osteoarthritis ( oa ) is the most prevalent disease of arthritis which induces the joint pain 
 and limits activity1 .
it is 
 multi - factorial disease ( e.g. , genetic , aging , obesity and mechanical stimuli)2,3,4,5,6 .
these factors contribute to oa progression 
 by either direct or indirect regulation the anabolic and catabolic pathways of the cartilage 
 extracellular matrix ( ecm)7,8,9 .
however , a 
 fundamental treatment strategy has not been established to markedly alter oa progression 
 because of our incomplete understanding of chondrocyte behavior . to maintain the ecm in articular cartilage
an inappropriate activity balance leads to ecm 
 degeneration and chondrocyte loss accompanied by apoptotic cell death during oa .
chondrocytes are aligned as regularly formed columns that are perpendicular to the joint 
 surface and organized into four zonal layers : superficial zone ( sz ) , middle zone ( mz ) , deep 
 zone ( dz ) and calcified zone ( cz)17,18,19 . 

therefore , the zone - specific turnover relies on the differentiation 
 phenotype and its localization .
for instance , parathyroid 
 hormone - related peptide ( pthrp ) and indian hedgehog ( ihh ) signaling play pivotal roles in 
 not only differentiation but also controlling chondrocyte localization20 , 21 .
ihh released 
 from hypertrophic chondrocytes in the dz drives differentiation in the same region of cells 
 and facilitates pthrp production by proliferative chondrocytes in the mz .
pthrp acts to both 
 facilitate proliferative chondrocyte division in the mz and inhibit the ihh production in 
 hypertrophic chondrocytes .
this negative feedback loop plays an important role in each zonal 
 homeostasis and their arrangement .
lin and colleagues revealed that high levels of ihh are 
 associated with oa pathogenesis in humans and animal model22 .
it remains unclear whether oa progression is related to chondrocyte alignment despite the 
 possibility of losing differentiation control triggered by cell death .
studies have shown 
 that chondrocytes are not aligned as columns in moderate oa according to qualitative 
 observations23 , 24 .
we have used a geographic information system ( gis ) for 
 quantitative assessment to determine the distribution of chondrocytes , which has been 
 difficult to evaluate by common qualitative assessment .
this useful system , which has been 
 developed to analyze various aspects of the causal relationship between the position and 
 space , can be applied to quantitative assessment .
furthermore , this geographic methodology 
 has been widely used in several epidemiology studies25,26,27 . in this study
, we analyzed the relationship between oa progression 
 and chondrocyte alignment using the gis .
specifically , the gis enabled evaluation of a cell 
 alignment pattern whether similar cell sizes were clustered , dispersed or random ( fig . 1fig .
1.schematic diagram of 
 spatial autocorrelationhigher positive z - scores indicate similar clustered 
 attributes ( cell size ) .
the more cell alignment becomes random , the closer the z - score 
 is to zero . ) . to 
 clarify the relationship
, we examined temporal changes in the chondrocyte location as an 
 important factor to maintain the articular cartilage in rats with surgically induced oa .
schematic diagram of 
 spatial autocorrelation higher positive z - scores indicate similar clustered 
 attributes ( cell size ) .
the more cell alignment becomes random , the closer the z - score 
 is to zero .
the study was approved by the animal committee of niigata university of health and welfare . 

thirty 10 weeks male wistar rats ( 369  21  g ) were purchased from clea japan , inc .
( tokyo , 
 japan ) and randomly housed at two animals per standard cage at 25  1  c , 55  5% humidity , 
 and a 12:12  h light : dark cycle .
the animals had free access to a standard diet and tap 
 water . at 13 weeks - old ,
the 
 medial meniscus ligament was resected to destabilize the medial meniscus on the right knee 
 joint as described previously for the destabilized medial meniscus ( dmm ) model28 .
a similar procedure was performed on left 
 knee for the sham group but without dissecting the medial meniscus ligament .
the animals were sacrificed at day 0 for the control ( n=6 ) and at 1 , 2 , 4 , or 8 weeks ( n=6 
 each ) after surgery under anesthesia .
tissue was fixed by 30  min perfusion with a fixation 
 solution ( 4% paraformaldehyde in 0.1  mol / l phosphate buffer , ph 7.35 ) via the abdominal 
 aorta .
both knee joint were harvested from the hind limbs and placed in the fixation 
 solution at 4  c for 24  h. each specimen was decalcified in 0.1  mol / l 
 ethylenediaminetetraacetic acid for 6wks at 4  c and then bisected frontally .
specimens were 
 gradually dehydrated with 70 , 80 , 90 , 95 and 100% ethanol for 24 hrs at 4  c .
frontal 5 m serial sections 
 were prepared from the middle part of knee joint .
the sections were stained with 
 safranin - o - fast green and hematoxylin and eosin ( h - e ) .
all histological images included 
 medial tibial plateaus ( mtps ) as observed by light microscopy ( dm1000led ; leica , germany ) , 
 and captured by a digital ccd camera ( dfc425 ; leica , germany ) .
the evaluation was performed according to the 
 osteoarthritis research society international ( oarsi ) scoring system within grade and 
 stage29 , 30 .
the scoring was performed by a single trained observer ( ht ) in a 
 blinded fashion .
h - e images stained images were used to measure the chondrocyte density and alignment .
the 
 mtp is divided into four weight - bearing areas , and the region of interest was the third area 
 from the anterior cruciate ligament insertion .
chondrocytes and total area were traced 
 manually at 400 magnification using imagej ( nih , usa ) .
chondrocyte densities were 
 calculated as the number of cells divided by total articular cartilage area . to assess 
 chondrocyte alignment , each chondrocyte coordinate from the center of mass and its size
thereafter , based on collected data of cell coordinates and sizes , 
 the spatial autocorrelation were calculated using gis software ( arc gis 10.2.1 ; esri , ca , 
 usa ) .
we examined the spatial autocorrelation interaction between the cell location and 
 associated attribute ( cell size ) .
1 ) . the more cell alignment becomes random , the closer the 
 z - score is to zero .
conversely , lager negative scores indicate a dispersed cell alignment . to 
 determine the z - score ,
we first calculated the moran s i by the following 
 formula : subsequently , a standardized normal variable was calculated as the z - score using the 
 following formula : e[i ] and v[i ] was calculated as follows : oarsi score are expressed as the median and iterquartile ranges .
cell density data of the 
 control as the standard were converted to cell density ratios .
data were analyzed by kruskal - wallis test followed by the 
 post - hoc steel test .
correlations between oarsi 
 scores and chondrocyte density , and oarsi scores and z - scores were examined by spearman s 
 partial rank correlation coefficient .
2.time 
 course of histological section ( medial tibial plateaus ) in the control ( a  c ) and at 1 
 week ( d  f ) , 2 weeks ( g  i ) , 4 weeks ( j  l ) , and 8 weeks ( m  o ) after dmm surgery : first 
 row ( 40 , scale bars=200 m ) ; second row ( 100 , scale bars=200 m ) ; third row ( 400 , 
 scale bars=50m)a representative safranin - o - stained section shows decreases in 
 staining , the number of cells , and cell regularity from 2 weeks after surgery . white 
 arrow shows subchondral bone cyst at 4 weeks after surgery . shows representative images of safranin - o - fast 
 green staining of mtps at 0 , 1 , 2 , 4 and 8 weeks after dmm surgery .
surface irregularity , 
 and less safranin - o staining and cellularity from the articular cartilage surface were 
 observed with longer morbidity .
there was less staining and dissipation of surface 
 chondrocytes from 2 weeks in the dmm group ( fig .
2i , l , 
 o ) . in particular , at 4 and 8 weeks in the dmm group , less staining of more than 
 three fourths and unequivocal dissipation of chondrocytes were observed at the lesion area 
 ( fig .
2l , o ) . however , there was chondrocyte 
 clustering in the proliferating zone only at 1 week in the dmm group ( fig .
2  j , m ) . additionally , a subchondral cyst 
 was confirmed immediately under the lesion at 4 weeks after surgery ( fig .
time 
 course of histological section ( medial tibial plateaus ) in the control ( a  c ) and at 1 
 week ( d  f ) , 2 weeks ( g  i ) , 4 weeks ( j  l ) , and 8 weeks ( m  o ) after dmm surgery : first 
 row ( 40 , scale bars=200 m ) ; second row ( 100 , scale bars=200 m ) ; third row ( 400 , 
 scale bars=50m ) a representative safranin - o - stained section shows decreases in 
 staining , the number of cells , and cell regularity from 2 weeks after surgery .
table 1table 
 1.temporal changes of oarsi scores , cell density ratio and 
 z - scores in medial tibial plateau0w1w2w4w8woarsi scorecontrol0 ( 00)sham0 ( 00)0 ( 00)0.25 
 ( 00.5)0 ( 00.75)dmm0.75 ( 0.51.38)*3.31 
 ( 0.946.06)*10.5 ( 9.3811.1)**13.25 
 ( 9.7515.63)**cell density ratio 
 ( /10,000 m)control100%sham114.4  34.3%117.2  35.1%110.2  45.6%109.2  24.7%dmm129.8  39.0%61.0  11.6%*60.9  16.9%*43.4  
 23.7%*z - scorecontrol6.27  1.04sham7.17  1.046.93  1.586.70  2.906.86  
 1.88dmm6.34 
  2.803.32  3.13 * 2.89  1.44 * 3.16  2.81*oarsi 
 scores are expressed as the median and iterquartile ranges .
cell density ratios are 
 expressed as mean  standard deviation compared with the control set at 100% .
* p<0.05 , * * p<0.01 vs. 
 control shows the alterations of oarsi scores , cell 
 density ratios and z - scores .
oarsi scores were an exponential increase over time from day 0 
 to 4 weeks after dmm surgery .
thereafter , there was a slight increase from 4 to 8 weeks in 
 the dmm group .
oarsi scores were significantly higher at 1 , 2 , 4 and 8 weeks in the dmm 
 group than in the control .
in contrast , there were no significant differences between 
 control and sham groups at any time point .
the alteration of cell density ratios of mtps 
 using h - e stain were a slight increase at 1 week in the dmm group compared with the control 
 but without significance . at 2 weeks in the dmm group ,
the cell density decreased suddenly 
 and sharply at 1 week in the dmm group .
except at 1 week in the dmm group , comparisons 
 between the control and dmm groups at 2 , 4 and 8 weeks showed significant differences .
to 
 investigate the cell alignment , we applied h - e stained images to quantitative analysis using 
 the spatial autocorrelation of gis technology .
relative to the control , there was a 
 significant decrease from 2 weeks to 8 weeks in the dmm group , respectively .
in contrast , no 
 significant differences were observed a between sham and control groups .
cell density ratios are 
 expressed as mean  standard deviation compared with the control set at 100% .
* p<0.05 , * * p<0.01 vs. 
 control the relationship between oarsi scores and chondrocyte density ratio in control , sham and 
 dmm groups was evaluated using the z - score corrected spearman s partial rank correlation 
 coefficient that takes into account the third variable .
assessment of the relationship 
 between oarsi scores and z - scores was performed using the cell density ratio corrected to 
 spearman s partial rank correlation coefficient .
it showed a poor negative correlation 
 between the oarsi score and chondrocyte density ratio ( r=0.41 ) ( fig . 3afig .
3.relationship between chondrocyte density , 
 alignment and oarsi scores(a ) adjusted correlation between the cell density 
 ratio and oarsi score .
oarsi score showed a poor negative correlation with the cell 
 density ratio ( r=0.41 ) .
similarly , 
 there was a negative correlation between the oarsi score and z - score ( r=0.62 ) ( fig .
relationship between chondrocyte density , 
 alignment and oarsi scores ( a ) adjusted correlation between the cell density 
 ratio and oarsi score .
oarsi score showed a poor negative correlation with the cell 
 density ratio ( r=0.41 ) .
this study demonstrated that the organization of chondrocytes deteriorates with oa 
 progression in dmm rats .
interestingly , all 
 timings of dramatic alterations , disordered the cell alignment , hypocellularity , and oa 
 progression , were similar . while oa progression occurred over time , the chondrocyte 
 alignment and cellularity were invariant from 4 to 8 weeks after surgery .
in addition , 
 chondrocyte alignment was highly associated with the severity of oa compared with cell 
 density .
hence , these results support spatial autocorrelation for articular cartilage 
 evaluation and the oarsi scoring system .
moreover , the chondrocyte alignment was principal 
 factor to maintain the ecm in articular cartilage .
accumulation of overloading at mtps induces upregulation of inflammatory mediator such as 
 interleukin ( il)-1 and il-6 in knee joint immediately after dmm surgery31 .
studies have shown that oa severity is associated with apoptosis , 
 cartilage thickness and ecm contents13 , 16 , 32,33,34 . 

our results also showed less chondrocytes at the surface lesion such as the sz and mz in 
 early stages of oa . considering these results
nevertheless , our data showed no chondrocyte loss at 1 week after surgery while 
 the degeneration progressed .
there was a time difference between the degree of degeneration 
 in articular cartilage and chondrocyte viability .
revealed that both 
 apoptosis and autophagy are co - localized with chondrocytes in the sz and mz during the early 
 stages of oa35 .
similarly , we also 
 confirmed that mammalian target of rapamycin ( m - tor ) , which is upstream of autophagy 
 inhibition , was not expressed in early stages of oa , whereas m - tor was expressed in 
 hypertrophic chondrocytes at the late stages of oa ( unpublished data ) .
indeed , a time lag 
 existed between ecm degeneration , such as the appearance of fibrillation , and chondrocyte 
 viability in the early stages of oa .
one of the pathways controlling 
 chondrocyte differentiations is the negative feedback loop of pthrp and ihh signaling37 .
it is certain that healthy chondrocytes 
 of articular cartilage are arranged as columns owing to an orderly differentiation program . 

studies suggest that aggravating oa may be caused by hypertrophic differentiation because of 
 upregulating hypertrophic makers such as runt - related transcription factor 2 ( runx2 ) , 
 collagen type x ( col x ) , matrix metalloproteinase 13 ( mmp13 ) and alkaline phosphatase 
 ( alp)38 .
it has also been reported that 
 ihh signaling proteins are highly upregulated in both oa patients and surgically induced oa 
 model animals , which is accompanied by upregulation of a disintegrin and metalloproteinase 
 with thrombospondin motifs 5 ( adamts-5 ) , runx2 , col x and mmp1322 .
it appears that chondrocyte disorder in oa results from 
 loss of cellularity in the sz and mz where it plays a role in inhibition of hypertrophic 
 differentiation .
furthermore , the cause of chondrocyte disorder is considered to involve in 
 gap junctions .
articular chondrocytes in the sz communicate interactively though the gap 
 junction protein connexin 43 for differentiation and metabolic homeostasis of the ecm31 , 39 , 40 .
thus , loss of cellular location is presumed to be a driving 
 factor in oa development .
it also implies that ensuring crosstalk by maintaining each cell 
 location is important to prevent oa development .
the decrease of cell 
 density and alignment was 38.9% and 47% at 2 weeks after surgery compare with the control . 

several studies have concluded that apoptosis is secondary to degeneration of articular 
 cartilage in early stages of oa12 , 13 , 41 .
other studies have shown that treatment of oa model animals with 
 rapamycin , an inhibitor of m - tor and activator of autophagy , reduces oa pathogenesis42 , 43 .
in addition , hypertrophic chondrocytes activate degeneration of ecm 
 and endochondral ossification44 . taken 
 together ,
note that 
 reversing the differentiation after such a condition is impossible in vivo at present . even 
 though treatments delay the oa progression or symptoms , it is presumed to be difficult 
 beyond this period .
. we should focus on not only intervention method but 
 also the period of appropriate therapeutic intervention .
three recent studies have 
 demonstrated that pre or early potential preventive effect on human and animal model 
 studies45,46,47 . in conclusion
, our findings suggest that chondrocyte alignment in articular cartilage due 
 to catabolic and anabolic reactions plays an important role in the oa progression . moreover , 
 the changes in chondrocyte density and alignment were not consistent .
hence , to prevent the 
 oa progression , we need to consider with appropriate therapeutic interventions .
 [ purpose ] chondrocytes in articular cartilage are aligned as columns from the joint 
 surface . 
 notably , loss of chondrocyte and abnormalities of differentiation factors give 
 rise to osteoarthritis ( oa ) . 
 however , the relationship between chondrocyte alignment and 
 oa progression remains unclear . 
 this study was performed to investigate temporal 
 alterations in surgically - induced oa rats . [ subjects and methods ] thirteen - week - old wistar 
 rats ( n=30 ) underwent destabilized medial meniscus surgery in their right knee and sham 
 surgery in their left knee . 
 specimens ( n=5 ) were collected at 0 , 1 , 2 , 4 and 8 weeks after 
 surgery . 
 histological analysis with osteoarthritis research society international ( oarsi ) 
 scores , cell density ratios , cell alignments and correlation between oarsi scores and cell 
 density / alignment was performed . 
 [ results ] oarsi scores were significantly higher at 1 , 2 , 
 4 and 8 weeks in the dmm group than in the control . 
 cell density ratios were decreased 
 significantly in the dmm group at 2 , 4 and 8 weeks compared with the control . 
 chondrocyte 
 alignment was decreased significantly in the dmm group at 4 and 8 weeks . 
 there were 
 negative correlations between oa severity and cell density / cell alignment . 
 [ conclusion ] 
 the results suggest a relationship between chondrocyte alignment and cartilage 
 homeostasis , which plays an important role in oa progression . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION