the articular cartilage consists of a high - specialized , low friction tissue that covers the epiphyses with the function of enabling bones do glide over each other and to absorb impacts within the joint without causing wear ; this cartilage has a poor capacity of renewal [ 13 ] .
its viscoelastic behavior depends on the intrinsic mechanical properties of the extracellular matrix , the presence of collagen gel in the matrix , and the flow of the interstitial water due to the load applied during the movement [ 4 , 5 ] .
the articular cartilage can suffer degeneration , beginning with a degradation of the cartilage itself ( chondral lesion ) that may reach and expose the subchondral bone ( osteochondral lesion ) .
the damages can be of traumatic origin , such as articular fractures , ligament , or meniscus lesions , or of inflammatory origin , as in autoimmune or metabolic diseases .
the articular cartilage exhibits a low - intrinsic ability of self - repair , this is due to a lack of vascularization and due to the low - metabolic activity of the mature chondrocytes , which in turn , limit the supply of growth factors , responsible for the cellular differentiation and proliferation .
furthermore , the lack of innervation delays the manifestation of clinical symptoms , facilitating a quiet advance of the articular degeneration . nowadays ,
treatment options are based on abrasion , microfractures , mosaicplasty , first- and second - generation autologous chondrocyte implantation , and allogeneic osteochondral grafting [ 916 ] .
these techniques have drawbacks such as morbidity of the donor area , reduced mechanical stability of the new tissue compared with the normal cartilage , genetic incompatibility between donor and recipient and high cost , among other problems , and none of these have so far proven to be effective enough to ensure complete long - term regeneration . in view of this , the development of osteochondral implants that mimic the physical , chemical , and mechanical properties of the articular cartilage has been proposed [ 4 , 17 , 18 ] .
such implants for osteochondral defects consist of scaffolds that must allow simultaneous growth of both cartilage tissue and subchondral bone .
they may neither be cytotoxic nor elicit inflammatory response of the host tissue ; otherwise they will not allow cartilage regeneration and will be encapsulated by fibrous tissue . among the materials used for
osteochondral defect repair , bioceramics , such as bioglass , hydroxyapatite , and calcium phosphate , occupy a prominent place .
noteworthy is also the use of synthetic biodegradable polymers such as poly(-hydroxy ester ) , poly(ethylene glycol ) ( peg ) , and poly(caprolactone ) ( pcl ) . these polymers exhibit the advantages of easy processing , low - processing costs , controlled biodegradability , and fda - approval . amongst them
, the polymeric hydrogels have proven to be a promising class of materials for osteochondral repair , since their physical and chemical characteristics can resemble that of the articular cartilage and the subchondral bone [ 4 , 2126 ] .
polyvinyl alcohol ( pva ) crosslinked by electron beam irradiation deserves special attention thanks to its chemical stability against biological fluids , good elasticity , adequate mechanical strength , hydrophilicity , physical properties similar to those of the cartilage , and low processing costs [ 2730 ] .
preliminary in vitro and in vivo tests of pva hydrogel have exhibited promising results [ 31 , 32 ] , qualifying pva for further tests .
thus , the aim of this study was to evaluate the in vivo performance of pva cross - linked by electron beam irradiation implanted in wistar rats ' knees by means of mechanical , chemical , and histological tests .
g / mol , 99% hydrolyzed and was prepared and homogenized at 70c for 1  h in magnetic stirrer .
the solution was transferred to a petri dish and kept at room temperature for 7 days resulting in 1  mm thick membranes ( figure 1 ) .
the membranes were acetalised by a chemical treatment composed of formaldehyde solution 40% ( w / w ) ( aldrich ) , concentrated sulfuric acid 50% ( w / w ) ( aldrich ) , and 300  g anhydrous sodium sulfate ( aldrich ) . the membranes were maintained in this solution under constant stirring at 70c for 24  h. after being washed and hydrated in running distilled water , the samples were crosslinked by electron beam irradiation at 25  kgy produced by a radiation dynamiton electron beam accelerator ( institute of energy and nuclear research , so paulo , brazil ) .
thereafter , the samples were hydrated and swelled in 0.9% sodium chloride ( nacl ) solution for 48  h and osteochondral implants ( plugs 2  mm in diameter and 1  mm in height ) were obtained by punched cut outs ( figure 2 ) .
the plugs were stored at low temperature in sterile falcon tubes containing 0.9% nacl until implantation .
forty - four male rats ( ~380  g ) were anesthetized by intravenous injection of 25  mg / kg pentobarbital according to the ethical protocol approved by the ethics committee in animal experimentation of the university of campinas , so paulo , brazil ( protocol n 1047 - 1/2008 ) .
the knee joint was accessed by a medial parapatellar incision and a twist drill fitted with a depth stop was used to produce cylindrical osteochondral defects ( 2  mm in diameter and 1  mm deep ) in the intercondylar region .
the animals were divided into 6 groups : 5 experimental groups with 8 animals each , for implantation of the pva samples ( 0 , 3 , 6 , 12 , and 24 weeks followup referred to as eg00 , eg03 , eg06 , eg12 , and eg24 , resp . ) and a  cartilage  control group ( cg ) with 08 animals , whose articular cartilage was kept intact . 

the hydrogel plugs were inserted under pressure into the defects with the aid of a trephine and sit flush with the surface of the adjacent articular cartilage ( figure 3 ) . the joint capsule and
the skin were sutured and the animals were placed in cages where they received paracetamol solution 25  mg / kg for 24  h. after that , only food and water were offered ad libitum until the animals were sacrificed .
six condyles from each eg and cg group ( n = 36 ) were randomly selected and stored in 0.9% nacl solution until the test , in order to avoid tissue autolysis .
the test was carried out on a universal testing machine emic dl300 ( curitiba , brazil ) . a load of 4.905  n ( 0.5  kgf )
was applied to the implant surface by a hemispherically ended indenter ( 1.4  mm diameter ) and kept for 180 seconds   and curves of indenter penetration depth versus time were plotted from test data that were continuously registered by the test machine software ( tesc 3.04 ) .
the creep modulus for the hydrogels in situ at 180 seconds after load application was calculated according to the equation below ( originally deduced for sheets of vulcanized rubber and successfully used by kempson et al .   for human articular femoral cartilage ) : 
 ( 1)e=9106p16r[1e(0.42  t / a)h]3/2,a=(2rhh2)[m ] .
three hydrogel plugs from each eg ( n = 12 ) and three nonimplanted plugs eg00 ( n = 3 ) were subjected to xrf analysis , in order to assess the incorporation of calcium ( ca ) , phosphorus ( p ) , and sulfur ( s ) by the pva .
after removal , the plugs were dried at room temperature , put into xrf polyethylene sample cups ( chemplex ) , and analyzed on an energy dispersive x - ray fluorescence spectrometer ( shimadzu edx 700 ) with rhodium x - ray tube and semiconductor si ( li ) detector ( energy resolution of 165  ev ( mn ka line ) , under the following conditions : 3  mm diameter incident beam collimator , x - ray tube voltage of 15  kv , exposure time of 100  s , and detector dead time of 25% [ 35 , 36 ] .
the software axil was employed to interpret the results and calculate the concentration of the elements in the samples .
after the mechanical test , fourteen samples from the eg group ( n = 40 ) were fixed in buffered 10% formaldehyde solution ( ph 7.5 ) for 48  h , decalcified in a solution of ethylenediaminetetraacetic acid disodium ( edta ) 7% ( w / w ) , hydrochloric acid 100% 140  ml ( w / w ) , sodium tartarate 0.15% ( w / w ) , sodium , and kaulium tartarate 0.9% ( w / w ) in distilled water for 15 days , washed in distilled water for removal of residual decalcifying solution , immersed in ethanol 70% , and finally embedded in paraffin .
semithin serial sections ( 4  m thick ) were cut on a rotatory microtome leica 2155 and stained with hematoxylin eosin ( he ) .
images were obtained with a leica dmlb 100  s microscope ( magnification 200  m ) .
the images were analyzed for the absence , moderate , or intensive presence of the following structures : granulation tissue ( gt ) , collagen fibers ( cf ) , mineralized bone matrix ( mb ) , neoformed bone tissue ( nb ) , and collapse of neoformed tissue ( cn ) .
the results obtained by om were evaluated according to the criteria : absent ( + ) , moderate ( + + ) , and ( + + + ) intensive as described in table 1 .

 figure 4 shows the average creep modulus and respective standard deviation for each experimental control group . 

figure 5 shows the average concentrations of calcium , phosphorus and sulfur according to follow - up time . 

figure 6 containing macroscopic images of egs after animals were sacrificed . 
 according to mow et al .  
implants , although inert when implanted , are involved by a fibrous capsule of connective tissue . in this study , this was confirmed by the absence of tissue formation on the pva , as observed ( figure 7 ) . 

as can be noticed , the implant was removed from all samples during the processing for histological analysis .
figures 9 , 10 , 11 , 12 , and 13 present mean scores obtained according to the criteria established in table 2 , these scores were given according to absence , moderate , or intensive presence of tissue on egs according to duration of followup .

 several studies have shown that the mechanical behaviors of the articular cartilage under in vitro and in vivo compression can not be compared with each other .
contrary to in vitro , in vivo tissue recovers its original dimensions after load removal thanks to a number of factors that define the elasticity and water absorption capacity of its complex structure , such as pressure , viscosity , temperature and macromolecular content of the interstitial fluid , and the concentration and type of dissolved ions [ 1 , 3 , 4 ] . 

figure 4 clearly shows that the average creep modulus of the implanted hydrogels increased over time .
the increase can be partially attributed to the regrowth of the subchondral bone at the defective bottom ( figures 6 and 7 ) , which provided a stiffer bed for the implant over time , thus restricting deformation and resulting in a higher calculated creep modulus .
comparing the creep modulus obtained in this study for rat cartilage ( figure 4 ) and that obtained by kempson et al .   for human femoral cartilage ( 8.410.6  mpa ) , the values obtained for the pva hydrogel , though relatively low , were still within the same order of magnitude .
another contribution to the increase of the creep modulus over time could be assigned to the absorption of calcium and phosphorus by the implant , evidenced by the results of the edxrf analysis ( figure 5 ) .
the edrfx analysis ( figure 5 ) revealed increasing concentrations of ca and p in the implants over time , probably due to absorption of calcium phosphate , and a steady concentration of s in comparison to the gc00 ( not implanted ) .
the consequences of the absorption of calcium phosphate are still unclear and deserve further investigations .
it is well known that all implants , although inert when implanted , are involved by fibrous capsule of connective tissue .
edrfx results indicated an increase in ca and p levels over follow - up time .
further studies are being carried out to determine if the analyzed elements were detected in or on the surface the pva . 
 according to the macroscopic results of the egs ( figure 6 ) ,
the integrity of the surface of pva and adjacent ca were maintained by the normal lubrication process . 
 as observed in the macroscopic images ( figures 7(a ) and 7(b ) ) , the pva implant kept its integrity , with no sign of wear , in all egs and in the adjacent ca .
the result indicates that the process of synovial fluid lubrication was maintained during the entire followup . from the om ( figure 8) , the quality of the interface between implant and the surrounding tissue ( subchondral bone ) was satisfactory , with no foreign body reaction .
the pva hydrogel could bear the load without rupture or wear and offered a suitable and stable environment for the formation of new bone at the interface with the host tissue .
the implant surface appears to have maintained its integrity due to the presence of synovial fluid .
that was possible because the electron beam crosslinking of the pva promoted the formation of a polymeric structure that was strong , yet still complacent and capable of absorbing and retaining synovial fluid , which kept low the friction between the articular surfaces and counter surfaces in all egs .
the absence of signs of implant rejection in almost all samples attests the good compatibility between the hydrogel and the tissues surrounding the artificial osteochondral defect ( figures 913 ) .
the pva hydrogel implant bore the load without wear or rupture , offered a stable and suitable environment for growing new bone at the interface with the host tissue , and did not disturb the natural articular lubrication .
the results of these mechanical , chemical , and histological analyses indicate pva hydrogel irradiated and acetalised as a promising biomaterial for repairing osteochondral defects .
 polyvinyl alcohol ( pva ) hydrogel plugs were implanted in artificial osteochondral defects on the trochlear groove of rat knees . 
 after 0 , 3 , 6 , 12 , and 24 weeks of followup , samples containing the implants were mechanically evaluated by creep indentation test , chemically , and histologically by optical microscopy . 
 the mechanical test pointed towards an increase of the implant creep modulus and the chemical analysis exhibited an increasing concentration of calcium and phosphorus within the implants over time . 
 optical microscopy showed no foreign body reaction and revealed formation , differentiation , and maintenance of new tissue at the defect / implant interface . 
 the absence of implant wear indicated that the natural articular lubrication process was not disturbed by the implant . 
 the performance of the irradiated and acetalized pva was considered satisfactory for the proposed application . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion