technological progress has made it possible for additive manufacturing technologies to be applied successfully in the medical sciences . one of these techniques is selective laser melting ( slm ) , which consists of producing irregular shape devices by fusing titanium alloy powder , such as ti6al7nb . although the surgical procedure of implant placement is of great importance , the clinical outcome stands in osseointegration , which represents the ultimate test in morphological and functional rehabilitation of the patient . the study of bone implants osseointegration should observe the device in a 3d setting in which it is present in the living tissues , considering that the histological examination , despite its great value , is just a 2d representation of the present situation . whatever the technology used to obtain the implants , foreign body rejection stands as one of the most difficult obstacles the human body has to overcome . osseointegration is a process that can be hard to induce , which is why , in this study , hydroxyapatite and sio2-tio2 coatings were applied on the implant surface , to enhance the osseointegration process . micro - ct analysis has proved itself to be appropriate in the measurement of certain in vivo / in vitro parameters such as mineral bone density and cortical bone thickness . bone mineral density ( bmd ) is defined as the volumetric density of calcium hydroxyapatite ( caha ) in a biological tissue in terms of g / cm . the combined density of a well - defined volume which contains a mixture of both bone and soft tissue , such as a selected volume of medullar trabecular bone in a femur or tibia , is measured as  bone mineral density  , or bmd . this parameter relates to the amount of bone within a mixed bone - soft tissue region . ti6al7nb alloy ( ati allvac , monroe nc , usa ) was used to create the sample implants , by selective laser melting technology ( realizer slm 250 machine , realizer gmbh , borchen , germany ) . the samples were designed with a cylindrical screw - type shape in order to have a good penetration in the bony structure of the rabbit femur , and perfect primary stability at insertion . the dimensions and properties of the implants were : 10 mm length and 3.3 mm diameter , with a controlled porosity of 2425% , determined through archimede s method iso 273899 . the implants were divided into three groups : one group uncoated , the second group with ha coating and the third one with sio2-tio2 coating ( figure 1 ) . the coating procedure was done by immersing the implants into a hydroxyapatite and sio2-tio2 solution . after that , they were dried in a special oven at 100c for 30 minutes . the thermal treatment was performed at 600c for 30 minutes for the implants infiltrated with ha and at 400c for 60 minutes for the implants immersed in sio2-tio2 ( rotaru et al . ) . eighteen new zealand white rabbit ( oryctolagus cuniculus ) were included in this study , divided into 3 groups of 6 individuals . all rabbits were of the same age ( six months ) and approximately the same weight , kept in standard conditions of temperature , humidity , day / night cycle and they all had the same access to food and water , ad libitum , throughout the experiment . the rabbits were anesthetized with a xylazine / ketamine cocktail using a dosage of 8 mg xylazine and 80 mg ketamine per kg of body weight . the study was approved by the ethical committee of the iuliu haieganu university of medicine and pharmacy , cluj - napoca , romania ( no . 407/03.12.2014 ) . the lateral aspect of the femur was shaved and disinfected with iodine solution . the femur approach was done through the muscle bodies without tampering with the muscle fibers . a periosteal scraper was used to fully expose the antero - lateral part of the femur . two cylindrical orifices were created at the proximal area of each femur , using cylindrical 10 mm long burs with ascending dimension : 1 mm - 2 mm  2.8 mm under continuous cooling with saline solution at 800 rotations / min and a 30 nm torque . in the left femur at the upper proximal area the ti6al7nb - ha implant was placed and in the inferior orifice the ti6al7nb - sio2-tio2 implant was inserted ( figure 2 ) . in the right the rabbits were sacrificed at one , three and six months intervals after the implants were placed , by a potassium chloride intravenous injection , after inducing general anesthesia . micro - ct was performed for each specimen in order to evaluate the position of the implant , the bone apposition between the threads of the implant , and the bmd at the implant site . the measurements were done using the ct - analyser ctan dedicated soft ( figure 3 ) . the samples were scanned with a bruker skyscan 1172 microct at 80 kv and 100 ma , using a 1 mm cu+al filter , a rotation step of 0.5 and a resolution of 20 m . before scanning rehydration by overnight storage in 0.9% physiological saline , was performed . the calibration of the machine was done with 8 mm diameter rods ( calibration phantoms ) . the samples were wrapped in paper tissue , loaded into a plastic tube , thenmoistened with saline , same as the bmd phantom rods . the obtained slices were reconstructed using the nrecon software ( bruker , belgium ) and analyzed using ctan ( bruker , belgium ) . the bone surrounding the implant was assessed by selecting it as the region of interest ( figure 3 ) and measuring the bone mineral density as compared to the scanned phantom rods . after formalin immersion for 2 days the samples were decalcified in an nitric acid solution for 3 days and then prepared for histological examination . the histological slices were colored with tricrom masson , which gives two separate colors for mineralized bone and for osteoid , thus an image analysis could be performed . the histological slices were obtained with a leica microtome cutting system with 4 micrometer thickness , and examined with a leica icc50hd cam microscope . panoramic image of the slice was used by merging images done at 50 magnification ( cs6 version photomerge ) . cleaning of the artifacts ( bone marrow , muscle fiber etc . ) was then performed in order to have the best result bone ( blue)/osteoid ( red ) figure 4 . by pixel quantification for each color , statistical analysis was performed using the medcalc statistical software version 15.2.1 ( medcalc software bvba , ostend , belgium ) . after formalin immersion for 2 days the samples were decalcified in an nitric acid solution for 3 days and then prepared for histological examination . the histological slices were colored with tricrom masson , which gives two separate colors for mineralized bone and for osteoid , thus an image analysis could be performed . the histological slices were obtained with a leica microtome cutting system with 4 micrometer thickness , and examined with a leica icc50hd cam microscope . panoramic image of the slice was used by merging images done at 50 magnification ( cs6 version photomerge ) . was then performed in order to have the best result bone ( blue)/osteoid ( red ) figure 4 . by pixel quantification for each color , statistical analysis was performed using the medcalc statistical software version 15.2.1 ( medcalc software bvba , ostend , belgium ) . the rabbits had no post - operative complications and the tissue specimens required for the intended analysis could be processed at state - of - the - art standards . micro - ct scan performed before the histological analysis of the specimens , showed no implant displacement or osteolysis around the implant threads . no inflammatory reaction or fibrous tissue was noticed for the implant site at 1 , 3 and 6 months interval . table i shows the average bmd for the three implant types , and the average percentage of mineralized bone present at the implant surface , seen at the histology examination . at one month both analyses done ( bmd and histological examination ) were showing a high degree of bone mineralization at the implant site , same as the results for 6 months . at 3 months period a demineralization process for the ti6al7nb sample was observed , both when calculating the mineralized bone by histology exam and the bmd by the micro - ct exam , while the values for the coated implants are much higher . the statistical analysis of the results for the three samples shows that there was a positive correlation between the bmd measurement and the histological results , which is highly statistically significant ( r=0.750 ; p=0.02 ) . one of the best treatment options in bone defect reconstruction is the use of 3d custom made implants . selective laser melting of titanium alloys is the best option to obtain an irregular shape implant , which can be biocompatible . the bone growth for a specific implant depends on the design of the implant , surface structure and coating material . osseointegration improvement of this type of alloys , and their clinical assessment , represents a challenging task . usually the degree of osseointegration is proven by the histology exam that shows the bone cells proliferation , direct bone - implant contact and the mineralization degree at the implant surface . micro - ct images allow us to quantify volumetric bone - implant contact , and bone mineral density ( bmd ) of a specific region that are of help in the osseointegration evaluation . usually , the titanium alloys and the different coatings used to enhance their osseointegration , were studied for a period of 2 , 4 , 6 and 12 weeks after implantation implantation , but none studied the behavior of the alloys for more that 18 weeks , thus we considerered that a longer period of time ( six months ) , would be more appropriate for integration assessment . it is a known fact that mineralization and demineralization balance require time to be achieved . this can explain why the demineralization process was continuous , for the ti6al7nb sample with a peak at about 3 months , followed by remineralization of the site within the following months , reaching a percentage of mineralized bone for 6 months comparable with the one at 1 month ( 94.67% vs. 93.83% ) . the remineralization process happened in the absence of any tempering force , the implants having a passive condition inside the femoral bone , without any stress applied directly . the niobium component of the studied alloy has the potential to stabilize the ti6al7nb samples in time , due to the niobium oxides ( nb2o5 ) that are formed at the surface of the implants . the mineralization process that occurs after 3 months can happen due to the surface oxide layer , which is less soluble and biocompatible , that forms in time . the micro - ct data , same as the histology examination , showed the same results in terms of bmd evaluation , proving the demineralization process that happens for the ti6al7nb samples . usually an implant is considered to be osseointegrated when bone develops directly on the implant surface , without any fibrotic tissue grown at the interface . clinically there is no mobility between the bone and the implant , under regular loading conditions , after an appropriate healing period . in our study we have not seen any fibrotic tissue at the bone - implant interface , also no inflammation was observed . micro - ct analysis is useful in understanding the circumstances that lead to a poor bone activity response , such as microcracks , plastic deformations or manufacturing deficiencies that can lead to failure ; in our study none of the above was seen . it has been clinically validated that titanium and its alloys present a superior biocompatibility and fewer foreign body reactions in comparison to other conventional materials . the bioinert nature of titanium is its principal quality which consigns itself biocompatibility properties , stating that it does not induce a positive reaction in the tissues , but more likely it conditions the absence of a negative reaction , even in the case of demineralization showed for the ti6al7nb implant , no inflammation or adverse reaction were seen at the histological analysis . implant surface roughness is also responsible for the proportion of osteoblasts proliferation , demonstrating its growth in the case of silicium dioxide sandblasted implants , that can explain the higher value of mineralized bone for the ti6al7nb - sio2tio2 than for the ti6al7nb implants . coating the implants with hydroxyapatite enhances their viability in time , and influences positively the cellular and protein adhesion , resulting in improved osseointegration . similar with our work , alzubaydi   showed in his study that the ha coated ti-6al-7nb screws after 18 weeks of the implantation seem to be well - tolerated by the bone since no adverse tissue reaction was evident . however , there was a faster reaction of bone towards the coated implants compared to the uncoated one . in another study , had observed that histologic evaluations of orthopedic implants revealed mineralization of interface bone directly onto the ha - coated implant surface , with no fibrous tissue layer interposed between the bone and ha visible at the light microscopic level . in vitro study done by brie ic on ti6al7nb with ha and sio2 coatings found that the behavior of osteoblasts depends on the type of implant and culture conditions . ti - sio2 scaffolds sustain osteoblast adhesion and promote differentiation with increased collagen and non - collagen protein production . ti - ha implants have a lower ability to induce cell adhesion and proliferation but an increased capacity to induce early mineralization . both types of infiltrates have their advantages and limitations , which can be exploited depending on local conditions of bone lesions that have to be repaired . the early mineralization process , present at the ti - ha coating could explain our results of high mineralized bone for the ti6al7nb -ha implants . the use of coated implants is of help whenever local or general condition could temper with the normal osseointegration process . for a good long - term outcome , the ti6al7nb implant need a period of stabilization of about 6 months , any stress applied in this period of time , especially at 3 months could lead to the onset of complications , even implant displacement . the ha coating represents the best option for the ti6al7nb alloy , that beside the osteoinductive and osteoconducting property ( rotaru el al . )   can induce early mineralization , that supports implant osseointegration . the histology examination of titanium based implants needs a long time period ( weeks ) in order to have a result , meanwhile the micro - ct examination can produce reliable results in a matter of hours , thus the micro - ct examination is easier and faster to be performed . further studies that will involve the loading of the implants or force applied to the implant site for the 6 months period of osseointegration should be taken into consideration .