document: it is known that bone mass density and architecture are intimately dependent upon mechanical factors . 
   several in vitro studies suggest that cell mechanical stimulation promotes proliferation and differentiation of osteogenic lineage [ 1 , 2 ] , in agreement with the results from several in vivo studies [ 35 ] . 
   stress shielding causes bone loss through remodeling due to decreased loading , particularly in noncemented prostheses [ 6 , 7 ] . 
   several solutions have been proposed for these two mechanical problems , namely , design variations and development of controlled - stiffness implants [ 8 , 9 ] . a new approach to this problem , with potential application to other orthopedic problems and other medical fields , would be the development of smart implants integrating systems for bone mechanical stimulation . 
   the present in vivo study explores the use of the converse piezoelectric effect to mechanically stimulate bone with resource to polyvinylidene fluoride films ( pvdf ) ( measurement specialties inc company , usa ) . 
   these films were used , after dip - coating to improve cell adhesion and assure electrical insulation , as a substrate for mc3t3 cells to explore the concept of using piezoelectric materials and converse piezoelectric effect to produce effective osteoblast mechanical stimulation . 
   the coating was performed by dip - coating at constant velocity of 0,238  mm / s . 
   the chosen material for coating was an acrylic , poly(methyl methacrylate ) ( pmma ) , ( perfex , international dental products , usa ) , used alone in the first three layers and a in forth layer along with 4% of bonelike ( 250500  m ) particles added ( kindly offered by inescporto ) . 
   the displacement was higher where the cells were seeded , in the central area of the coated devices , in the order of 700  nm along the z - axis , in a semisinusoidal fashion . 
   on the substrates submitted to dynamic conditions , stimulation was done with an alternating sinusoidal current ( ac ) , of 5  v , at 1  hz and 3  hz for 15 minutes at each frequency . in this exploratory in vivo study , 
   the same coating procedures and daily stimulation regimens were applied . the biological response around the osteotomies 
   was assessed through histology and histomorphometry in undecalcified sections , and through histochemistry and immunohistochemistry in decalcified sections , namely , through masson 's trichrome , and labeling of osteopontin ( opn ) , proliferating cell nuclear antigen ( pcna ) , and tartrate - resistant acid phosphatase ( trap ) . 
   the actuator device was composed of a microboard containing an ultra - low power 16-bit microcontroller ( ez430-rf2500 , texas instruments , usa ) , powered by lithium battery and encapsulated in pmma and a set of six actuators composed of polyvinylidene fluoride ( pvdf ) and silver electrodes , electrically insulated by dip - coating as described elsewhere . 
   each actuator measured 15  mm width and 40  mm length , with an active area of 12  31  mm . 
   the actuator device was programmed to apply a 5  v square wave of 1  hz and 3  hz for 15 minutes at each frequency , once every 24 hours . 
   finite numerical method studies and electronic speckle pattern interferometry process ( espi ) studies have shown the lower deformation levels occurred in the clamped region of the films , for a potential differential of 5  v , and the higher deformation occurred in the opposite extremity of the film . a control device , similar but static was also developed . a sterilization method was developed and validated based on iso 11737 - 1 and iso 14937 , using a series of decontaminating solutions ( 10% sodium hypochlorite for 20 minutes , without agitation , washing in sterile water , and immersion in 10% hydrogen peroxide for 30 minutes , without agitation , followed by rinse in sterile water and drying under laminar flux ) , before 2 hours of exposure to uv ( in two different positions ) . 
   the actuator device was implanted in the left hind limb , and a control device was implanted in the right hind limb , of a 4-year - old merino ewe , with 45  kg body - weight . 
   the animal was premedicated with atropine ( 0.7  mg / kg ) , xylazine ( 0.1  mg / kg ) and butorphanol ( 0.01  mg / kg ) , and anesthesia induced with thiopental sodium 5% ( 5  mg / kg ) by intravenous injection . the anesthesia was maintained through isoflurane with spontaneous ventilation . 
   the stifle joint of both legs was prepared with a solution of povidone - iodine and ethanol 70% , after being shorn and shaved . 
   two longitudinal incisions were made on the craniomedial border of the femur and at tibial crest level . 
   the tibial bone was exposed by reflecting deep medial fascia , the vastus medialis and cranial part of the sartorius muscles and the periosteum , and two osteotomies were made using an especially metal designed guide to make two regular and well - orientated osteotomies using an oscillating saw . 
   the bone was continuously irrigated with a sterile saline solution during the process of low - speed drilling and cutting . 
   the same procedure was followed with a different design guide for the distal femoral physis , where four osteotomies were done . 
   a stab wound was made in the fascia lata , and the extremities of the piezoelectric films passed through it , and then , the active area of each piezoelectric film was placed inside the slot osteotomy cavity . the actuator position inside the tibial bone can be clearly viewed in figure 1(a ) , obtained post - mortem . 
   the portion of the device containing the microprocessor and the power supply was left in the nearby subcutaneous space . 
   the actuator device was implanted in the left hind limb , and control on the right hind limb . after placing the implants , 
   incisions were closed routinely with a continuous - pattern suture in the retinaculum and fascia , using absorbable surgicryl 2/0 ( smi ag , belgium ) and in the skin with silk 0 ( smi ag ) . 
   postoperative x - ray images were obtained thirty days after implantation , before sacrifice , to ensure the proper evolution of the bone cuts and to detect signs of infection / inflammation ( figure 2 ) . 
   one week after implantation calcein ( 15  mg / kg ; sigma , usa ) was injected subcutaneously and one week prior to sacrifice the same procedure was done with alizarin complexone ( 25  mg / kg ; sigma , usa ) . 
   the present study was authorized by competent national authorities and conducted accordingly to felasa 's guidelines for animal care . 
   both hind limbs were dissected , the implanted materials and surrounding tissue removed and fixed in 4% paraformaldehyde for two weeks . 
   bone samples were cut transversally to the long axis of the bone , each including a piezoelectric film and the surrounding bone ( figure 1(a ) ) . 
   soft tissues ( local lymph nodes and samples of the fibrous capsule surrounding the implants ) were routinely processed and embedded in paraffin . 
   undecalcified bone samples of each of the implants were included in resin ( technovit 9100 , heraeus kulzer , germany ) according to the manufacturer 's instructions and 80  m thick sections cut with a saw microtome ( leica 1600 , germany ) parallel to the piezoelectric film long axis 
   sections were then appropriately processed for routine staining ( giemsa eosin ) , mounted for fluorescence microscopy . 
   for histomorphometric studies , the interface between the bone and implant was divided in four distinct areas : z1 , z2 , z3 , and z4 , from cortical towards bone surrounding the free extremity of the piezoelectric film ( figure 1(b ) ) . 
   pictures were taken from the bone surrounding both sides of the film in areas z1 to z3 and z4 , using a 40x magnification ( figure 3 ) . 
   for immunohistochemistry and histochemistry , bone sections were decalcified in formic acid 5% for 3 weeks , dehydrated in ethanol , cleared in xylene , and embedded in paraffin wax and 3  m sections cut . 
   after deparaffinization and rehydration , immunohistochemistry sections were treated with 3% hydrogen peroxide for 10 minutes . 
   primary antibodies for tartrate - resistant acid phosphatase ( trap ) ( thermo scientific , usa , mouse monoclonal antibody , ab-1 , clone 26e5 , cat . 
   no.ms-1768 ) , proliferating cell nuclear antigen ( pcna ) ( neomarkers , usa , mouse monoclonal antibody , ab-1 , clone pc10 , cat . 
   no.rb-9097 ) were diluted to 1  :  20 , 1  :  200 , and 1  :  50 , respectively . 
   prior to immunostaining , the sections were pretreated for antigen retrieval at 100c in 10  mm citrate buffer , ph 6 , for 20 minutes in microwave oven , followed by cooling for 30 minutes at room temperature . for double staining of pcna and osteopontin , 
   immunohistochemistry was done using a double staining kit picture ( zymed laboratories inc , usa ) , according with the manufacturer 's instructions . 
   trap immunohystochemistry was performed with resource to kit picture - max polymer ( invitrogen , usa ) . 
   slides were counterstained with mayer 's hematoxilin , dehydrated , mounted with entellan ( merck , germany ) , and coverslipped . for all sections , 
   as negative controls adjacent sections were incubated : ( a ) without primary antibody and ( b ) with rabbit / rat normal serum ( similar concentration as that of primary ) . 
   masson trichrome was done using a kit ( bio - optica , italy ) , followed by dehydration and mounting . 
   observation and pictures were done using optic ( olympus bx41 ) and fluorescence microscope equipped with wb fluorescence cube ( olympus u - lh100hg ) . 
   total bone area , immature bone area , and mature bone area were measured using sigma scanpro 5 ( systat software inc . 
   the results , in percentage of total bone area , mature bone , and immature bone , were determined for each piezoelectric film in the pre - determined areas z1 to z4 . 
   normal distribution of the results was verified using the kolmogorov - smirnov test , homogeneity of variance assessed through the levene test , and differences between groups tested using one - way anova ( at a 0.05 level ) . 
   the statistical analysis was done using software originpro 8 ( originlab corporation , usa ) . 
   total bone area around the actuators was significantly higher , when comparing to static controls ( 39.91  14.08% versus 27.20  11.98% ) ( figure 4 ) . 
   the increment of bone occupied area was due to new bone formation , and in actuators the area occupied by woven bone and osteoid was 64.89  19.32% of the total bone area versus 31.72  14.54% in static devices ( figure 5 ) . 
   the chosen staining technique , giemsa eosin , stains older bone in a darker shade of pink , whilst more recently deposed bone is of a lighter pink color and frequently adjacent to much less prominent osteoid ( dark blue ) areas ( figures 5(a ) and 5(b ) ) . 
   taking into account the close relation in between osteoid and woven , recently formed bone , the authors chose not to make separated measurements that could be misleading . 
   the significant differences arise from the bone areas surrounding the two distal thirds of the devices ; around the devices ' clamped area , no differences were found between actuated and static devices ( figure 6 ) . 
   when assessing the mineral apposition rate in the distal third of the films , with the aid of the fluorochrome labeling , the mineral apposition rate was significantly higher around actuated devices ( 4.44  1.67  m / day ) than around static devices ( 2.70  0.95  m / day ) ( figure 5 ) . 
   a fibrous capsule was obvious both in actuators and static devices , with no statistical significant differences in capsule thickness between the two groups ( 289.59  131.20  m in actuated films versus 293.93  84.79  m ) . 
   immunohistochemistry failed to detect differences in trap expression between the two groups , but there is a marked elevation in opn detection around actuators , in z3 and z4 ( figure 7 ) . 
   opn is also strongly labeled in the fibroblast layers closer to the pvdf films , both in actuators and static controls , and this is the area where positive pcna labeling was found more often . 
   pcna labeling was also positive in some of the preosteoblastic cells in layers apposed to opn - positive columnar osteoblasts . 
   the existence of micro - movements at the interface , depending on the magnitude , may enhance osteogenesis or impair it and promote the formation of fibrous tissue [ 11 , 12 ] . considering the deformation levels of the actuator devices , under the experimental conditions , the generated strains were low , but the results suggest they are enough to elicit response . 
   mineral apposition rate and total bone mass are significantly higher around actuated films when compared to static controls , and these differences arise from around the areas of the films known to have higher displacement values . 
   the increased opn expression we found around actuators when comparing to static controls is most likely associated with mechanical stress , and it was not accompanied by a proportional increase in trap . 
   opn is a multifunctional noncollagenous glycoprotein present in bone matrix , expressed in various degrees by proliferating preosteoblasts , osteoblasts , and osteocytes but also by fibroblasts , osteoclasts , and macrophages [ 1315 ] . 
   opn production is known to be increased in association with mechanical loading [ 15 , 16 ] , and its deficiency significantly decreases bone fracture toughness , leading to heterogeneous mineral distribution , since opn is known to bind strongly to hydroxyapatite , and cell surface as well [ 17 , 18 ] . 
   we observed that all the devices were separated from neighboring bone by a fibrous capsule . 
   this is probably due to the material itself , since there were no differences between actuators and static controls . 
   it would be mandatory to develop and test a material with improved biocompatibility to evaluate accurately the bone material interface . 
   one other issue arises from reflecting upon the material and the coating properties and behavior . 
   the in vitro study previously developed shows evidence of adequate electric insulation for 48 hours in the experimental conditions . 
   however , the in vivo environment is not as controlled and , in addition to a longer period of time , the implantation procedure itself may induce damage on the coating . if coating damage occurs , one should consider the possible effects of the material ( pvdf and silver nitrate particles from the electrode ) and electrical current . 
   previous studies have shown cytotoxic silver nitrate activity , dependent on the concentration [ 19 , 20 ] . 
   the clinical advantages of applying electrical and electromagnetic fields in the treatment of fractures and mal - unions have been long known although the mechanisms leading to the clinical response are not well understood [ 2123 ] . 
   electric fields as low as 0.1  mv / cm promote osteoblast proliferation , with cell metabolic activity being influenced by the intensity of the electric field , and cell signaling based on the voltage - dependent calcium channels [ 24 , 25 ] . 
   electric and electromagnetic fields also exert effects on fibroblasts , enhancing proliferation , protein synthesis and influencing cell alignment , by calcium - independent mechanisms [ 2629 ] . 
   the fact that we found no significant differences in fibrous capsule thickness surrounding the devices suggests it is likely that the electric insulation remained satisfactory , but further studies would be needed to prove this . 
   however , if an electric field was being created locally , increased bone formation along the whole actuator could also be expected . 
   instead , new bone formation follows a pattern in accordance with what is known to be the areas of increased motion and deformation . 
   this study was performed in one animal only but using an adequate number of devices ( total of twelve ) . 
   the sheep was chosen as an animal model considering the bone biology similarities with the human species and the size of the devices . 
   the results are very clear in evidencing qualitative and quantitative statistically significant differences when comparing static and actuated films , but it would be necessary to enlarge the animal study and to study response with longer implantation times . 
   however , from an ethical point of view , it would be desirable to do so after developing a material other than pvdf , more biocompatible . 
   in conclusion , the present in vivo study suggests that in fact piezoelectric materials and the converse piezoelectric effect may be used to effectively stimulate bone growth at the bone implant interface , eliciting an early response . 
   piezoelectric materials have the advantages of enabling a wide range of deformation / displacement , frequencies , and regimens . 
   therefore , they are excellent candidate components of actuators for clinical purposes . however , new materials should be developed , aiming better biocompatibility .
summary: this in vivo study presents the preliminary results of the use of a novel piezoelectric actuator for orthopedic application . 
   the innovative use of the converse piezoelectric effect to mechanically stimulate bone was achieved with polyvinylidene fluoride actuators implanted in osteotomy cuts in sheep femur and tibia . 
   the biological response around the osteotomies was assessed through histology and histomorphometry in nondecalcified sections and histochemistry and immunohistochemistry in decalcified sections , namely , through masson 's trichrome , and labeling of osteopontin , proliferating cell nuclear antigen , and tartrate - resistant acid phosphatase . 
   after one - month implantation , total bone area and new bone area were significantly higher around actuators when compared to static controls . 
   bone deposition rate was also significantly higher in the mechanically stimulated areas . in these areas , osteopontin increased expression was observed . 
   the present in vivo study suggests that piezoelectric materials and the converse piezoelectric effect may be used to effectively stimulate bone growth .