. limitations of osteochondral grafts include limited donor site , morbidity , questionable cell viability , and fibrocartilage formation in between osteochondral plugs .
cell - based strategies have explored chondrocytes and mesenchymal stem cells ( mscs ) with extensive basic science and preclinical studies .
however , clinical strategies have focused on autologous chondrocyte implantation , yet the concept itself is not ideal ; it is a 2-step procedure with donor site morbidity , the quantity of harvested chondrocytes is limited , and chondrocytes dedifferentiate into fibroblasts when cultured ex vivo .
because of these limitations , the focus in cartilage repair is shifting toward bone marrow mesenchymal stem cells ( bm - mscs ) and biodegradable scaffolds with or without growth factors .
bm - mscs are available in larger quantities , are easier to isolate and expand , and demonstrate strong chondrogenic potential .
platelet - rich preparation such as platelet - rich plasma ( prp ) and platelet - rich fibrin glue ( pr - fg ) is a novel biological scaffold that has not been widely tested by itself as an msc carrier in clinical chondrogenesis .
the -secretory granules of platelets contain transforming growth factor ( tgf)-1 and insulin growth factor-1 ( igf-1 ) , which stimulate cartilage regeneration .
it is expected that the biological effect of multiple growth factors on tissue regeneration is greater than that of a single growth factor .
pr - fg has also been shown to provide sustained release and protection against proteolytic degradation of endogenous growth factors .
because the average baseline blood platelet count in an individual is 200,000  75,000/l , a platelet concentrate count of 1,000,000/l ( 5-fold ) has been commonly described for therapeutic platelet - rich preparations .
concentrations of growth factors in pr - fg are found to be in supraphysiological levels ( 5- to 27-fold , according to different processing methods ) .
the concentrations of tgf-1 and igf-1 in pr - fg are in the range of 90 to 400 ng / ml and 50 to 200 ng / ml , respectively .
this pilot study was conducted for the following aims : first , to test the hypothesis that pr - fg can be used clinically as a scaffold to deliver cultured mscs for cartilage repair .
the 2nd aim is to report on the clinical results 1 y after implantation of mscs - pr - fg .
the study included 5 patients : 4 men and 1 woman ( range = 21 - 37 y ; mean = 25.4 y ) .
inclusion criteria were a full - thickness cartilage lesion in weightbearing areas of the femoral condyle ( outerbridge grade iii or iv ) , size 3 to 12 cm , disabling symptoms not responding to conservative treatment , age between 18 and 50 y , and patients agreeing to comply with strict rehabilitation .
exclusion criteria were complex ligamentous knee instability , kissing lesions , axial malalignment ( > 10 varus / valgus ) assessed clinically , osteochondral neoplasia , inflammatory joint disease , immune suppression , and symptomatic vascular / neurologic disorder of the lower extremities .
all of the patients provided written informed consent according to regulations and after approval of the ethical committee and were operated on by the same surgeon ( the 1st author ) .
characteristics of the 5 patients with cartilage defects treated with bone marrow mesenchymal stem cells transplanted in platelet - rich fibrin glue note : ocd = osteochondritis dissecans ; oa = osteoarthritis ; mfc = medial femoral condyle ; lfc = lateral femoral condyle ; rt = right ; lt = left ; na = non applicable ; acl = anterior cruciate ligament ; aclr = anterior cruciate ligament reconstruction ; mf = microfracture . for clinical evaluation
, we used 2 scores collected by an independent observer : the lysholm score ( subjective ) and the revised hospital for special surgery knee score ( subjective and objective ) .
in addition , plain x - rays ( ap , lateral  tunnel views ) and magnetic resonance images ( mris ; t1 weighted , t2-fat - suppressed weighted , proton density imaging ) of the involved knee were performed using a 1.5 t mri machine ( philips , amsterdam , the netherlands ) and evaluated by an independent senior musculoskeletal radiologist .
the scores , x - rays , and mris were taken preoperatively and 6 and 12 mo postoperatively .
second - look arthroscopy was performed after 12 mo on 2 patients who consented to the procedure .
the repair tissue was rated according to the international cartilage repair society ( icrs ) arthroscopic grading system .
bone marrow ( 20 ml ) was aspirated from the iliac crest under aseptic standard operative procedures and placed in heparinized tubes .
cell cultures were performed at the molecular biology and tissue engineering unit at the same university .
briefly , nucleated cells were isolated with a density gradient ( ficoll - paque ; ge healthcare , waukesha , wi ) and resuspended in culture medium ( delbecco s modified eagle s medium ) supplemented with 10% fetal bovine serum ( fbs ) and 1% penicillin - streptomycin ( 10,000 g / ml ) .
fbs used in this study was purchased from reliable countries without bovine virus traceability , where it is furthermore subjected to serial filtration and terminal sterilization with gamma irradiation and followed by bacterial and viral testing using validated procedures to ensure pathogen freedom .
cells were incubated at 37 c in 5% co2 for ~14 d. culture medium was changed every 2 to 3 d. when cells reached 80% to 90% confluence , cultures were washed twice with phosphate - buffered saline ( pbs ) , and the cells were trypsinized with 0.25% trypsin in 1 mm edta for 5 min at 37 c .
after centrifugation , cells were resuspended in medium and subcultured for ~10 d , reaching an average count of 15  10 .
mscs in culture were characterized by their adhesiveness and fusiform shape , by flow cytometry for msc surface markers ( cd34 , cd45 , and cd73 ) and for cd29 gene expression by reverse transcriptase polymerase chain reaction . on the day of implantation
, cells were trypsinized , collected , and resuspended in 1 ml pbs and transferred to the operating room in sterile tubes .
potential immunogenicity from fbs proteins was furthermore minimized by repeated copious irrigation of the msc pellet with pbs before final resuspension and transfer to the operating room .
platelet concentrate was prepared in the hospital blood bank ( el - shabrawishi hospital blood bank , cairo , egypt ) according to standardized protocols previously reported elsewhere .
briefly , using a cell saver centrifuge system , 500 ml volume of blood from healthy donors was collected into 70 ml of citrate - phosphate - dextrose anticoagulant .
this was delivered into the 125-ml disposable bowl at 60 ml / min , with the centrifuge spinning at 2039 g until the red blood cells reached 1.25 cm from the collar of the bowl .
the centrifuge speed was then reduced to 368 g , and a blood flow rate of 60 ml / min was used to continuously collect the prp , from which 1 ml of platelet - rich concentrate was used with fg to form pr - fg .
this method has been shown to yield a platelet count of 7.7  1.1  10/ml . in a 2nd - stage implantation procedure , under a tourniquet , the knee joint was exposed via medial or lateral parapatellar arthrotomy .
multiple 3- to 4-mm - deep perforations in the subchondral bone were performed using a 1.2 kirschner wire at 3- to 4-mm intervals .
an autologous periosteal flap was harvested from the anteromedial ipsilateral proximal tibia and tailored to the defect size .
the flap was sutured to surrounding cartilage with the cambium layer facing toward the subchondral bone using absorbable 4 - 0 sutures , leaving the 12 oclock position open for introduction of the bm - mscs / pr - fg mixture .
allogenous pr - fg was supplied as 1 ml platelet concentrate , 1 ml fibrinogen , and 1 ml thrombin in separate syringes from the aforementioned blood bank .
bm - mscs were mixed with the pr - fg , and the mixture was left to gel and then introduced through the 12 oclock position and subsequently sutured and sealed by fg ( fig .
the inoculum density of bm - mscs at the time of implantation was 2  10 cells / cm .
autologous mesenchymal stem cell ( mscs ) platelet - rich fibrin glue ( pr - fg ) implantation .
( a ) cartilage defect curetted to a peripheral stable rim of cartilage and subchondral bone drilled .
( c ) defect covered with periosteal flap , sutured , and then sealed with fibrin glue .
( d ) mscs mixed with pr - fg intraoperatively to be introduced through the 12 oclock position of the defect .
weightbearing was restricted , whereas passive , active - assisted , and active range - of - motion exercises were initiated toward a full range equal to the contralateral knee . after 4 to 6 wk , partial weightbearing was initiated as tolerated . by 8 wk ,
statistical analyses were done using statistical package for social studies ( spss ) software , version 11 for windows .
the clinical scores were shown as the mean  standard deviation at 3 time points : preoperatively and 6 and 12 mo postoperatively .
data were analyzed statistically by friedman s test and the wilcoxon signed - rank test .
none of the cell cultures had bacterial or fungal infection , nor were there any perioperative complications in any of the patients .
details of the clinical scores of the 5 patients preoperatively , 6 and 12 mo postoperatively , as well as the arthroscopic scores of the 2 patients who underwent follow - up arthroscopy at 1 y statistically significant difference . compared with preoperative status , all patients experienced marked improvement in symptoms and knee function , allowing them to return to normal daily activities .
all 4 patients without preexisting radiographic signs of advanced degenerative changes returned to their previous sporting levels .
clinical scores 6 mo and 12 mo postoperative were statistically different from preoperative scores ( p < 0.05 ) .
the difference between the 6- and 12-mo clinical scores did not reach statistical significance ( p = 0.18 ; fig .
2 ) . mean clinical scores : preoperative ( dark gray column ) , 6 mo postoperative ( light gray ) , and 12 mo postoperative ( black ) .
( a ) lysholm ( lysh ) scores 6 mo ( lysh 6 m ) and 12 mo ( lysh 12 m ) postoperative were statistically different ( * ) from preoperative scores ( p < 0.05 ) .
the mean 6 and 12 mo postoperative lysholm scores improved from 41.2  13.14 preoperatively to 81.6  11.93 at 6 mo ( p = 0.042 ) and 86.0  9.25 at 12 mo ( p = 0.042 ) .
( b ) revised hospital for special surgery knee ( rhssk ) scores 6 mo ( rhssk 6 m ) and 12 mo ( rhssk 12 m ) postoperative were also statistically different ( * ) from preoperative scores ( p < 0.05 ) .
the mean 6 and 12 mo postoperative rhssk scores improved from 53.8  15.39 to 79.0  11.77 at 6 mo ( p = 0.043 ) and 83.8  9.78 at 12 mo ( p = 0.043 ) .
no statistically significant difference was found between scores at 6 and 12 mo postoperative ( p = .18 ) .
radiologically , patient 1 had more advanced degenerative changes at the time of surgery ( fig .
3 ) than the other 4 patients , and his preoperative scores were also initially lower . six- and 12-mo follow - up mri of the operated knee showed partial defect fill , complete incongruity of the repair tissue with native cartilage , and lower signal intensity in relation to the surrounding subchondral bone .
x - rays of the patient at 6 and 12 mo showed no evidence of filling of the bony element of the defect nor restoration of the congruity of the femoral condyle .
however , the patient reported significant relief of symptoms and was able to return to his laborious job after being incapacitated because of knee pain before surgery .
( a ) preoperative ap radiograph of the knee of case 1 showing medial femoral condyle osteochondral erosion ( black arrow ) , narrowing of the medial joint line , and osteophyte formation .
( b ) preoperative ap radiograph of the knee of case 4 showing preserved joint line with no radiographic evidence of advanced degenerative joint changes .
the patient had an osteochondral lesion in the lateral femoral condyle that was apparent on magnetic resonance imaging ( see fig .
mri of patient 2 showed partial defect filling with incomplete congruity of the repaired articular surface .
x - rays at 6 and 12 mo showed persistent radiolucency and sclerosis at the margins of the defect . arthroscopic evaluation of the repair tissue showed 75% repair of defect depth , 75% graft integration , 25% with a border > 1-mm wide , and a fibrillated surface all through .
the patient had described the outcome as  good .  patients 3 , 4 , and 5 described their outcomes as  excellent ,  and all showed complete defect fill with no overgrowth or periosteal hypertrophy and congruity of the repair tissue with the native cartilage on 6 to 12-mo follow - up mri ( fig .
x - rays showed complete filling of the defects with restoration of the congruity of the femoral condyles and no evidence of degenerative changes . in patient 4 , 2nd - look arthroscopy after 12 mo showed the graft surface to be level with the surrounding cartilage , a demarcating border < 1 mm at some zones ( ~25% of the circumference ) , and an intact smooth surface ( fig .
the patient returned to high - impact sports after 9 mo . magnetic resonance image ( mri ) of sample case 4 .
( a ) preoperative sagittal magnetic resonance imaging ( mri ) proton density sequence ( pd ) showing an osteochondral lesion ( white arrow ) in the lateral femoral condyle .
this lesion was found to be completely detached on arthroscopy performed during bone marrow aspiration .
( b ) sagittal mri ( pd ) at 12 mo postoperative showing isointense signal of the repair cartilage ( white arrow ) compared with the native cartilage , complete filling of the defect , congruent surface of the repair cartilage , no delamination , and no subchondral bone marrow edema .
case 4 : arthroscopic view of the knee 12 mo postoperative , with the arthroscopic probe pointing to grossly appearing , smooth , hyaline - like repair tissue completely filling the defect .
numerous preclinical studies have reported development of hyaline - like tissue from autologous culture - expanded bm - mscs , and some have shown superior functionality of bm - mscs over chondrocytes .
pr - fg as a cell carrier has the advantage of being biodegradable , autologous and contains chondrogenic growth factors with sustained release profiles . in this pilot study , we found that all patients with osteochondral defects who were reconstructed with bm - mscs transplanted on pr - fg experienced significant improvement in their functional knee scores and mri findings as early as 6 mo and maintained over 12 mo postoperatively .
although clinical improvement will be seen after most cartilage repair techniques in this short time period , this remains true only for small - sized defects ( < 2 cm ) , not large - sized defects , as included in this small case series ( average = 5.8 cm ) .
an example is patient 5 , who had microfracture with anterior cruciate ligament reconstruction that did not relieve his cartilage - related symptoms as early as 6 mo postoperative .
there were notable variations in the results among patients with and without advanced degenerative changes in the knee prior to the surgery .
this suggests that the condition of native cartilage surrounding a chondral defect affects the outcome of repair , possibly because of the decreased number and functionality of available chondrocytes as well as the decreased proteoglycan / collagen content of degenerative cartilage , impeding integration of repair tissue .
this emphasizes early intervention in treating cartilage defects before the progression of degenerative changes that hinder better results .
although patient 2 had the smallest defect size ( 3 cm ) in this case series , the relatively inferior outcome in this case compared with patients 3 to 5 could be attributed to a number of causes : first , the ongoing subchondral bone pathology due to osteochondritis disssecans evident as radiolucency with surrounding bone sclerosis on plain x - rays at 1-y follow - up might have impeded proper tissue vascularization , with subsequent msc death and poor graft integration .
second , the chondrogenic potential of bm - mscs in this patient might have been quantitatively and qualitatively less than other patients .
have recently reported that msc chondrogenic potential shows both individual and age - dependent variations .
although the age inclusion criterion for the study was 18 to 50 y , the patients in this pilot study were of relatively younger age ( range = 21 - 37 y ; mean = 25.4 y ) .
this might have influenced the outcome of this cartilage repair procedure because the qualitative and quantitative difference in the metabolic activity of cells in the repair tissue is age dependent .
it is well documented that mscs age as they undergo more population doublings , and their synthetic activity and chondrogenic potential decline . the biopsy - induced injury and whether it heals spontaneously or further jeopardizes the repair limited its ethical justification to patients and remains a point of debate .
this is of particular importance if long - term follow - up of the repair tissue is intended that should not be violated .
mri was used to evaluate the reparative tissue and is considered a good noninvasive method that was found to correlate well with 2nd - look arthroscopy and histology of cartilage repair tissue .
although these concerns can not be completely eliminated , a phase 1 clinical study employing culture - expanded stem cells in fbs - containing media ( at higher concentrations than used in our study ) reinjected in patients with liver insufficiency similarly reported no complications or specific side effects related to the procedure .
msc implantation on pr - fg is a new technique and required an initial small number of patients enrolled in the pilot study , consequently limiting the sample size of this study . because the sample size was small , we used nonparametric statistical methods , which do not require the assumptions of normality and equal variances ( i.e. , standard deviations ) .
friedman s test was used to compare observations repeated on the same subjects as a group .
it is similar to the parametric repeated - measures analysis of variance and is used to detect differences in treatments across multiple test attempts .
the wilcoxon signed - rank test was used to test for repeated measurements on a single sample .
it can be used as an alternative to the paired student s t test when the population can not be assumed to be normally distributed .
nonparametric tests are more conservative and are appropriate for hypothesis testing when the sample size is small . because the sample size was small and there was no randomly assigned control group , a further randomized controlled clinical trial of this treatment modality , with larger numbers of patients and longer follow - up periods , is necessary to achieve even higher levels of validity .
this pilot study demonstrates feasibility and proof of principle that bm - mscs pr - fg can be used clinically for the treatment of articular cartilage defects .
this is consistent with basic science and preclinical in vivo studies reported in this field .
we used standardized clinical outcome scores to assess the results of this treatment modality and mri for assessment and follow up of the repair .
the 2nd - look arthroscopies performed on 1 patient with partial defect fill by mri and 1 patient with complete defect fill by mri confirm the mri assessments and support the use of mri as a noninvasive postoperative assessment tool .
we conclude from this study that the transplantation of autologous culture - expanded bm - mscs in pr - fg shows great promise in the treatment of full - thickness articular cartilage defects , particularly large - sized defects ( > 4 cm ) .
we have demonstrated that pr - fg successfully fixed the cultured mscs within the defects and provided them with a suitable environment to synthesize a hyaline - like grossly appearing cartilaginous matrix .
the positive 1-y clinical outcomes support further randomized controlled clinical trials of this treatment modality with larger numbers of patients and longer follow - up periods , as previously stated .
 objective : to test the hypothesis that platelet - rich fibrin glue ( pr - fg ) can be used clinically as a scaffold to deliver autologous culture - expanded bone marrow mesenchymal stem cells ( bm - mscs ) for cartilage repair and to report clinical results 1 y after implantation of mscs pr-fg.patients and methods : autologous bm - mscs were culture expanded , placed on pr - fg intraoperatively , and then transplanted into 5 full - thickness cartilage defects of femoral condyles of 5 patients and covered with an autologous periosteal flap . 
 patients were evaluated clinically at 6 and 12 mo by the lysholm and revised hospital for special surgery knee ( rhssk ) scores and radiographically by x - rays and magnetic resonance imaging ( mri ) at the same time points . 
 repair tissue in 2 patients was rated arthroscopically after 12 mo using the international cartilage repair society ( icrs ) arthroscopic score.study design : case series ; level of evidence 4.results:all patients symptoms improved over the follow - up period of 12 mo . 
 average lysholm and rhssk scores for all patients showed statistically significant improvement at 6 and 12 mo postoperatively ( p < 0.05 ) . 
 there was no statistically significant difference between the 6 and 12 mo postoperative clinical scores ( p = 0.18 ) . 
 icrs arthroscopic scores were 8/12 and 11/12 ( nearly normal ) for the 2 patients who consented to arthroscopy . 
 mri of 3 patients at 12 mo postoperatively revealed complete defect fill and complete surface congruity with native cartilage , whereas that of 2 patients showed incomplete congruity.conclusion:autologous bm - msc transplantation on pr - fg as a cell scaffold may be an effective approach to promote the repair of articular cartilage defects of the knee in human patients . 
Introduction
Patients and Methods
Results
Discussion