structural deterioration in corresponding media. Characterization data are pre- affected joints [1, 2]. sented in Fig. 1 and Additional file 1. Viability and pro- Cell therapy using stem cells has become a large field liferation of cryopreserved cells was confirmed by the of research focusing on the development of effective maintenance of similar cell doubling time (DT) after treatments for this disease [3]. Mesenchymal stem cells thawing. MSC gene expression of MHC-I and MHC-II is (MSCs) are studied as a possible tool for cell therapy not showed in Fig. 1. Moderate expression of MHC-I and only by their “stem” properties and differentiation poten- low expression of MHC-II were reported, according to tial but also by their trophic and immunomodulatory previous studies [18]. properties [4, 5]. Horses commonly suffer from OA and osteochondrosis, being diseases of great concern for equine clinicians [6]. In addition, horses are considered Clinical assessment the most appropriate animal model for testing the clin- No visual evidence of synovitis was observed in the con- ical effects of MSC-based therapies for humans joint trol joints in any of three injections. Some articular dis- injuries [7]. tension was detected on treated joints in all While both autologous and allogeneic MSCs therapies experiments. The visual exam showed a marked disten- have been used for the treatment of several diseases [8], sion only in two joints injected with autologous MSCs the use of allogeneic MSCs has gained relevance due to (Injection 1). Mild synovial effusion after the first allo- their shorter ex vivo expansion time [9] and the possibil- geneic pool of MSCs administration (Injection 2) was ity to be selected according to their characteristics to observed, whereas the allogeneic reinjection (Injection optimize the treatment (higher immunomodulatory cap- 3) provoked only a slight distention. In addition, a slight acity, rate of growth in culture, etc). increase in local temperature was observed (Fig. 2), ran- The administration of a single dose and repeat doses ging from 0.5 to 3 °C after Injection 1 in the treated of allogeneic-derived MSCs obtained from one donor joints compared with contralateral joints injected with has been tested in equine under different conditions Lactated Ringer’s Solution (LRS) as control. In Injec- [10–12]. Although it has been demonstrated that repeat tions 2 and 3, the average temperature increase in injections of MSCs can enhance the benefit of these cells treated joints respect their controls was less than 1 °C. in different pathology models and administration routes [13, 14], it remains unclear if allogeneic MSCs can pro- voke an immunoresponse [15, 16]. Using single-donor allogeneic MSCs has some constraints, such as the donor selection or the number of cells obtained under culture conditions. Therefore, the use of MSCs pooled from several donors could be advantageous. To our knowledge, safety of repeat intra-articular ad- ministrations of allogeneic bone-marrow derived MSCs (BM-MSCs) pooled from several donors has not been yet studied in horses. Neither their safety profile can be extrapolated from allogeneic-single administration of one-donor MSCs [10, 17]. Hence, in this work we evalu- ate the clinical and inflammatory response to the admin- istration of autologous and repeat doses of allogeneic BM-MSCs pooled from several donors in tarso-crural and radio-carpal equine healthy joints. Fig. 1 Mean ± standard error (S.E.) of relative mRNA expression Results (y axis) of 6 antigens surface markers (light grey) and 2 major MSC isolation, differentiation and characterization histocompatibility complex molecules (dark grey) (x axis) for BM-MSCs Approximately 80 x 106 BM-MSCs in third passage were (n = 6) examined by RT-qPCR. The BM-MSC used in this study were positive for CD90, CD105, CD73, CD166 and MHC-I and negative for successfully obtained from the bone marrow aspirate of CD34 and CD45. MHC-II was expressed at low level each horse. Gene expression of the surface marker Ardanaz et al. BMC Veterinary Research (2016) 12:65 Page 3 of 9 In all cases, the normal values were restored between 3 day 10 (Additional file 2). Ultrasonography findings were and 10 days and differences were not statistically signifi- compatible with transient slight synovitis in treated cant at any time for any Injection. animals receiving Injections 1, 2 and 3. Ultrasonographic Throughout the study none of the animals showed signs of synovitis disappears by10 days post-inoculation. lameness in trot, except one horse after Injection 1. The Re-injected joints (Injection 3) showed the lowest signs lameness gradually decreased over time and absent at of synovitis (Fig. 3). Fig. 2 Mean ± standard deviation of difference in local temperature and synovial fluid parameters in Injections 1, 2 and 3. Mean ± standard deviation (SD) of difference in local temperature and synovial fluid parameters analyzed for autologous (Injection 1), allogeneic (Injection 2) and repeat allogeneic (Injection 3) mesenchymal stem cell (MSC) injected joints compared to controls. a Difference in local temperature (°C) between MSC injected joints (TR.) and control (CTRL.) joints (local temperature in MSC injected joints – local temperature in control joints) at each time point for Injection 1 (A1), Injection 2 (A2) and Injection 3 (A3). b Total protein concentration (g/dL) at each time point for Injection 1 (B1), Injection 2 (B2) and Injection 3 (B3). c White blood cell count (cells/μl) at each time point for Injection 1 (C1), Injection 2 (C2) and Injection 3 (C3). d Neutrophil count (cells/μl) at each time point for Injection 1 (D1), Injection 2 (D2) and Injection 3 (D3). Asterisk symbol (*) indicates statistically significant difference (p <0.05) between mean ± SD in control and MSC treated joints at each time point. Controls are represented as dotted lines and treated as solid lines Ardanaz et al. BMC Veterinary Research (2016) 12:65 Page 4 of 9 Fig. 3 Longitudinal ultrasound images of representative MSC-injected tarso-crural and radio-carpal joints after Injections 1, 2 and 3. The transducer was longitudinally placed on the lateral plantar pouch of the tibiotarsal joint or in the dorsal aspect of the carpus, parallel and medial to the extensor carpi radialis tendon, at the level of the radio-carpal joint. Images are recorded previous to the IA injection of BM-MSCs (T0), and 1 day (T1) and 10 days (T10) after BM-MSC injection. Distance between the bone surface and the skin was measured in two points at every time. Slight distension and effusion was observed 1 day after all BM-MSC injection, which was normalized by the 10th day. Ta = talus, Ti = tibia, R = radius, RC = radio-carpal bone, * = synovial fluid (effusion) in tarso-crural or radio-carpal joint, white arrow indicates the bone surface Assessment of synovial fluids treated joints was evident on day 1 after the three Injec- TP, WBC and NC values at each time-point are represented tions, being statistically significant for Injections 1 and 3. in Fig. 2 and Additional file 3 . This NC increment suddenly decreased at 48 h post- injection and remained low until reaching normal refer- Total protein (TP) ence range on day 10. A non-significant transient increase of TP values (<2gr/dL) was observed in control joints injected with LRS. Discussion Significant differences in TP values between treated In our work, we have evaluated the clinical and inflam- and control joints were detected post-injection on days matory responses of healthy equine joints after intra- 1 and 2 (Injection 1), days 2 and 5 (Injection 2) and articular injections of autologous and allogeneic single days 1, 2, 3 and 5 (Injection 3). The largest TP increase and repeat pool of BM-MSCs. BM-MSCs were used due was detected 24 h post-Injection 3. In all cases the TP to their regenerative performance and its ability to inte- progressively decreased reaching normal-healthy values grate into the soft tissue joint [19, 20] as well as their (<2gr/dL) on the 10th day post-inoculation. anti-inflammatory and immunomodulatory properties [4]. Equine species was selected due to its availability as White blood cells count (WBC) human joint disease model [21] and its own relevance in A moderate rise in WBC was observed in all control veterinary medicine [22]. Healthy joints were chosen to joints (Additional file 3). WBC counts increased in all approach articular immunoresponse to repeat allogeneic treated joints. The increase was statistically significant injections of BM-MSCs. Behavior and properties of after inoculation on days 1, 2 and 5 after Injections 1 MSCs, such their migratory pattern [23] or the safety of and 3. No significant differences in WBC values were therapeutic strategies based on MSCs [24, 25], are usually found after Injection 2 at any time point. WBC values prior studied in healthy animals. The conditions in healthy diminished to normal reference ones in all cases by the and inflamed joints are different (i.e. vascularization, 10th day. leukocyte traffic), so the response of healthy joints to repeat allogeneic MSCs cannot be directly extrapolated. Neutrophil count (NC) Despite these differences, performing a preliminary study Control joints showed a slight increase in NC after the in healthy joints seems mandatory since if a negative three Injections (Additional file 3). The NC increase in response in normal joints would have been observed, this Ardanaz et al. BMC Veterinary Research (2016) 12:65 Page 5 of 9 would discourage the use of repeat allogeneic doses of parameters. Our results are similar to other clinical evi- MSCs in altered joints. dence recently described after injection of autologous There are a few of very recent studies comparing the and allogeneic MSCs in equine healthy joints [10, 11]. immune response to autologous and allogeneic mesen- Similarly, reports using an equine synovitis model have chymal stem cells intra-articular injections in horses, showed that allogeneic MSCs induce an initial mild self- both in normal joints [10, 26, 27] and inflamed joints limiting inflammatory reaction when administered [28]. However, none of these studies assess the use of a within the inflamed joint, but MSCs reduced the syn- pool of allogeneic donors and the re-inoculation of joints ovial fluid cell populations in this model [28]. Slight with a second dose of cells. The use of repeat doses of lameness was noted only in one animal 24 h post autolo- MSCs could be very important in order to potentiate the gous MSC administration (Injection 1). This animal also MSC therapeutic effect, as it has been demonstrated in showed more marked signs of synovitis at this time. other species and diseases [13, 14]. Using a pool of allo- Allogeneic MSCs injection did not lead to any grade of geneic MSC donors is also interesting in order to avoid lameness in any horse at any time. Mild to severe lame- individual variations and limited number of MSCs in ness 24 h post injection of allogeneic CB-MSCs in in- some cases. flamed joints has been reported, but lameness did not The BM-MSCs showed a normal growth pattern, with correlate with synovitis induced by MSCs injection [28]. a proliferation rate and viability similar to other studies We assessed the articular inflammatory response after [29, 30]. After cryopreservation, the cells showed a a second inoculation of the allogeneic equine MSC normal growth pattern indicating no effect on growth. (Injection 3). No hypersensitivity response was observed. According to previous reports [31], equine BM-MSCs The re-injection caused a transient inflammatory reac- displayed similar gene expression of the surface markers. tion similar to that detected after Injections 1 and 2. MSCs used in our study were in passage 3 to avoid the Synovial parameters (TP, WBC and NC) changes were risk of morphological or functional changes that might more evident than clinical signs, but spontaneously occur at long-term culture [10]. returned to baseline by 10 days in all cases. Intra-articular injections were performed using autolo- Although the cause of inflammation produced by the gous and allogeneic pools of BM-MSCs. Allogeneic MSC injection of BM-MSCs is still unknown [26], there are therapy is an interesting strategy using “off-the-shelf” different factors that might be taken into account. The product. Allogeneic therapy avoid autologous limitations production, expansion and infiltration procedures were such as the scarcity of MSCs, especially in cases where carried out in aseptic conditions, leaving out a possible MSC are low and the quality might be compromised as septic origin that was supported by no detection of clin- it happens in aged individuals and disease, and also in ical or cytological evidence of a possible septic arthritis. situations where autologous MSCs might have the same Besides the inflammation was self-limiting and resolved genetic defects as the patient [9, 32]. In addition, allo- without any medication, which is incompatible with the geneic use of MSCs offers the possibility of selecting the development of iatrogenic joint sepsis [33]. There is con- most suitable donors according to their proliferation troversy about the cause of this joint inflammation. rate, their differentiation ability or their immunoregula- Some authors point out that any articular injection may tory properties. cause at least a slight synovitis, or even a kind of reactive The allogeneic BM-MSC pool offers the advantage of synovitis could occur after joint injection of any type of availability of elevated number of MSCs for the treat- product [33–35]. Other authors suggest that the inflam- ment of acute orthopedic lesions without the inherent matory response that follows administration of MSCs period (4–6 weeks) associated with isolation and expan- may be due to the exquisite sensitivity of healthy equine sion of autologous MSCs [26]. Furthermore, the prolifer- joints to the introduction of pro-inflammatory cytokines ation rate and chondrogenic differentiation capacity of that may be present in cell culture, or even to chemical MSCs might be different because of individual-related or biologically active substances present in the culture factors [11] and also their immunomodulatory proper- medium used, such as fetal bovine serum [26]. Recently, ties, so the use of several donors could attenuate this it has been demonstrated that equine allogeneic MSCs variability. The results of this study show that intra- are capable of eliciting antibody responses in vivo, which articular inoculation of equine BM-MSC, independently might be cross-reactive with donor MHC types different of their origin (autologous or allogeneic pool), or the to the recipient [36]. In our experimental conditions, number of doses, produces a transitory inflammatory re- our results suggest that the significant increase in syn- action that becomes restored to normal values 10 days ovial parameters detected during the first few days after later, without any anti-inflammatory or analgesic drug LRS injection could be an inflammatory effect of re- administration. This temporary inflammation was de- peated arthrocentesis. Similar results were found by tected in both clinical and synovial (TP, WBC and NC) Jacobsen et.al. [37] and Sanchez Teran et.al. [38]. Ardanaz et al. BMC Veterinary Research (2016) 12:65 Page 6 of 9 Differential inflammatory responsiveness of the joints All procedures were carried out under Project Licence have also been discussed [11]. Indeed, the distal inter- (PI 09/12) approved by the in-house Ethic Committee for phalangeal joint and tarso-crural joint are more suscep- Animal Experiments from the University of Zaragoza. The tible to reactive arthritis following intra-articular care and use of animals were performed in accordance with injection [37]. In this context, differences in the inflam- the Spanish Policy for Animal Protection RD53/2013, matory response between autologous BM-MSCs and which meets the European Union Directive 2010/63 on the the allogeneic pool of BM-MSC treated joints could protection of animals used for experimental and other be due to inter-individual and inter-joint variability. scientific purposes. Certainly, the autologous injection was made in the tarso-crural joint and allogeneic doses were adminis- Study design tered in the radio-carpal joint, which seems to be less Three batches of injections (Injections 1, 2 and 3) were prone to inflammatory reaction than the tarso-crural performed in all horses by a single blinded researcher joint [34]. (Nekane Ardanaz: NA). Joint treatments were randomly In agreement with other similar trials, our results assigned. The same researcher also carried out the as- showed higher WBC and NC increases than those ob- sessment of the clinical and inflammatory response on served for protein values [10, 26]. This difference may days 0, 1, 2, 3, 5 and 10 after each intra-articular admin- be due to the kinetics of these two inflammation istration of the tree Injections. Horses were not ban- markers [26, 39]. This trend has also been observed in daged or medicated with any anti-inflammatory, other works analyzing the response of equine synovial antibiotic or analgesic drugs in order to not interfere fluid to intra-articular injection of different inflammatory with the inflammatory response, and to provide an un- stimuli [40]. altered clinical response to the MSC injections through- out the experiment. Conclusion Injection 1: All horses were blindly injected in the Our experiments have shown a transitory inflammatory tarso-crural joints. One of the joints was inoculated response in all injected equine healthy joints to the ad- with 3 mL of Lactated Ringer’s solution (LRS), which ministration of autologous, and single and repeat doses was used as control vehicle. The contralateral joint was of allogeneic MSCs pooled from several donors. This treated with 25x106 autologous BM-MSCs suspended situation resolved spontaneously within 10 days post- in 3 mL of LRS. inoculation. The different conditions in healthy and in- Injection 2: Ten days after the Injection 1 each horse flamed joints do not allow to directly extrapolating our was blindly inoculated in their radio-carpal joints. One results to an articular damage situation. Despite this, we of the joints was injected with 3 mL of LRS (control) suggest based on these findings that repeat intra- and the contralateral was inoculated with a pool of articular administration of an allogeneic pool of BM- 25x106 allogeneic BM-MSCs (5x106 BM-MSCs from MSCs could be used as a safe strategy, enhancing the each donor) diluted in 3 mL of LRS. For each individual MSCs availability. The absence of a hypersensitivity re- horse, the BM-MSC pool used was derived from all sponse to the second allogeneic BM-MSCs injection other animals in the study but excluded BM-MSCs could also be considered an important contribution to from the animal in question. the in vivo transplantation of MSCs, since several injec- Injection 3: The same procedure described for tions could potentiate the therapeutic benefit of these Injection 2 was replicated 10 days later, once the cells. These results could notably contribute to the de- inflammatory parameters after Injection 2 returned to velopment of stem cell based therapies for equine and normality. human joint diseases. MSC isolation, culture, expansion, cryopreservation, differentiation and characterization Methods Horses were sedated with romifidine (0.04 mg/kg Animals IV) and butorphanol (0.02 mg/kg IV) and were Six crossbreed saddle gelding horses aged from 3 to placed in a restrain stock. Approximately 60 mL of 7 years were used in this study. Absence of musculoskel- bone marrow (BM) aspirate from the 5th sternebrae etal abnormalities in the tarso-crural and radio-carpal from each horse were aseptically collected in heparinized joints was determined by clinical and radiological exam- syringes [41]. Bone marrow derived mesenchymal stem ination, including absence of lameness, palpation, range cells (BM-MSCs) were obtained from the BM aspirates. of motion, static and dynamic flexion tolerance and ab- BM aspirates were diluted 1:3 with PBS and then lay- sence of radiological abnormalities. ered over Lymphoprep (Atom) and centrifuged for Ardanaz et al. BMC Veterinary Research (2016) 12:65 Page 7 of 9 20 min at 1700 g. The mononucleated cell population et al. [31, 42]. All reactions were carried out in triplicate layer above the Lymphoprep was aspirated and washed in a total volume of 10 μl with 2 μl of cDNA as template twice with PBS (Gibco). The pellet was resuspended in and Fast SYBR Green Master Mix (Applied Biosystems). 10 mL growth medium, consisting of low glucose The amplification consisted of 40 cycles of 3 s at 95 °C Dulbecco Modified Eagle’s Medium (DMEM) and sup- and 30 s at 60 °C. For analyzing and presenting the gene plemented with 10 % fetal bovine serum, 1 % glutamine expression data, a relative expression method was used: and 1 % streptomycin/penicillin (all from Sigma- the comparative Ct method was used to give the levels Aldrich). Cells were counted, plated at a density of 2 × of gene expression. Expression level of every gene in 106 nucleated cells/cm2 in 6-well plates and incubated at each sample was normalized through a normalization 37 °C, 5 % CO2. Cells were washed twice with PBS after factor (NF), calculated as the geometric mean of the 24, 48 and 72 h of incubation and were maintained in quantity of 2 housekeeping genes, GAPDH and B2M growth medium until reaching approximately 80 % con- [43]. The data of the gene of interest is presented as fluence. The cells were then detached by treating with mRNA expression level relative to housekeeping genes, 0.25 % trypsin-EDTA (Sigma Aldrich), counted in a using the cycle threshold (Ct) values to calculate the Neubauer counting chamber using trypan blue staining, expression. and plated in T75 or T175 flasks (Becton Dickinson) at Primer Express 2.0 software was used to design 5000 cells/cm2. The cells were trypsinised repeatedly primers based on known equine sequences. Information until the third passage and then were cryopreserved in about primers is shown in Additional file 4. 106 aliquots with freezing medium consisting on 90 % Proliferation rates, i.e. cell doubling times (DT), was FBS and 10 % DMSO. Approximately 106 cells from calculated before freezing and after thawing using the passage three were thawed at 37 °C and plated in a formulae CD = ln (Nf/Ni)/ln2 and DT = CT/CD, where T75 flask for three days to re-adjust the culture con- Nf indicates final number of cells; Ni indicates initial ditions prior to being characterized and used on the number of cells, CT indicates culture time and CD indi- different Injections. cates cell doubling number. Cells were characterized as BM-MSCs by tri-lineage For the experimental treatments, MSCs were detached differentiation and gene expression profile of cell surface from flasks by treating with 0.25 % trypsin-EDTA. MSCs markers. The tri-lineage differentiation ability was were then rinsed three times with PBS and subsequently, assessed plating cells at 20.000 cells/cm2 or 2.500 cells/ three times with Lactated Ringer’s Solution in order to cm2 in 12-well plates and cultured for 7 or 14 days in remove any residuary FBS. osteogenic or adipogenic medium, respectively. For chondrogenic differentiation, approximately 500.000 Clinical determinations cells were pelleted in 15 mL conical tubes and cultured Evidence of synovial effusion and joint distention were visu- in chondrogenic medium for 21 days. Differentiation ally assessed. One of the classical signs of inflammation is ability was evaluated for each lineage by specific staining, heat, which is exchanged between injured tissues and the using Alizarin Red S (Sigma-Aldrich), Oil Red O and environment [44]. Heat was quantified by measuring the Alcian Blue for osteogenic, adipogenic and chondrogenic skin temperature at each time point by non-contact infra- differentiation, respectively. Methodology and compos- red laser thermometer (GIM 530 MS Intelligent Multi ition of differentiation media were described by Ranera Purpose Infrared Thermometer, Thermolab). Static and dy- et al [31]. namic lameness examinations were performed following Phenotype of the BM-MSCs was determined by Real the parameters established by the American Association of Time quantitative polymerase chain reaction (RT-qPCR). Equine Practitioners (AAEP) [45]. Ultrasonography mea- Total RNA was isolated from approximately 106 cells surements were carried out in triplicate, using the same using the RNA spin mini (GE Healthcare Lifesciences, anatomical references. The evaluations of the articular sur- LittleChalfont, UK) and DNAse turbo (Ambion, Foster face, the thickness between skin and articular surface and City, California, USA.) kits. Afterwards, reverse tran- the relaxation of the synovial recesses were performed scription of 1 μg of total RNA to complementary DNA using an Ultrasound machine (HDI-3500, ATL) and a was performed using the Superscript kit (Invitrogen, 10 MHz linear transducer. All the parameters in the clinical Carlsbad, CA, USA). All kits were used according with assessment were evaluated by a researcher blinded to the the manufacturer’s instructions. The expression levels of Injections administered. genes coding for MSC surface markers (CD90, CD105, CD73 and CD166), haematopoietic markers (CD34 and Assessment of synovial fluids CD45) and immunogenic molecules (MHC-I and MHC- Synovial fluid (2 mL) samples from each control and II) were analyzed using a StepOne Real Time PCR Sys- treated joints were taken at the predefined time points. tem device (Applied Biosystems) as described by Ranera Total protein values (TP) were measured by refractometry Ardanaz et al. BMC Veterinary Research (2016) 12:65 Page 8 of 9 [46] using a RHB-32 Hand-held brix refractometer Acknowledgements (Spectrum Technologies). White blood cells (WBC/μL) This study was supported by the Ministerio de Economía y Competitividad, España (AGL2011-28609) and by the Gobierno de Aragón (Grupo de and neutrophil (NC/μL) countings were carried out using Investigación LAGENBIO). L. Barrachina was supported by a doctoral grant the Neubauer chamber. Joints with synovial values of TP from the Gobierno de Aragón. The authors thank Dr. Barba from Laboratorio <2 g/dL, WBC <1500 cells/μL and NC 250 neutrophil/μL de Análisis Veterinarios, Móstoles (Madrid) for the assistance in white blood cells (WBC) count. were considered as healthy [47]. All the parameters assessed in the synovial fluid were evaluated by a re- Author details 1 searcher blinded to the Injections administered. Servicio de Cirugía y Medicina Equina, Hospital Veterinario, Universidad de Zaragoza, C/Miguel Servet, 177, Zaragoza 50013, Spain. 2Laboratorio de Genética Bioquímica LAGENBIO, Universidad de Zaragoza, C/Miguel Servet, Statistical analysis 177, Zaragoza 50013, Spain. 3Departamento de Cirugía Ortopédica y Traumatología, Hospital Universitario Lozano Blesa, Universidad de Zaragoza, Statistical analysis was performed using the SPSS 15.0. San Juan Bosco, 15, Zaragoza 50009, Spain. 4Departament de Medicina i Differences between control and treated joints at each Cirugia Animal, Universidad Autónoma de Barcelona, Edifici H, UAB, 08193 time point were analyzed by Student’s t test. Significance Bellaterra, Barcelona, Spain. level was set at P < 0.05 for all analyses. Received: 30 June 2015 Accepted: 23 March 2016 Availability of supporting data Supplementary data associated with this article can be References 1. Walker-Bone K, Javaid K, Arden N, Cooper C. Regular review: found, in the online version, at doi: medical management of osteoarthritis. British Medical Journal. 2000;321(7266):936–40. 2. Hunter DJ, Felson DT. Osteoarthritis. British Medical Journal. Additional files 2006;332(7542):639–42. 3. Ilic D, Polak J. Stem cell based therapy–where are we going? Lancet. Additional file 1: BM-MSC characterization data, part 2. BM-MSCs used 2012;379(9819):877–8. in this studied were differentiated to adipogenic (A), osteogenic (B) and 4. Bartholomew A, Sturgeon C, Siatskas M, Ferrer K, McIntosh K, Patil S, Hardy chondrogenic (C) lineages. Negative controls (no differentiation induc- W, Devine S, Ucker D, Deans R, et al. Mesenchymal stem cells suppress tion) for each differentiation assay are presented below the differentiation lymphocyte proliferation in vitro and prolong skin graft survival in vivo. data from each lineage. (TIF 1300 kb) Experimental Hematology. 2002;30(1):42–8. Additional file 2: Lameness score data. Lameness scores for each 5. Ankrum JA, Ong JF, Karp JM. Mesenchymal stem cells: immune evasive, not animal at each time-point. Both control limb (receiving LRS as vehicle immune privileged. Nature Biotechnology. 2014;32(3):252–60. control) and MSC limb (receiving autologous or allogeneic MSCs) were 6. Schluetera A, Ortha M. Equine osteoarthritis: a brief review of the disease evaluated within groups of Injections 1, 2 and 3. (DOCX 17.5 KB) and its causes. Equine and Comparative Exercise Physiology. 2004;1(4):221–31. Additional file 3: Values of clinical and synovial parameters. Mean ± 7. Cellular TaGTAC: Cellular products for joint surface repair In FDA Center for standard deviation (SD) of local temperature and synovial fluid analysis Biologics Evaluation and Research..In.; 2005. for autologous (Injection 1), allogeneic (Injection 2) and repeat allogeneic 8. Schnabel LV, Fortier LA, McIlwraith CW, Nobert KM. Therapeutic use (Injection 3) mesenchymal stem cell (MSC) injected joints and their of stem cells in horses: Which type, how, and when? Veterinary Journal. controls. Numeric values for local temperature (°C), total protein 2013;197(3):570–7. concentration (g/dL), White blood cell count (cells/μl) and neutrophil 9. Hare JM, Fishman JE, Gerstenblith G, DiFede Velazquez DL, Zambrano JP, count (cells/μl) at each time point for Injection 1, Injection 2 and Suncion VY, Tracy M, Ghersin E, Johnston PV, Brinker JA, et al. Comparison Injection 3 are provided. Asterisk symbol (*) indicates statistically of allogeneic vs autologous bone marrow-derived mesenchymal stem cells significant difference (p <0.05) between control and MSC treated joints at delivered by transendocardial injection in patients with ischemic each time point. (DOCX 18 kb) cardiomyopathy: the POSEIDON randomized trial. The Journal of the Additional file 4: Data of primers used for RT-qPCR. Cell surface markers American Medical Association. 2012;308(22):2369–79. analyzed by RT-qPCR. GenBank accession numbers of the sequences used 10. Pigott JH, Ishihara A, Wellman ML, Russell DS, Bertone AL. Inflammatory for primers design. Primers (F: Forward and R: Reverse) and length of the effects of autologous, genetically modified autologous, allogeneic, and amplicon in base pair (bp). (DOCX 18.0 KB) xenogeneic mesenchymal stem cells after intra-articular injection in horses. Veterinary and Comparative Orthopaedics and Traumatology. 2013;26(6):453–60. Competing interests 11. Boone L. Intra-articular administration of allogeneic equine bone marrow The authors declare that they have no competing interests. None of the derived mesenchymal stem cells. Athens, Georgia: University of Georgia; authors has any financial or personal relationships that could inappropriately 2013. influence the content of the paper. 12. Carrade DD, Affolter VK, Outerbridge CA, Watson JL, Galuppo LD, Buerchler S, Kumar V, Walker NJ, Borjesson DL. Intradermal injections of equine Authors’ contributions allogeneic umbilical cord-derived mesenchymal stem cells are well CR, IMB, PZ and FJV conceived and supervised the experiments. CR obtained tolerated and do not elicit immediate or delayed hypersensitivity reactions. funding for the study. NA, AR, FJV, AV, JA and MP carried out clinical aspects Cytotherapy. 2011;13(10):1180–92. of the experiments. NA, LB, ARR and BR carried out the laboratorial work 13. van Velthoven CTJ, Kavelaars A, van Bel F, Heijnen CJ. Repeated including cell cultures, isolation, growing and differentiation of cells. AS, LB, Mesenchymal Stem Cell Treatment after Neonatal Hypoxia-Ischemia Has ARR and BR developed specific methodology for expression analysis. CR, FJV, Distinct Effects on Formation and Maturation of New Neurons and LB, JA and MP contributed with the interpretation of data. LB, AS and ARR Oligodendrocytes Leading to Restoration of Damage, Corticospinal Motor performed statistical analysis. PZ, IMB, JA and MP assisted in the experiments Tract Activity, and Sensorimotor Function. Journal of Neuroscience. with both theoretical and technical guidance throughout the entire work. All 2010;30(28):9603–11. the authors had extensive scientific discussion throughout this study. All the 14. Hatsushika D, Muneta T, Nakamura T, Horie M, Koga H, Nakagawa Y, Tsuji K, authors participated in manuscript writing. All authors read and approved Hishikawa S, Kobayashi E, Sekiya I. Repetitive allogeneic intraarticular the final manuscript. injections of synovial mesenchymal stem cells promote meniscus Ardanaz et al. BMC Veterinary Research (2016) 12:65 Page 9 of 9 regeneration in a porcine massive meniscus defect model. Osteoarthritis 34. Bertone AL. Noninfectious arthritis. In: Ross MW, Dyson SJ, editors. Diagnosis Cartilage. 2014;22(7):941–50. and management of lameness in the horse. 2nd ed. St. Louis, Missouri: 15. Schnabel LV, Pezzanite LM, Antczak DF, Felippe MJ, Fortier LA. Equine bone Elsevier; 2011. p. 657–90. marrow-derived mesenchymal stromal cells are heterogeneous in MHC 35. Ross M, Dyson S. Diagnosis and Management of Lameness in the Horse class II expression and capable of inciting an immune response in vitro. Elsevier Inc.; 2002. Stem Cell Res Ther. 2014;5(1):13. 36. Pezzanite LM, Fortier LA, Antczak DF, Cassano JM, Brosnahan MM, Miller D, 16. Carrade DD, Lame MW, Kent MS, Clark KC, Walker NJ, Borjesson DL. Schnabel LV. Equine allogeneic bone marrow-derived mesenchymal stromal Comparative Analysis of the Immunomodulatory Properties of Equine cells elicit antibody responses in vivo. Stem Cell Research & Therapy. 2015;6: Adult-Derived Mesenchymal Stem Cells. Cell Med. 2012;4(1):1–11. 54. 17. Fortier LA. Making progress in the what, when and where of regenerative 37. Jacobsen S, Thomsen MH, Nanni S. Concentration of serum amyloid A in medicine for our equine patients. Equine veterinary journal. serum and synovial fluid from healthy horses and horses with joint disease. 2012;44(5):511–2. American Journal of Veterinary Research. 2006;67(10):1738–42. 18. Carrade DD, Borjesson DL. Immunomodulation by Mesenchymal Stem Cells 38. Sanchez Teran AF, Rubio Martinez LM, Villarino N, Sanz M. Synovial serum in Veterinary Species. Comparative Medicine. 2013;63(3):207–17. amyloid A after repeated intraraticular administration of amikacin into 19. Murphy JM, Fink DJ, Hunziker EB, Barry FP. Stem cell therapy in a caprine equine healthy joints. In: Large animal sessions ECVS proceedings. model of osteoarthritis. Arthritis and rheumatism. 2003;48(12):3464–74. 2012. p. 2012. 20. Koga H, Muneta T, Nagase T, Nimura A, Ju Y-J, Mochizuki T, Sekiya I. 39. Lindegaard C, Gleerup K, Thomsen M, Martinussen T, Jacobsen S, Andersen Comparison of mesenchymal tissues-derived stem cells for in vivo P. Anti-inflammatory effects of intra-articular administration of morphine in chondrogenesis: suitable conditions for cell therapy of cartilage defects in horses with experimentally induced synovitis. American Journal of rabbit. Cell and tissue research. 2008;333(2):207–15. Veterinary Research. 2010;71(1):69–75. 21. Chu CR, Szczodry M, Bruno S. Animal models for cartilage regeneration and 40. Kay AT, Bolt DM, Ishihara A, Rajala-Schultz PJ, Bertone AL. Anti-inflammatory repair. Tissue Engineering Part B: Reviews. 2010;16(1):105–15. and analgesic effects of intra-articular injection of triamcinolone acetonide, 22. Buckwalter JA, Brown TD. Joint injury, repair, and remodeling: roles in post- mepivacaine hydrochloride, or both on lipopolysaccharide-induced traumatic osteoarthritis. Clin Orthop Relat Res. 2004;423:7–16. lameness in horses. American journal of veterinary research. 23. Wood JA, Chung DJ, Park SA, Zwingenberger AL, Reilly CM, Ly I, Walker NJ, 2008;69(12):1646–54. Vernau W, Hayashi K, Wisner ER, et al. Periocular and Intra-Articular Injection 41. Kasashima Y, Ueno T, Tomita A, Goodship AE, Smith RKW. Optimisation of of Canine Adipose-Derived Mesenchymal Stem Cells: An In Vivo Imaging bone marrow aspiration from the equine sternum for the safe recovery of and Migration Study. Journal of Ocular Pharmacology and Therapeutics. mesenchymal stem cells. Equine veterinary journal. 2011;43(3):288–94. 2012;28(3):307–17. 42. Ranera B, Ordovas L, Lyahyai J, Bernal ML, Fernandes F, Remacha AR, 24. Minguell JJ, Pereira A, Bartholomew P, Lasala GP. The Intrathecal Infusion of Romero A, Vazquez FJ, Osta R, Cons C, et al. Comparative study of equine Mesenchymal Stem Cells into Healthy Rabbits is Safe and Devoid of bone marrow and adipose tissue-derived mesenchymal stromal cells. Neurological or Clinical Complications. J Stem Cell Res Ther. 2011;1(104):1–5. Equine Veterinary Journal. 2012;44(1):33–42. 25. Kol A, Wood JA, Holt DDC, Gillette JA, Bohannon-Worsley LK, Puchalski SM, 43. Kolm G, Klein D, Knapp E, Watanabe K, Walter I. Lactoferrin expression in the Walker NJ, Clark KC, Watson JL, Borjesson DL. Multiple intravenous injections horse endometrium: relevance in persisting mating-induced endometritis. of allogeneic equine mesenchymal stem cells do not induce a systemic Veterinary immunology and immunopathology. 2006;114(1–2):159–67. inflammatory response but do alter lymphocyte subsets in healthy horses. 44. Bowman KF, Purohit RC, Ganjam VK, Pechman Jr RD, Vaughan JT. Stem Cell Research & Therapy. 2015;6:73. Thermographic evaluation of corticosteroid efficacy in amphotericin B- 26. Carrade DD, Owens SD, Galuppo LD, Vidal MA, Ferraro GL, Librach F, induced arthritis in ponies. Am J Vet Res. 1983;44(1):51–6. Buerchler S, Friedman MS, Walker NJ, Borjesson DL. Clinicopathologic 45. Anonymous: American Association of Equine Practitioners (AAEP). Guide for findings following intra-articular injection of autologous and allogeneic veterinary service and judging of equestrian events: definition and placentally derived equine mesenchymal stem cells in horses. Cytotherapy. classification of lameness. In.; 1991. 2011;13(4):419–30. 46. Korenek NL, Andrews FM, Maddux JM, Sanders WL, Faulk DL. Determination of total protein concentration and viscosity of synovial fluid from the 27. Pigott JH, Ishihara A, Wellman ML, Russell DS, Bertone AL. Investigation of tibiotarsal joints of horses. American journal of veterinary research. the immune response to autologous, allogeneic, and xenogeneic 1992;53(5):781–4. mesenchymal stem cells after intra-articular injection in horses. Veterinary 47. Kevin Corley J. The Equine Hospital Manual. Publisher Wiley-Blackwell; June Immunology and Immunopathology. 2013;156(1–2):99–106. 2008. 28. Williams LB, Koenig JB, Black B, Gibson TW, Sharif S, Koch TG. Equine allogeneic umbilical cord blood derived mesenchymal stromal cells reduce synovial fluid nucleated cell count and induce mild self-limiting inflammation when evaluated in an LPS induced synovitis model. Equine Vet J. 2015;47(5)503–632. 29. Martinez-Lorenzo MJ, Royo-Canas M, Alegre-Aguaron E, Desportes P, Castiella T, Garcia-Alvarez F, Larrad L. Phenotype and chondrogenic differentiation of mesenchymal cells from adipose tissue of different species. Journal of orthopaedic research : official publication of the Orthopaedic Research Society. 2009;27(11):1499–507. 30. Colleoni S, Bottani E, Tessaro I, Mari G, Merlo B, Romagnoli N, Spadari A, Galli C, Lazzari G. Isolation, growth and differentiation of equine Submit your next manuscript to BioMed Central mesenchymal stem cells: effect of donor, source, amount of tissue and and we will help you at every step: supplementation with basic fibroblast growth factor. Veterinary research communications. 2009;33(8):811–21. • We accept pre-submission inquiries 31. Ranera B, Lyahyai J, Romero A, Vazquez FJ, Remacha AR, Bernal ML, • Our selector tool helps you to find the most relevant journal Zaragoza P, Rodellar C, Martin-Burriel I. Immunophenotype and gene • We provide round the clock customer support expression profiles of cell surface markers of mesenchymal stem cells derived from equine bone marrow and adipose tissue. Veterinary • Convenient online submission Immunology and Immunopathology. 2011;144(1–2):147–54. • Thorough peer review 32. Chen FH, Tuan RS. Mesenchymal stem cells in arthritic diseases. Arthritis Res • Inclusion in PubMed and all major indexing services Ther. 2008;10(5):223. 33. Stashak T. Adams: claudicacion en el caballo, 5 edn: Intermedica; 2005. • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit