BACKGROUND
osteoarthritis  is a common degenerative disease in veterinary medicine that affects various tissues surrounding joints such as articular cartilage, subchondral bone, synovial membrane, and ligaments, although cartilage is the main tissue afflicted by oa  <cit> .
however, restoration of the diseased articular cartilage in patients with oa is still a challenge for researchers and clinicians.
currently, several therapeutic regenerative strategies have investigated whether autologous mesenchymal stem cells  have significant effects on regeneration and/or maintenance of articular cartilage in oa  <cit> .
autologous msc therapy is based on the isolation of these cells from tissues such as fat or bone marrow  <cit>  and then after culture expansion, they are administered back to the patient  <cit>  on account of the demonstrated affinity that mscs have for damaged joint tissue, such as cruciate ligaments, menisci, and cartilage  <cit> .
in veterinary medicine, previous studies evaluating autologous admsc therapy showed clinical improvement in horses and dogs with orthopedic conditions  <cit> .
recent investigations have shown that growth factors contained in platelet-rich plasma  act as vehicles that could act as potentiators or even extend the effect of mscs  <cit> .
this may be explained by the antiinflammatory effect of prgf, which could help the mscs differentiate in a more favorable environment  <cit> .
at this point, knowing the onset and duration of action mscs have in canine oa should be highly interesting, as well as further understanding of their efficacy.
if previous subjective studies seem to show certain effects, our research hypothesis should be that this effect could be detected and quantified by kinetik devices such as force platforms.the aim of this study was to use force platform kinetic analysis to demonstrate that the effect of a single intraarticular injection of admscs alone is more limited with respect to time, compared with admscs + prgf in  <dig> dogs with hip oa.
the obtained results were compared with other medical strategies in canine oa and the advantages and disadvantages discussed.
RESULTS
the body weight of enrolled dogs ranged from  <dig> to 56 kg , and ages were  <dig> to 8 years .
the mean  value for walking velocity of both sound  and diseased groups of dogs was  <dig>  ±  <dig>  m/s.
no significant difference in walking velocity existed between dogs .
pvf and vi mean values are summarized in table  <dig>
pvf ml: peak vertical force in the more-lame limbs.
pvf ll: peak vertical force in the less-lame limbs.
vi ml: vertical impulse in the more-lame limbs vi ll: vertical impulse in the less-lame limbs.
pvf s: peak vertical force in the control group.
vi s:vertical impulse in the control group.
data are shown for each day of observation.
analysis of pvf
more-lame limbs analysis showed that differences in % pvf between d <dig> and d <dig> were significant .
between the other periods, differences were of no significance .
compared with the control group, % pvf at d <dig>  d <dig> and d <dig> is significantly less .
in comparison, at d <dig> difference was non significant .
less-lame limbs analysis showed non significant differences at all study periods .compared with control group, % pvf at d <dig>  d <dig> and d <dig> is significantly greater .
at d <dig> this difference was non significant  .
vi analysis
more-lame limbs analysis showed that differences in % vi between d <dig> and d <dig> were significant .
with the other periods, differences were not significant .
compared with the control group, % vi at d <dig>  d <dig> and d <dig> is significantly less .
in comparison, at d <dig> difference became non significant .compared with the control group, significant differences at d <dig> and d <dig> were found  .
association between more and less-lame limbs
the fitted linear mixed model shows a negative association between % pvf in less-lame limb and more-lame limb .
in % vi the same was found .
the validity of the model fit was assessed by testing normality and homoscedasticity of the residuals.
both assumptions could be accepted: the shapiro-wilk test for normality and levene’s test for homoscedasticity were not significant .
discussion
the aim of the current study was to test the effectiveness of admscs in lame oa dogs using a force platform.
the ground reaction forces-related aspects of the gait, such as the pvf and vi, which represent maximal weight bearing and distribution of forces through time, respectively.
allowing for the objective measurement of the clinical impact of admsc treatment on the function of the limb during the stance phase of walking.
force platform analysis could be complemented with kinematic studies since three-dimensional kinematic alterations in hip dysplasia have been well described  <cit> .
the lack of a direct relationship between radiographic evidence of oa and force platform findings is well known; in the current study, diseased dogs were selected both on the basis of the presence or absence of radiographic signs of severe oa  and for clear lameness determined by platform gait analysis objectively  <cit> .
some authors  <cit>  reported that force platform gait analysis at trot was much more sensitive than at walk for low-grade hind limb lameness, but not for severe lameness.
based in previous experience  <cit> , in the oa dogs group lameness was evident by direct observation, even at walk.
although each dog had bilateral lameness, the authors believed that it would only be possible to obtain confident data from the more-lame limbs , in order to avoid a possible bias caused by inconsistent weight redistribution to the less affected contra-lateral hind limb.
in fact, mean values showed how less-lame limbs seemed to perform better than limbs from the control group over the whole study period.
a substantial improvement was observed both in pvf and vi values, but only in the first month post inoculation of admscs; afterwards the animals seemed to worsen returning to the pretreatment condition.
although some authors  <cit>  found independent evolution of both pvf and vi values, in this study it was showed how these two parameters progressed consistently during the different study periods; the authors believe that this occurred due to the limited effect of the treatment in terms of time; after d <dig>  beyond this point, animals clearly returned to the initial state.
selection of a control group is determinant to test the efficacy of a treatment for lameness; in fact, when a treated group could improve its lameness, a lame non-treated or placebo control group could worsen, making these animals unable to provide fixed reference data  <cit> .
in accordance with this, the experimental study developed here used a control group of sound dogs that could provide fixed reference data.
various adipose tissue donor sites have been reported in revised literature: retroperitoneal adipose tissue  <cit> , lateral thoracic area  <cit> , gluteous fat  <cit>  or inguinal region  <cit> .
the authors preferred this location to others for its easier access, abundant quantities of fat, absence of surgical complications and production of a non-visible scar.
with regards to the role that other adjuvants associated to admsc could play increasing the effect, some results published in  <dig> showed that the use of mscs deposited in a fibrin matrix would be useful  <cit> .
more recently, another study  <cit>  objectively demonstrated how the effect of the association admsc + prgf-endoret was prolonged beyond six months; however the current study showed evidence that admscs alone have a significant but short effect in terms of time.
based on these results, it is likely that modulation of the matrix or cells will need to be accomplished to observe long-term benefit of mscs for cartilage repair  <cit> .
not only could adjuvants play a role in the duration of effect of mscs therapy; joint idiosyncrasy, and more specifically primary pathologies that cause joint instability such as hip dysplasia or cranial cruciate ligament rupture, they could also noticeably shorten the effect because of the constant reactivation of the associated oa.
presence of side-effects in msc therapy has also been recently investigated: comparing intra-articular injections of autologous, allogeneic and xenogenic mscs in horses, only a moderate acute inflammatory response was developed, less evident in autologous cells  <cit> .
in this study, only one dog experimented a transitory worsening after injection.
this fact could probably be attributable to incorrect technique in the administration of the cells, because the researcher had to perform various attempts to reach the articular space.
this study objectively demonstrated that a single intraarticular administration of admscs alone decreases pain and lameness in dogs with oa during a period less than three months, at which point pvf and vi values returned to being similar to the pre-treatment status.
these results differ with those previously published  <cit>  where improvement was still present after six months of treatment with mscs associated to prgf endoret®.
the identical study design suggests that prgf strongly plays a potentiating role, potentiating the effect of msc therapy, as shown by other authors  <cit> .
in  <dig>  <cit>  a multicentre study with msc alone showed subjective improvement during a 3-month period using different scoring systems, although the results from the current study were supported by objective kinetic data, the different study design  could explain these differences.
on the other way, the authors are of the opinion that a follow up of six months could be considered as a standard for testing the evolution of medical or surgical treatments; in fact although the dogs in the current study seemed to improve during the first month after treatment, this effect was reverted before three months after treatment.
this fact should induce researchers to objectively determine when a new cycle of treatment should be useful to stop a relapse  <cit> , and clinicians to set out a therapeutic strategy that should be based in msc associated to prgf, since a much longer-lasting effect has been demonstrated  <cit> .
regarding statistical analysis, more complex models could have been considered but the authors chose this one because it offers an adequate compromise between complexity and ability to represent the relationships between the considered variables  <cit> .
CONCLUSIONS
the measurement of pvf and vi during a follow-up period of six months demonstrated quantitatively that the efficacy of admsc therapy alone is significant in terms of improvement of hip joint lameness due to oa, although this effect decreased progressively between d <dig> and d <dig>

 osteoarthritis is a common degenerative disease in veterinary medicine that affects various tissues surrounding joints such as articular cartilage, subchondral bone, synovial membrane, and ligaments, although cartilage is the main tissue afflicted by oa. however, restoration of the diseased articular cartilage in patients with oa is still a challenge for researchers and clinicians. currently, several therapeutic regenerative strategies have investigated whether autologous mesenchymal stem cells have significant effects on regeneration and/or maintenance of articular cartilage in oa. autologous msc therapy is based on the isolation of these cells from tissues such as fat or bone marrow and then after culture expansion, they are administered back to the patient on account of the demonstrated affinity that mscs have for damaged joint tissue, such as cruciate ligaments, menisci, and cartilage. in veterinary medicine, previous studies evaluating autologous admsc therapy showed clinical improvement in horses and dogs with orthopedic conditions. recent investigations have shown that growth factors contained in platelet-rich plasma act as vehicles that could act as potentiators or even extend the effect of mscs. this may be explained by the antiinflammatory effect of prgf, which could help the mscs differentiate in a more favorable environment. at this point, knowing the onset and duration of action mscs have in canine oa should be highly interesting, as well as further understanding of their efficacy. if previous subjective studies seem to show certain effects, our research hypothesis should be that this effect could be detected and quantified by kinetik devices such as force platforms.the aim of this study was to use force platform kinetic analysis to demonstrate that the effect of a single intraarticular injection of admscs alone is more limited with respect to time, compared with admscs + prgf in dogs with hip oa. the obtained results were compared with other medical strategies in canine oa and the advantages and disadvantages discussed.  the body weight of enrolled dogs ranged from to 56 kg, and ages were to 8 years. the mean value for walking velocity of both sound and diseased groups of dogs was  ±   m/s. no significant difference in walking velocity existed between dogs. pvf and vi mean values are summarized in table  pvf ml: peak vertical force in the more-lame limbs. pvf ll: peak vertical force in the less-lame limbs. vi ml: vertical impulse in the more-lame limbs vi ll: vertical impulse in the less-lame limbs. pvf s: peak vertical force in the control group. vi s:vertical impulse in the control group. data are shown for each day of observation. analysis of pvf more-lame limbs analysis showed that differences in % pvf between d and d were significant. between the other periods, differences were of no significance. compared with the control group, % pvf at d d and d is significantly less. in comparison, at d difference was non significant. less-lame limbs analysis showed non significant differences at all study periods.compared with control group, % pvf at d d and d is significantly greater. at d this difference was non significant. vi analysis more-lame limbs analysis showed that differences in % vi between d and d were significant. with the other periods, differences were not significant. compared with the control group, % vi at d d and d is significantly less. in comparison, at d difference became non significant.compared with the control group, significant differences at d and d were found. association between more and less-lame limbs the fitted linear mixed model shows a negative association between % pvf in less-lame limb and more-lame limb. in % vi the same was found. the validity of the model fit was assessed by testing normality and homoscedasticity of the residuals. both assumptions could be accepted: the shapiro-wilk test for normality and levene’s test for homoscedasticity were not significant. discussion the aim of the current study was to test the effectiveness of admscs in lame oa dogs using a force platform. the ground reaction forces-related aspects of the gait, such as the pvf and vi, which represent maximal weight bearing and distribution of forces through time, respectively. allowing for the objective measurement of the clinical impact of admsc treatment on the function of the limb during the stance phase of walking. force platform analysis could be complemented with kinematic studies since three-dimensional kinematic alterations in hip dysplasia have been well described. the lack of a direct relationship between radiographic evidence of oa and force platform findings is well known; in the current study, diseased dogs were selected both on the basis of the presence or absence of radiographic signs of severe oa and for clear lameness determined by platform gait analysis objectively. some authors reported that force platform gait analysis at trot was much more sensitive than at walk for low-grade hind limb lameness, but not for severe lameness. based in previous experience, in the oa dogs group lameness was evident by direct observation, even at walk. although each dog had bilateral lameness, the authors believed that it would only be possible to obtain confident data from the more-lame limbs, in order to avoid a possible bias caused by inconsistent weight redistribution to the less affected contra-lateral hind limb. in fact, mean values showed how less-lame limbs seemed to perform better than limbs from the control group over the whole study period. a substantial improvement was observed both in pvf and vi values, but only in the first month post inoculation of admscs; afterwards the animals seemed to worsen returning to the pretreatment condition. although some authors found independent evolution of both pvf and vi values, in this study it was showed how these two parameters progressed consistently during the different study periods; the authors believe that this occurred due to the limited effect of the treatment in terms of time; after d beyond this point, animals clearly returned to the initial state. selection of a control group is determinant to test the efficacy of a treatment for lameness; in fact, when a treated group could improve its lameness, a lame non-treated or placebo control group could worsen, making these animals unable to provide fixed reference data. in accordance with this, the experimental study developed here used a control group of sound dogs that could provide fixed reference data. various adipose tissue donor sites have been reported in revised literature: retroperitoneal adipose tissue, lateral thoracic area, gluteous fat or inguinal region. the authors preferred this location to others for its easier access, abundant quantities of fat, absence of surgical complications and production of a non-visible scar. with regards to the role that other adjuvants associated to admsc could play increasing the effect, some results published in showed that the use of mscs deposited in a fibrin matrix would be useful. more recently, another study objectively demonstrated how the effect of the association admsc + prgf-endoret was prolonged beyond six months; however the current study showed evidence that admscs alone have a significant but short effect in terms of time. based on these results, it is likely that modulation of the matrix or cells will need to be accomplished to observe long-term benefit of mscs for cartilage repair. not only could adjuvants play a role in the duration of effect of mscs therapy; joint idiosyncrasy, and more specifically primary pathologies that cause joint instability such as hip dysplasia or cranial cruciate ligament rupture, they could also noticeably shorten the effect because of the constant reactivation of the associated oa. presence of side-effects in msc therapy has also been recently investigated: comparing intra-articular injections of autologous, allogeneic and xenogenic mscs in horses, only a moderate acute inflammatory response was developed, less evident in autologous cells. in this study, only one dog experimented a transitory worsening after injection. this fact could probably be attributable to incorrect technique in the administration of the cells, because the researcher had to perform various attempts to reach the articular space. this study objectively demonstrated that a single intraarticular administration of admscs alone decreases pain and lameness in dogs with oa during a period less than three months, at which point pvf and vi values returned to being similar to the pre-treatment status. these results differ with those previously published where improvement was still present after six months of treatment with mscs associated to prgf endoret®. the identical study design suggests that prgf strongly plays a potentiating role, potentiating the effect of msc therapy, as shown by other authors. in a multicentre study with msc alone showed subjective improvement during a 3-month period using different scoring systems, although the results from the current study were supported by objective kinetic data, the different study design could explain these differences. on the other way, the authors are of the opinion that a follow up of six months could be considered as a standard for testing the evolution of medical or surgical treatments; in fact although the dogs in the current study seemed to improve during the first month after treatment, this effect was reverted before three months after treatment. this fact should induce researchers to objectively determine when a new cycle of treatment should be useful to stop a relapse, and clinicians to set out a therapeutic strategy that should be based in msc associated to prgf, since a much longer-lasting effect has been demonstrated. regarding statistical analysis, more complex models could have been considered but the authors chose this one because it offers an adequate compromise between complexity and ability to represent the relationships between the considered variables.  the measurement of pvf and vi during a follow-up period of six months demonstrated quantitatively that the efficacy of admsc therapy alone is significant in terms of improvement of hip joint lameness due to oa, although this effect decreased progressively between d and d 
ten lame dogs with severe hip oa and a control group of sound dogs were used for this study. regenerative medicine using mesenchymal stem cells alone or combined with plasma rich in growth factors is a rapidly growing area of clinical research and is currently also being used to treat osteoarthritis. force platform analysis has been consistently used to verify and quantify the efficacy of different therapeutic strategies for the treatment of oa in dogs including msc associated to prgf, but never with ad-msc alone. the aim of this study was to use a force platform to measure the efficacy of intraarticular admsc administration for limb function improvement in dogs with severe oa. results were statistically analyzed to detect a significant increase in peak vertical force and vertical impulse in treated dogs. intraarticular admsc therapy objectively improved limb function in dogs with hip oa.
 regenerative medicine using mesenchymal stem cells alone or combined with plasma rich in growth factors is a rapidly growing area of clinical research and is currently also being used to treat osteoarthritis. force platform analysis has been consistently used to verify and quantify the efficacy of different therapeutic strategies for the treatment of oa in dogs including msc associated to prgf, but never with ad-msc alone. the aim of this study was to use a force platform to measure the efficacy of intraarticular admsc administration for limb function improvement in dogs with severe oa.  ten lame dogs with severe hip oa and a control group of sound dogs were used for this study. results were statistically analyzed to detect a significant increase in peak vertical force and vertical impulse in treated dogs. mean values of pvf and vi were significantly improved within the first three months post-treatment in the oa group, increasing 9% and % body weight, respectively, at day after this, the effect seems to decrease reaching initial values.  intraarticular admsc therapy objectively improved limb function in dogs with hip oa. the duration of maximal effect was less than 3 months. osteoarthrosishipadipose-derived mesenchymal stem cellsmscforce platform 