joint contracture causes functional disturbances in patients activities of daily living , 
 decreased movement ability , and impairs the joint function , with high incidence1 , 2 . 

therefore , preventing the development of joint contracture is a key goal of physical 
 therapists and rehabilitation specialists .
unfortunately , medical professionals are still 
 struggling to find definitive therapies for the prevention and management of joint 
 contracture .
although a number of factors can lead to the development of contractures3 , differentiating between them in a clinical 
 setting can be difficult .
orthopedic surgeons generally classify contractures on the basis of changes 
 causing restriction of the normal range of motion of a joint , a rigidity resulting from some 
 extracapsular cause3,4,5 . in physical therapy , 
 classifying cases in terms of the area causing the condition has little practical purpose . 
 therefore , joint immobility causing restricted range of motion can be treated as a case of 
 contracture2 .
changes in the cartilage and the synovial membrane have been reported as intracapsular 
 joint sequelae resulting from contracture6,7,8,9 . to date
, significant discrepancies exist 
 among reports , and thus , the current state of research is inadequate for drawing meaningful 
 conclusions .
we previously clarified organic changes in the joint composition and structure 
 resulting from contracture10 , 11 : fibrosis of the cartilage surface , atrophy of adipose 
 tissue in the synovial membrane , fibrosis in the lower layers of the synovial membrane , 
 microvasculature expansion , and blood congestion .
furthermore , evans et al.6 and yaoita7 investigated the effects of natural healing processes on these 
 organic changes and reported that restrictions of range of motion and structural changes 
 both recovered within a comparatively short time period .
this study aimed to investigate histopathological changes to clarify temporal effects on 
 restrictions to range of motion and joint components after joint immobilization in a rat 
 knee - contracture model .
fifty - four 8-week - old male wistar rats ( body weight 234269  g ) were used in this study .
the 
 rats were randomly divided into two groups : a fixation group ( n = 36 ) and a control group ( n 
 = 18 ) .
in the fixation group , right hindlimb knee joints were immobilized in full flexion by 
 plaster casts , which were removed after four weeks .
the research protocol followed kanazawa university animal experiment 
 regulations and kinjo university experimental animal handling regulations .
all animal 
 handling and experimentation were undertaken with the approval of the ethics committees of 
 both universities ( kanazawa university : ap-091379 and kinjo university : approval no .
rats from each group were 
 raised in cages ( similar to fixation group rats ) up to ages corresponding to the 
 subdivisions used for the fixation group .
they were labeled as 0wc , 4wfc , 8wfc , 16wfc , 
 24wfc , and 32wfc , corresponding to the times of examination after cast removal : 0 , 4 , 8 , 16 , 
 24 , and 32 weeks , respectively .
fixation group rats were immobilized with plaster casts according to a modified version of 
 previously described methods10 , 12 , 13 .
pentobarbital 
 sodium ( 5  mg/100  g body weight ) anesthesia was administered through the abdominal cavity . 

manchester , nh , usa ) were fitted 
 to the animals backs . to prevent scratches and abrasions to the right hind limb resulting 
 from the plaster casts ,
the right hindlimb was covered with a layer of gauze that centered 
 on the knee .
the casts extended from the pelvic girdle to the ankle joint , with the hip 
 joint at maximal extension , the knee joint at maximal flexion , and the ankle joint at 
 maximal plantar flexion .
the cast area was exposed from the distal ankle to the foot to 
 confirm the absence of swelling and congestion .
considering the bone growth during the cast 
 period , the kneecap was exposed as well .
rats were able to freely move in their cages using their left hind limb and both forelimbs . 

if swelling was 
 observed in the fixed limb , the fixation procedure was repeated , and casts were immediately 
 replaced to prevent continuance . if casts were observed to slip out of place or become 
 loose , they were also replaced as soon as possible .
the cast period was set at four weeks on 
 the basis of the findings of previous studies , which confirmed that histological changes 
 were evident in the joint structure and content at this point .
following the cast period , fixation group rats were randomly divided into six subgroups , 
 with six rats in each group : these were labeled 0wf , 4wf , 8wf , 16wf , 24wf , and 32wf , 
 corresponding to the examination times after cast removal : 0 , 4 , 8 , 16 , 24 , and 32 weeks , 
 respectively .
the 0wf and 0wc groups underwent immediate assessments ( described below ) to 
 measure the extension angle of their knee joints , to assess restrictions to range of motion , 
 and to create histological specimens for analysis of the joints .
the restriction of knee joint extension 
 angle was measured and recorded on the basis of a preliminary experiment .
an intra - abdominal 
 pentobarbital sodium anesthetic was administered ( 5  mg/100  g body weight ) , and the rats were 
 laid on their sides . using one hand to hold the femur in place , researchers used the other 
 hand to hold the leg at 35  mm from the knee joint and guide the knee into maximal extension , 
 the feeling experienced at the end of the range of motion . using a digital camera , images of 
 the principal axis of extension of the knee joint ( the line running from the greater 
 trochanter to the lateral condyle of the femur ) and the movement axis ( the line running from 
 the head of the fibula to the lateral malleolus ) were captured in the sagittal plane .
the 
 images were loaded onto a computer , and the angles of the principal and movement axes were 
 measured using three - point measuring instrument software ( version 1.1.2 ) .
this protocol was 
 used for all assessments . in addition , a digital force gauge ( imada dps-5 ) was attached at 
 35  mm from the knee joint , which was then extended to the angle measured .
three technicians , each with more than five years of clinical 
 experience , performed each procedure five times .
the average value was 0.33  0.05 n. on the 
 basis of these preliminary findings , all measurements of the restriction of knee joint 
 extension angle were recorded by first attaching a digital force gauge at 35  mm from the 
 knee joint , followed by application of 0.3 n of extension force and recording of the 
 resulting angle . for the fixation group ,
these measurements were conducted before the 
 experiment , immediately after cast removal and then weekly until the experiment was 
 concluded .
individual differences in the development of contractures were assessed using retention 
 rate calculations , which were as follows : retention rate = [ ( the measured angle at each 
 time - period )  ( the angle at development of contracture ) ]  100 . at the end of each experimental period ,
a group of six animals was euthanized with a 
 pentobarbital sodium overdose , and their hindlimbs were severed at the hip joint .
tissues 
 were fixed over a 72-h soaking period in a 10% buffered neutral formalin solution , and then 
 decalcified using the planck - rychlo solution for 72  h at 4  c .
thereafter , an area 
 approximately 10  mm above and below the knee joint was removed .
tissues from the center of 
 the kneecap , moving toward its inner regions in the sagittal plane , were removed . to 
 preserve this removed region , a 5-mm margin was cut and stored in an embedding cassette .
it 
 was subsequently neutralized in a 5% anhydrous sodium sulfate solution for 72 h , rinsed 
 under running water for 30  min , and soaked for approximately 3  h in 100% alcohol to remove 
 any remaining fat .
thereafter , samples were processed in an automatic 
 dehydrating - and - paraffin - wax - embedding device into paraffin blocks and sliced into 3-m 
 thick sections using a sliding microtome ( leica sm2000r ) .
after these organizational 
 segments had been attached to a slide glass and dehydrated , they were stained with 
 hematoxylin - eosin ( he ) stain and enclosed in a hydrophobic mounting agent , to complete the 
 histological specimen preparation .
the specimens were observed using a microscopic digital 
 camera ( olympus dp50 ) connected to an optical microscope ( olympus bx-51 ) to examine the 
 entire knee joint structure .
although it is possible to classify cartilage on the basis of 
 the degeneration it has undergone in cases of osteoarthritis14,15,16 , histological classification of cartilage degeneration resulting 
 from immobilization is a relatively new area of study .
therefore , to quantify histological 
 changes in the cartilage , we classified samples into one of the following four groups : group 
 i , fibrosis and degeneration could not be identified in outer layers ; group ii , 
 irregularities and fibrosis could be identified on the cartilage surface ; group iii , 
 thinning and transformation into fibrous pseudocartilage could be identified ; and group iv , 
 destruction and damage could be identified in the cartilage .
for each group fitted with plaster casts , a comparative examination was conducted with the 
 control group using fisher s exact test using the statistical software , r ( version 2.15.1 ) . 

following the cast period , knee joint extension angles revealed following restrictions : 
 73.2  9.9 in the 0wf group , 71.4  8.3 in the 4wf group , 71.2  8.1 in the 8wf group , 
 68.8  7.4 in the 16wf group , 73.6  7.9 in the 24wf group , and 70.2  11.2 in the 32wf 
 group . using retention rate calculations ,
each group showed improvement over time , and the 
 retention rate in the 4wf group was 7.1  3.8% at the end of the study . excluding the 4wf 
 group ,
retention rates by the fifth week after cast removal were as follows : 8wf , 1.4  
 1.1% ; 16wf , 1.7  0.6% ; 24wf , 0.5  1.1% ; and 32wf , 1.2  1.2% . furthermore , retention rates 
 of all groups were 0% by the sixth week .
knee extension angles were measured over the 
 subsequent weeks , but none of the individual subjects in any group revealed any recurrence 
 of their restrictions ( table 1table 1 .
changes in knee extension angle restrictionsimmediateremovalweek following fixation removal1th2nd3rd4th5th6thgroup 4wf100%69.414.6%42.510.4%19.65.9%7.13.8%group 8wf100%74.45.9%50.54.8%20.38.5%7.85.3%1.41.1%0%group 16wf100%71.16.0%46.09.6%16.26.9%6.71.7%1.70.6%0%group 24wf100%76.68.8%41.48.4%16.04.5%7.53.2%0.51.1%0%group 32wf100%75.67.5%44.63.7%22.04.3%10.53.0%1.21.2%0% ) . considering the relationship between age and histological changes , case 
control comparisons 
 matched by age produced the following data ( table 
 2table 2 .
abnormal findings in joint cartilage and the frontal synovial membranefibrosis ofcartilagedegenerationof cartilagethinning ofcartilageadhesion offrontal tibiaatrophy ofadipocytefibroblasthyperplasiagroup 0wf600566group 4wf600366group 8wf600366group 16wf620366group 24wf631166group 32wf652066numbers within the table indicate the number of specimens presenting the conditions 
 out of a total of 6 . , fig .
( he stain , scale bar left : 1  mm , center and right : 100 m ) .
the 
 0wf group : adhesions of fibrous hyperplasia tissue between the frontal tibial area and 
 the frontal synovial membrane were noted (  , left ) .
adipocyte atrophy and fibroblast hyperplasia 
 appeared , as well as expansion and blood congestion in the microvasculature ( right ) . 

there were no clear 
 differences in the extent of any of the conditions between the 4wf and 0wf groups .
the space between adhesions 
 was more expanded than in the 4wf group (  , left ) .
the 16wf group : adhesions covered 
 an even larger area than in the 8wf group ( left ) . some hyaline cartilage had been 
 replaced with fibrous cartilage tissue ( center ) .
findings related to conditions in the 
 frontal synovial membrane were identical to those of the 0wf group ( right ) .
the 24wf 
 group : spaces between adhesions were bigger than in the 16wf group (  , left ) . 
 cartilage degeneration and thinning were identified ( center ) .
findings related to 
 conditions in the frontal synovial membrane were identical to those of the 0wf group 
 ( right ) .
the 32wf group : no adhesions were evident ( left ) . cartilage degeneration and 
 thinning were identified ( center ) .
findings related to conditions in the frontal 
 synovial membrane were identical to those of the 0wf group ( right ) ) . in the articular cartilage of the 0wf group ,
fibrosis was identified on the surface 
 of all cartilage specimens , and in five of six specimens , adhesion of the forward synovial 
 membrane and fibrous structure of the hyperplasia was observed in the frontal tibial area . 

all specimens 
 revealed adipocyte tissue atrophy in the frontal synovial membrane , fibroblast hyperplasia 
 in the lower layers , and expansion and blood congestion in the microvasculature .
no 
 significant changes were observed between 0wf , 4wf , 8wf , 16wf , 24wf and 32wf groups in terms 
 of adipocyte tissue atrophy and size , fibroblast hyperplasia , and microvasculature 
 changes .
numbers within the table indicate the number of specimens presenting the conditions 
 out of a total of 6 .
histological findings ( he stain , scale bar left : 1  mm , center and right : 100 m ) .
the 
 0wf group : adhesions of fibrous hyperplasia tissue between the frontal tibial area and 
 the frontal synovial membrane were noted (  , left ) .
adipocyte atrophy and fibroblast hyperplasia 
 appeared , as well as expansion and blood congestion in the microvasculature ( right ) . 

there were no clear 
 differences in the extent of any of the conditions between the 4wf and 0wf groups .
the space between adhesions 
 was more expanded than in the 4wf group (  , left ) .
the 16wf group : adhesions covered 
 an even larger area than in the 8wf group ( left ) . some hyaline cartilage had been 
 replaced with fibrous cartilage tissue ( center ) .
findings related to conditions in the 
 frontal synovial membrane were identical to those of the 0wf group ( right ) .
the 24wf 
 group : spaces between adhesions were bigger than in the 16wf group (  , left )
findings related to 
 conditions in the frontal synovial membrane were identical to those of the 0wf group 
 ( right ) .
the 32wf group : no adhesions were evident ( left ) . cartilage degeneration and 
 thinning were identified ( center ) .
findings related to conditions in the frontal 
 synovial membrane were identical to those of the 0wf group ( right ) although findings in the 4wf group were largely identical to those of the 0wf group , 
 adhesions were only found in three out of six samples .
the same numbers of adhesions were 
 observed in the 8wf group and the 4wf group , but the gap between fibers tended to become 
 larger .
in addition , the results of articular cartilage examination in the 16 wf were the same as 
 for the 4wf and 8wf groups , but two of six specimens revealed replacement of the original 
 hyaline cartilage with of a fibrous - type cartilage .
although adhesions of the frontal tibial 
 area were identified in three of six specimens , gaps between fibers greater than those in 
 the 8wf group , and fibrous structures were sparser .
however , 
 only a partial adhesion was observed in the frontal tibial area of just one of six 
 specimens .
the gap between fibers was even greater than those observed in the 16wf group . in 
 three of six samples ,
original cartilage was replaced with fibrous - type structures , and one 
 of these revealed additional thinning in the cartilage .
fibrosis of the articular cartilage but no adhesion was observed in the frontal tibial area 
 in the articular cartilage samples of the 32wf group .
however , the original cartilage was 
 replaced with fibrous - type structures in five of six cases , and the degeneration was quite 
 severe ; two of these revealed additional thinning the cartilage .
all adhesions had 
 disappeared in this group . finally , inflammatory cell infiltration was observed in all groups . on the basis of 
 histological classification of the cartilage in the cast group compared with the control 
 group , a significant difference was observed between both groups ( table 3table 3 .
in this study , six weeks after cast removal , all rats showed complete recovery in their 
 range of knee joint extension . however , although the degree of contracture - associated 
 adhesions decreased over time , a replacement of existing cartilage with fibrous - type 
 structures along with thinning of the cartilage was observed .
moreover , at all stages , there 
 were persistent changes in the frontal synovial membrane , atrophy and differences in the 
 size of adipocyte tissues , fibroblast hyperplasia in lower layers , and expansion and blood 
 congestion in the microvasculature .
therefore , although the range of knee joint extension 
 improved , the articular cartilage and frontal synovial membrane remained damaged .
evans et al.6 evaluated the restoration 
 of mobility in immobilized joints following the onset of contracture using an 
 internal - fixation knee - arthrodesis contracture model in rats .
their study reported that 
 provided , the fixation lasted no longer than 30 days , changes in the range of motion were 
 reversible .
yaoita7 and maoka17 also reported that range of motion and 
 organizational structures were restored comparatively quickly following short - term fixation 
 of 30 days or less in a rat knee contracture model .
although we found some differences in 
 the time required to restore mobility and used a different fixation method , the complete 
 restoration of mobility we observed agrees with prior research .
the hyaline cartilage forms a structure around joints and functions as a load - bearing 
 buffer , reducing friction between bones in the course of normal joint movement , and playing 
 a role in maintaining mobility .
hyaline cartilage does not contain blood cells , lymph ducts , 
 or nerves , and does not directly experience inflammation18 . in a broader evaluation of joint changes resulting from contracture , evans et al.6 examined histological changes in rats with 
 internal fixation knee arthrodeses .
they reported that after 30 days of fixation , there was 
 a proliferation of intra - articular connective tissue as well as fibrosis and blood 
 congestion under the kneecap and in the intercondylar fossa .
they also observed cartilage 
 compression at contact points of the femur and tibial condyles , and miniaturization of 
 chondrocytes , and they concluded that intra - articular connective tissue hyperplasia and 
 adhesions within the cartilage were the cause of the restricted range of motion .
these 
 adhesions did not completely cover the cruciate ligament of the knee and its surroundings , 
 rather they originated from fibrosis of the synovial membrane .
similar to results of the study conducted by evans et al.6 , yaoita et al.7 , 
 using internal fixation for the knee joint in rats , revealed thinning of cartilage tissue , 
 disordered cell arrangement , and necrobiosis after 30 days of fixation . because of these 
 structural changes , fibrous connective tissues and adipocytes filled the intra - articular 
 area from the rear of the joint , extending the joint surface forward , parallel to the femur , 
 narrowing the articular space .
both the studies cited here discussed the process used to 
 induce contracture in detail and highlighted resulting histological changes .
however , their 
 descriptions of the recovery process after the removal of fixation was limited to general 
 statements about the range of motion being restored to its pre - experimental levels , and the 
 joint being otherwise normal .
we could find no other study in the literature which described the progress of contracture 
 and histological joint changes over time .
moreover , the hyperplasia we observed in the 
 membrane tissue of the outer layer of articular cartilage is consistent with our previously 
 published reports10,11,12,13 and could have resulted from joint immobilization .
in addition , 
 adhesions within the frontal synovial membrane and the frontal tibial area were consistent 
 with the findings of watanabe et al.10 
 during long - term cast fixation .
although these adhesions may be consistent with the work of 
 evans et al . and
this could be explained by differences in the fixation methods and observation 
 areas among the studies , but the true reasons remain unclear and require further 
 investigation . in the present study , as the time interval progressed ,
adhesions diminished ; moreover , by 
 32 weeks after cast removal , they had completely disappeared .
yaoita , who noted that free movement after removal of fixation resulted 
 in mechanical stress , though it is equally possible that the adhesions may have simply 
 detached .
in contrast with the resolution of adhesions , the number of cases , wherein normal 
 cartilage was replaced with fibrous structures and thinning , increased as time 
 progressed .
ely 
 et al.8 evaluated changes in joint 
 components resulting from cast fixation in adult dogs , and reported a narrowing of the space 
 between opposite cartilage structures along with thinning of the cartilage in load - bearing 
 areas . in addition , hagiwara et al.19 
 evaluated knee articular cartilage thickness in rats after internal fixation at 150 , and 
 reported that the thickness increased in movement areas , although it remained unchanged in 
 contact and non - contact areas .
the timing of the appearance of cartilage thinning in the 
 study of hagiwara et al . clearly differs from that reported by ely et al . and yaoita . 

furthermore , the cartilage thickening observed by hagiwara et al . was not observed in any 
 sample in the present study .
first , because adhesions 
 connect viscera and tissue that should be separated20 , the detachment of these adhesions through mechanical stress may 
 have caused bleeding and inflammation21 . 
 as a result
, the normal repair process may have caused the replacement of normal cartilage 
 with fibrous tissue structures .
second , the articular cartilage derives nutrition from 
 synovial fluid , and if this nutritional supply were obstructed by surface tissue 
 hyperplasia , it might cause degeneration .
this could then result in a decline in cartilage 
 regeneration , necrobiosis , and decreasing thickness , resulting in the replacement of 
 cartilage with fibrous tissues through the recovery process21 .
these qualitative changes to the articular cartilage did not occur 
 immediately after the onset of the contracture but after an amount of time had passed .
this 
 cartilage degeneration is a new finding , and future biochemical analyses will be required to 
 clarify its causes .
takemura et al.12 analyzed changes in 
 the frontal synovial membrane resulting from contracture with regard to contractures induced 
 by cast fixation .
joint immobilization resulted in adipocyte tissue atrophy and size 
 differences in the frontal synovial membrane , fibroblast hyperplasia in the lower layers , 
 and expansion and blood congestion in the microvasculature .
watanabe et al.10 and matsuzaki et al.11 reported identical findings , and these were unsurprisingly 
 confirmed by our study .
these changes may have occurred because of a decrease in the 
 mechanical stress resulting from the contracture . to date , very few articles have prospectively considered the recovery process of the 
 frontal synovial membrane following immobilization .
and yaoita 
 provided detailed descriptions of the recovery process , they only made general statements 
 about histological improvement .
conversely , takemura et al . and matsuzaki et al . failed to 
 observe any improvement in frontal synovial membrane atrophy . in this study , we observed 
 adipocyte tissue atrophy and size differences in the frontal synovial membrane , fibroblast 
 hyperplasia in the lower layers , and expansion and blood congestion in the microvasculature . 

moreover , this occurred at all the examination periods ( up to and including 32 weeks after 
 removal of cast fixation ) .
even after the passage of a long time interval , no trends toward 
 improvement were identified in the findings . to the best of our knowledge ,
although articles 
 have reported the posterior synovial membrane changes that result from joint immobilization , 
 our report is the first to observe atrophy of adipose tissue in the frontal synovial 
 membrane .
in addition , atrophy of structural fat such as the corpus adiposum infrapatellar 
 as well as bone marrow is not thought to easily occur in such cases11 . therefore , future histological and biochemical 
 investigations may be necessary to clarify how this occurs .
the results of our present study are in agreement with previous research that joint 
 mobility may completely recover after contracture .
we further add that this may occur 
 through time alone , and that damage to the articular cartilage and frontal synovial membrane 
 did not improve within 32 weeks after fixation , the end of our observation period .
briefly , 
 free motion merely improves mobility and does not result in any organic improvements in the 
 joint itself .
when physical therapists passively mobilize joints to improve range of motion 
 following contractures , they often experience a different sensation toward the end of the 
 range of motion .
this may result from an increased coefficient of friction because of the 
 fibrosis and degeneration in the cartilage surface .
the inadequate consideration of these 
 issues in reports to date means future research focusing more closely on appropriate medical 
 interventions to limit organic changes is required .
furthermore , future research must 
 respond to advances in our understanding of the fundamental scientific basis of physical 
 therapy .
   [ purpose ] the purpose of our study was to clarify temporal effects on restrictions to 
 range of motion and the histopathological changes of joint components after joint 
 immobilization in a rat knee - contracture model . 
 [ subjects ] fifty - four male wistar rats 
 were randomly divided into two groups : a fixation group , and a control group . 
 [ methods ] in 
 the fixation group , unilateral knee joints were immobilized at full flexion using a 
 plaster cast for 4 weeks . at four weeks 
 the animals were randomly divided into six 
 subgroups , corresponding to the time of examination after cast removal : 0 , 4 , 8 , 16 , 24 , 
 and 32 weeks . for comparison , 
 control group animals of corresponding age were also 
 examined . 
 [ results ] although movement restrictions of the knee joint had completely 
 recovered 6 weeks after the cast removal , cartilage and synovial membrane structures did 
 not completely recover . 
 [ conclusion ] these findings have not previously been reported , and 
 as they form an addition to the fundamental scientific foundations of physical therapy , 
 further research must examine these findings from a variety of perspectives . 
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION