osteoarthritis ( oa ) is the most common chronic joint disorder , and it causes detrimental 
 effects on the quality of life and functional status .
these are characterized by 
 progressively occurring cartilage destruction , osteophyte formation , and subchondral 
 sclerosis1 , 2 .
the histopathological findings of oa show that homeostasis between 
 the destruction and repair mechanisms of the joint cartilage is disturbed by the increased 
 expression of proinflammatory cytokines ( il-1 , tnf alpha ) , matrix metalloproteinases , 
 aggrecanases , nitric oxide , and prostaglandins .
this causes degradation of the joints along 
 with the insufficiency in the synthesis of growth factors ( gfs ) , collagens , proteoglycans , 
 and anti - inflammatory cytokines ( il-4 , il-10)3 , 4 .
conservative treatments have been reported to increase the quality of life of patients 
 particularly in the early phases , when the pathophysiology of the disease does nt 
 change5 .
the effects of these treatments 
 are short term and their local and systemic side effects cause frequent problems .
therefore , 
 recent studies have focused on stimulating cartilage healing processes through 
 administration of growth factors ( gf ) , cytokine inhibitors , matrix metalloproteinase 
 inhibitors , or il-1 receptor antagonists5 , 6 .
platelet - rich plasma ( prp ) is an autologous concentration of a high number of platelets in 
 a small volume of plasma , and it is prepared by centrifugation of blood .
platelets contain 
 significant amounts of cytokines and growth factors which are capable of stimulating 
 cellular growth , vascularization , proliferation , tissue regeneration , and collagen 
 synthesis .
delivery of high concentrations of cytokines and gfs to damaged tissues by prp is 
 considered to have a beneficial effect on tendon and cartilage tissue regeneration7 , 8 . in 
 some in vitro and in vivo studies ,
anti - inflammatory and reparative effects of prp on 
 cartilage , tendon , and ligament recovery have been shown9,10,11 ; however , there is no consensus on eligible patient selection , the 
 number and frequency of injections , the preparation technique , or the appropriate platelet 
 concentration5 . in knee oa ,
prp injections aim to promote cartilage repair and relieve osteoarthritic 
 symptoms , potentially delaying the need for joint replacement surgery12 .
some studies have reported a reduction in prp efficacy in 
 moderate and advanced ( kellgren lawrence grade 34 ) knee osteoarthritis , as this group of 
 patients have higher pain and functional impairment , which require more medical 
 attention13 , 14 . in some studies , it was suggested that in terms of prp activity , 
 oa and chondropenia level is more critical than platelet number and function4 .
grade 4 oa generally requires surgical 
 treatments such as tibial osteotomy and total knee replacement . in the present study the 
 effects of prp administration to control the disease activity of grade 3 knee oa either with 
 one injection , two injections two weeks apart , or three injections separated by 2-week 
 intervals on the patients pain , quality of life and physical activity levels were 
 investigated .
patients in the age range of 4075 years who visited our physical medicine and 
 rehabilitation outpatient clinic between may 2014october 2014 because of single knee pain 
 for a minimum 6 months were recruited for this study .
oa was diagnosed according to the 
 american college of rheumatology ( acr ) criteria15 .
radiological assessment was conducted by standing anteroposterior 
 and lateral knee radiography according to kellgren- lawrence grading system16 .
a total of 102 patients identified with 
 grade 3 knee osteoarthritis ( with multiple osteophytes , definite joint space narrowing , 
 sclerosis and bony deformity ) were studied .
the exclusion criteria were bilateral 
 symptomatic knee oa ; age older than 75  years ; receiving physical therapy , intra - articular 
 steroid , hyaluronic acid or prp injections in the last 6 months ; recent history of severe 
 trauma of the affected knee ; active infection , inflammation or tumor existence around the 
 knee ; history of diabetes mellitus , severe cardiovascular diseases , coagulopathies , 
 malignant , immunosuppressive , collagen vascular or autoimmune disorders ; hb values of < 
 11  g / dl or platelet values of < 150,000 per micro- liter ; receiving treatment with 
 anticoagulant or antiplatelet medications or systemic corticosteroids 10 days before 
 injection , or use of nsaids 5 days before injection ; genu varum or valgus greater than 5 
 degrees ; pregnancy , or breastfeeding .
after receiving the approval of our hospital s ethics committee , the aims and methods of 
 prp therapy as well as the benefits and the possible adverse effects of study participation 
 were presented to the patients in a written form . only the participants who signed a written 
 consent form were included in the study .
the study participants attended a screening visit 
 that included recording of medical history , physical examination , laboratory testing 
 ( complete blood count , erythrocyte sedimentation rate , c - reactive protein , coagulation 
 profile , routine biochemistry ) , and a survey of medication use .
subjects age , gender , 
 height and weight were recorded and their body mass index ( bmi ) was calculated .
the participants were randomized by block randomization into three groups : 34 participants 
 in group 1 received a single injection , 34 participants in group 2 received two injections 
 two weeks apart , and 34 participants in group 3 received three injections of prp separated 
 by 2-weeks interval .
one patient in group 1 , 2 patients in group 2 , and 1 patient in group 3 
 did not complete the follow - up period due to personal reasons . to prepare 45 cc prp with platelet concentration of 46 times the average normal value ,
a 
 3040 cc venous blood sample from antecubital vein was collected in a sterile sodium 
 citrated tube using an 18 g needle to avoid traumatizing platelets .
approximately 1  ml of 
 whole blood was separated for a complete blood count .
then , the blood with anticoagulant was 
 centrifuged twice : first at 1,800  rpm for 15 minutes to separate erythrocytes ; then at 
 3,500  rpm for 5 minutes to concentrate platelets .
finally , 0.0425  ml of 10% calcium chloride per 1  ml of prp was added to the final product to 
 activate the platelets .
prp in a sterile condition was injected by a physician using a classic lateral approach 
 with a 22 g needle with the subjects in a supine position with the knee in full extension . 

since some studies have indicated that a local anesthetic may have toxic effects on 
 chondrocytes and affect platelet activation by modifying the ambient ph , a local anesthetic 
 agent was not used before the injection17 .
the second and third injections were administered under the same 
 conditions as the first injection .
after the injections patients were told to actively flex 
 and extend their knees a few times to allow the prp to spread throughout the joint before 
 gelling .
patients in each of the three groups were discharged to home after 1520 minutes of 
 rest with instructions to have rest , to limit weight bearing and to use cold packs 34 times 
 a day for 10 minutes for 72 hours . during the follow - up period ,
the patients were asked to 
 take acetaminophen only when necessary , or acetaminophen with codeine for persistent pain . 

the patients were instructed to not take them in the 48 hours before an assessment .
patients 
 were prohibited from using other analgesics , nsaids , steroids or medications which might 
 have influenced platelet count or function .
exercise or physical treatment was not allowed 
 during the study period to eliminate synergistic effects .
patients were evaluated before the treatment and at the 1 month , 3 months and 6 months 
 after the treatment with a visual analog scale ( vas ) for pain , the western ontario and 
 mcmaster universities arthritis index ( womac ) , and the timed- up and go test ( tug ) . 

post - injection measurements were recorded by a different physician to ensure a blinded 
 status .
vas assessment was done with numbers from  0  to  10  , equidistantly marked on a 10  cm 
 line .
the patients were explained that  0  meant they were experiencing no pain ,  5  
 moderate pain and  10  unbearable pain , and they were asked to mark the appropriate score on 
 the line describing their own pain during rest and physical activity18 .
the womac osteoarthritis index is a disease - specific 
 questionnaire for the disease , which assesses pain , stiffness and physical functions of oa 
 patients .
it consists of 24 questions in total : 5 on pain , 2 on stiffness and 17 on physical 
 functions .
individual subgroups scores or the total score can be calculated . a likert scale 
 ( 1 : none , 2 : low , 3 : medium , 4 : high , 5 : very high ) is used to assess all parameters on the 
 womac oa index .
high womac scores are indicative of intense pain and stiffness and 
 impairment of the physical function . in our study , the patients womac sub - scores ( pain , 
 stiffness , function ) and total womac score were calculated . the turkish validity and 
 reliability of the turkish version of the womac index
the timed up and go test ( tug ) was 
 measured from when the patients rose from sitting with their feet on the floor and their 
 arms resting on the armrest of a chair .
the patients were asked to stand up without using 
 their arms , walk for three meters , turn around , walk back and sit down .
three measurements were 
 taken and the best value was recorded and categorized as : < 10 seconds = freely mobile , 
 1019 seconds = mostly independent , 2029 seconds = variable mobility , > 30 = impaired 
 mobility20 . the mean and
the means of age 
 and bmi of the groups were analyzed by one way anova followed by the bonferroni post - hoc 
 test . repeated - measures anova is used to compare the means of three or more matched groups
. the 
 term repeated - measures strictly applies to treatments repeatedly administered to each 
 subject , and the term randomized block is used for randomly assigned treatments within a 
 group ( block ) of matched subjects . a repeated - measures experimental design can be very 
 powerful , as it controls factors that cause variability between subjects .
if the matching is 
 effective , the repeated - measures test will yield a smaller p value than ordinary anova .
the 
 repeated - measures test is more powerful because it separates between - subject variability 
 from within - subject variability21 .
data were analyzed using repeated anova and multiple comparisons ( the bonferroni test ) 
 test .
the spss statistical program was used to perform statistical analyses and values of 
 p<0.05 were considered significant .
patients were randomly and equally 
 divided into three treatment groups , and 98 ( 15 male and 83 female ) patients completed the 
 follow - up period ( group 1 : n= 33 , group 2 : n=32 , group 3 : n=33 ) ( fig . 1fig .
the groups were homogenous in terms of age , gender and bmi ; the results are presented in 
 table 1table 1.the mean age and bmi of the patientsvariabletreatment groupsgroup - i meansdgroup - ii meansdgroup - iii meansdage ( years)53.66.754.95.455.25.7bmi ( k / m)24.92.325.11.625.51.9bmi : body mass index .
vas scores , tug scores and womac total and sub - scores were significantly better than 
 preinjection scores in all of the three treatment groups during the follow - up period 
 ( p<0.001 ) .
table 2table 2.the mean and standard error of meam ( sem ) values and the test values according to 
 the repeated values for different four timestreated groupsfollow - upsoutcome measuresvas meansemw.total mean semw.pain mean semw.stiffness mean semw.function mean semtug mean sem1pretreatment7.70.191.42.017.90.56.50.167.01.413.00.21 month5.60.281.72.114.90.55.00.261.81.612.80.23 months6.50.289.35.620.75.05.40.263.11.413.00.26 months7.20.287.61.916.90.46.10.264.61.413.10.22pretreatment7.71.281.63.017.20.56.60.258.02.012.70.21 month3.30.265.92.412.60.43.90.249.42.012.10.23 months4.81.269.62.313.70.44.50.251.32.012.40.26 months6.40.274.52.415.20.55.50.253.72.012.50.23pretreatment8.41.289.91.718.90.37.10.263.81.312.60.11 month2.40.167.41.611.90.33.40.252.01.311.60.13 months3.01.269.81.812.50.33.80.253.51.411.70.16 months4.51.275.11.714.10.24.90.256.01.411.70.3vas : visual analog scale , w : womac , tug : timed up and go test , sd : standard 
 deviation presents the mean and standard deviation values and comparisons of the treated 
 groups during the follow - up period , and includes the results of repeated anova and multiple 
 comparisons .
vas : visual analog scale , w : womac , tug : timed up and go test , sd : standard 
 deviation the mean differences , the sem of mean differences , p value and 95% confidence intervals of 
 two groups were compared and the mean differences of group 1-group 2 , group 1-group 3 , and 
 group 2-group 3 in vas and tug scores were found to be significant ( p<0.001 ) . also the 
 mean differences of group 1-group 2 and group 1-group 3 in womac total , womac pain , womac 
 stiffness and womac function scores were also found to be statistically significant 
 ( p<0.001 , table 2 ) .
no significant 
 complications were observed other than transient increases in local pain or swelling during 
 the treatment and follow - up periods .
one of the major results of this study was the effectiveness of prp treatment for pain and 
 physical function in grade 3 knee oa .
however the effectiveness of a single injection was 
 found to be significantly lower than that of two or three injections . in this study , during 
 the follow up period , significant improvements were observed in the vas , womac and tug 
 values of all of the three groups compared to their pre - injection values , and they showed a 
 tendency of gradual decrease over time . according to the study of kon et al . which examined prp effectiveness on the knee joint , 
 better results were achieved in patients with a low degree of cartilage degeneration9 .
chang s meta - analysis showed that prp 
 effectiveness was higher at the degenerative chondropathy stage , and the effect decreased at 
 two or lower doses when degeneration was worse5 . in a study administering a single injection of prp , a longer 
 sustained period of pain relief
noted in a study of 14 patients 
 cartilage thicknesses measured by ultrasonography , that responses to prp decreased as the 
 level of oa and chondropenia increased4 . 

as joint degeneration increases , factors such as decrease of viable cells , muscle function 
 deficiency , joint instability due to increased ligament laxity , decrease in anabolic 
 response to growth factors , loss of chondrocyte and thinning of cartilage plate may diminish 
 the effectiveness of prp12 . despite poorer results , patients with advanced oa still benefit from prp . in a comparitive 
 study of prp and hyaluronic acid ( ha ) in grade 13 knee oa
, the prp group showed 
 significantly better results after 6 months and the worst results were observed in 
 ha - treated subjects with grade 3 knee oa23 .
speculate that , additional biological mechanisms , not 
 currently known , are responsible for the improvement of oa symptoms after prp treatment6 . in the advanced stages of oa
, prp might not 
 have a direct effect on the chondrocyte anabolic process , but an anti - inflammatory effect 
 through the regulation of joint homeostasis and the cytokine level6 , 24 .
. showed that prp administered three times at weekly 
 intervals to patients with grade 3 and 4 knee oa reported improvements in their quality of 
 life , and reduced levels of pain , and had increased cartilage thickness as measured by 
 ultrasonography at the 6-month follow up25 .
there is not enough data regarding the effectiveness of prp in the regeneration of 
 substantial and irreversible bone and cartilage damage26 .
accordingly , objective studies conducted using magnetic resonance 
 imaging or arthroscopic methods will be valuable in this regard .
to our knowledge , there are 
 no studies in the literature which have compared different doses of prp administered to 
 patients with grade 3 knee osteoarthritis . in the present study ,
3 prp injections separated 
 by 2-week intervals were found to be more effective for the improvement of pain and mobility 
 than 2 injections in grade 3 oa patients ; however , no significant differences were observed 
 in the womac values .
a significant effect was observed in the early period after a single 
 injection of prp , but the effect decreased in a short time .
based on the present results , we 
 recommend 2 or 3 injections of prp for patients with moderate knee oa , and physicians 
 decisions should be based on various factors such as the level of pain , level of activity , 
 cost - effectiveness , and bmi .
we further speculate that repeating the application after 6 
 months may further relieve symptoms for a longer period and delay oa progression . in the studies conducted so far
, the lack of standardization of prp dosing regimens makes 
 it difficult to compare outcomes of studies for the evaluation of clinical 
 effectiveness27 . the amount and 
 effectiveness of platelet concentration and
the gfs related to platelets in prp content will 
 vary according to prp preparation techniques .
for example , using an activator , the existence 
 of leukocytes in prp content , application frequency , and platelet number range are currently 
 debated issues .
in addition , the follow - up period in many studies was short term , and there 
 were no control groups5 , 27 .
prp preparation technique in this study was standardized 
 by our transfusion medicine department and no commercial filters were used .
the platelet 
 numbers injected in this study were between 1.1 billion1.4 billion , 46 fold higher than 
 the baseline value , a number similar to that used in many studies , and it was also within 
 the recommended range28 .
the absence of a control group and the relatively small 
 patient numbers were the limitations of the study . considering the evidence , this minimally invasive injection procedure appears to be safe 
 and effective , and since prp injections biologically change the articular cartilage , they 
 may be a worthwhile treatment option even in moderate knee osteoarthritis .
further studies 
 are required with larger sample sizes with longer follow - ups and objective outcome 
 measures . in conclusion
, prp is an effective and reliable treatment for functional status and pain 
 for grade 3 oa , and a minimum of two injections appears to be appropriate .
 [ purpose ] to assess the effects of different numbers of platelet - rich plasma ( prp ) 
 applications on pain and physical function in grade 3 knee osteoarthritis ( oa ) . 
 [ subjects 
 and methods ] a total of 102 patients with grade 3 knee oa were randomly divided into three 
 groups : group 1 received a single injection of prp , group 2 received two injections of prp 
 two weeks apart , group 3 received three injections of prp at 2-weeks intervals . 
 all 
 patients were evaluated with a visual analog scale ( vas ) , the western ontario and mcmaster 
 universities arthritis index ( womac ) , and the timed - up and go test ( tug ) before the 
 treatment and at 1 , 3 and 6 months after the treatment . 
 [ results ] ninety - eight patients 
 ( 15 males , 83 females ) completed the study . 
 the mean ages of the patients were 53.56.6 , 
 54.95.3 , and 55.15.6  years in group 1 , group 2 , and group 3 , respectively . 
 statistically 
 significant improvements were noted in all of the evaluated measures in all of the groups . 
 
 the mean differences of group 1-group 2 and group 1-group 3 womac total , womac pain , womac 
 stiffness , and womac function scores were statistically significant . 
 [ conclusion ] prp is 
 an effective treatment for functional status and pain in moderate knee osteoarthritis and 
 a minimum of two injections is appropriate . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION