Evaluating the osseointegration of nanostructured titanium implants in animal models : Current experimental methods and perspectives ( Review )

The aim of this paper is to review the experimental methods currently being used to evaluate the osseointegration of nanostructured titanium implants using animal models. The material modifications are linked to the biocompatibility of various types of oral implants, such as laser-treated, acid-etched, plasma-coated, and sand-blasted surface modifications. The types of implants are reviewed according to their implantation site (endoosseous, subperiosteal, and transosseous implants). The animal species and target bones used in experimental implantology are carefully compared in terms of the ratio of compact to spongy bone. The surgical technique in animal experiments is briefly described, and all phases of the histological evaluation of osseointegration are described in detail, including harvesting tissue samples, processing undemineralized ground sections, and qualitative and quantitative histological assessment of the bone-implant interface. The results of histological staining methods used in implantology are illustrated and compared. A standardized and reproducible technique for stereological quantification of bone-implant contact is proposed and demonstrated. In conclusion, histological evaluation of the experimental osseointegration of dental implants requires careful selection of the experimental animals, bones, and implantation sites. It is also advisable to use larger animal models and older animals with a slower growth rate rather than small or growing experimental animals. Bones with a similar ratio of compact to spongy bone, such as the human maxilla and mandible, are preferred. A number of practical recommendations for the experimental procedures, harvesting of samples, tissue processing, and quantitative histological evaluations are provided.


I. INTRODUCTION
The history of the implantology dates back to Egyptian times, more than 1350 years before the famous, recently deceased Per-Ingvar Branemark developed a threaded implant design made from pure titanium that raised the popularity of implants to a new level. 1,2He observed bone growing in such close proximity to the titanium implant that it had effectively adhered to the metal.Based on these observations, he and his colleagues later defined the concept of osseointegration as a direct and stable anchorage of an implant through the formation of bony tissue without the growth of fibrous tissue at the bone-implant interface. 3o be considered successful, an osseointegrated oral implant has to meet certain criteria in terms of function, tissue physiology, and user satisfaction. 4These requirements include immobility in any direction; average radiographic marginal bone losses of less than 1.5 mm during the first year of function and less than 0.2 mm annually thereafter; no radiographic evidence of peri-implant radiolucency; and a) Author to whom correspondence should be addressed; electronic mail: vaclav.babuska@lfp.cuni.cz6][7] Aesthetic requirements and patient and dentist satisfaction with the implant prosthesis should also be considered criteria for success. 8or osseointegration of dental implants, the correct surface modifications, shape, and materials are critical. 9In this review, we will try to generalize our own experiences with testing and evaluating nanostructured titanium dental implants.

II. MATERIALS AND TREATMENT OF DENTAL IMPLANTS
1][12][13][14][15][16][17][18][19] While commercially pure titanium has sufficient biocompatibility but relatively poor strength, titanium alloys have superior strength but contain potentially toxic or allergenic ingredients. 20,21ecause of their positive properties, such as the easy formation of a stable oxide, high corrosion resistance, and fast passivation, titanium and its alloys were among the first metals to be used for the manufacture of medical implants. 22he long-term stability of titanium implants depends on establishing direct bone-implant contact (BIC) without a fibrous connective tissue interface.Loosening or failure of the implant can be caused by inflammation and bone resorption induced by wear debris in the form of titanium particles from the implant that enter into the surrounding tissues. 23herefore, it is essential to improve the biocompatibility and wear resistance of a titanium implant for its successful longterm survival. 24o improve the biocompatibility, wear resistance, and osseointegration of a titanium implant for its successful long-term survival, intensive development in the processing of bulk, fully dense nanostructured metals and alloys began.The fabrication of such materials based on severe plastic deformation (SPD) methods appears very interesting and useful.Nanostructured materials processed using SPD methods were first developed and investigated by Valiev et al. [25][26][27] A promising and efficient method for the production of bulk ultrafine grained or nanostructured material is equal channel angular (ECA) pressing.The ECA pressing method involves the deformation of massive billets via pure shear followed by another possible deformation.The method was further developed and applied as an SPD method for processing structures with submicron and nanometric grain sizes.Nanostructured titanium produced in this way has both excellent biocompatibility and extraordinary mechanical properties. 20,28

B. Types of oral implants
Several types of implants are available, and implant selection for a particular patient depends on a number of factors.The size and condition of the patient's natural jawbone is probably the main factor.Jawbones can be wide and deep or narrow and shallow, with many variations in between.In general, oral implants can be categorized into three main groups. 21

Endosseous implants
Endosseous implants [Fig.1(a)] are surgically inserted into the jawbone.They are molded or shaped to fit in a cavity in the jaw rather than sit on top of the jaw.These implants are currently the most frequently used type.Based on their shape, function, surgical placement, and surface treatment, they could be further divided into several subcategories.
Root form implants, which are also called cylindrical or screw-type implants, resemble the shape of the natural root of a tooth and have a surface area that is designed to promote good attachment to the bone.They are the most widely used design and most popular type of dental implant.A root form implant is formed as a self-tapping screw, which gives it good surface area for maximum fusion to living bone.These implants used to be called endosseous or endosteal implants, meaning they are placed in the bone.The bone grows in and around the implant, creating a strong structural support.This bond can be even stronger than the original teeth.However, this type of implant can only be used if there is sufficient width and depth to the jawbone.It may be necessary to graft additional bone to the jawbone before dental implants can be considered.
Plate form implants, also known as blade form implants, are flat rectangles that take the form of a long, narrow strip of titanium inserted between the jawbone and the gum.They will fuse with the jawbone, providing a foundation for a number of new prosthetic crowns.They are usually used when there is insufficient living bone structure because either the jawbone is too narrow or not deep enough or certain vital anatomical structures prevent the placement of conventional implants.
Ramus frame implants are another type of endosseous implants.They are designed only for a toothless lower jaw and are surgically inserted into the jaw bone in three areas, including the left and right distal area of the jaw (the approximate area of the wisdom teeth) and the chin area in the front of the mouth.The part of the implant that is visible in the mouth after implantation looks similar to a subperiosteal implant.This type of implant is usually indicated for a severely resorbed, toothless lower jaw bone that does not offer enough bone height to accommodate a root form implant as the anchoring device or when the jaws are resorbed to a point where even a subperiosteal implant will not suffice.Once integrated, ramus frame implants can stabilize and protect weak jaws and help to prevent them from fracturing.

Subperiosteal implants
Subperiosteal implants [Fig.1(b)] are designed to sit on top of the bone, but under the gums, fusion takes place between the jawbone and the subperiosteal implant.They are an option in cases of advanced bone loss when the condition of the jawbone is such that an insert is not possible and a bone graft is not feasible.

Transosseous implants
Transosseous implants [Fig.1(c)] are designed for people who have very little bone in their lower jaw and no lower teeth.These implants are no longer commonly used because they require an extraoral surgical approach for placement that includes extensive surgery, general anesthesia, and hospitalization.They are used only in the mandible and involve the insertion of two metal rods from below the chin through the chin bone until they are exposed inside the mouth.The rods that can be seen inside the mouth are used to attach a denture.
Today, most clinicians prefer to use bone grafts and one of the other endosseous implant methods described above.Subperiosteal and transosseous implants are more or less historically obsolete and are listed only for clarity.

C. Characterization of surfaces
The important reactions of implants occur at the interface between the material and bone, and their surface properties determine their suitability. 29Surface modification plays a very important role because of the enhanced properties, which include cytocompatibility, osseoinduction, bone ingrowth, and antibacterial characteristics. 30,31In addition, the coating may also prevent release of metal ions from the implant. 32Such suitable surfaces are expected to have beneficial effects on the implant's long-term success.There are different methods of titanium-implant surface treatment.

Laser treatment
The laser beam radiates electromagnetic energy that interacts with the titanium, taking it from a solid state to a plasma state.The extremely concentrated energy pulses of the laser allow for microfabrication of the implant surface without any dangerous effects, such as thermal changes of material properties that can induce microfractures or alteration of the metal structure.This controlled microablation is obtained using a low power setting.An important goal of laser treatment of an implant surface is to produce a surface with thousands of hemispheric pores for bone apposition. 33,34

Acid etching
Acid etching of titanium is of particular interest because it creates a microtextured surface (a fine rough surface with micropits of 1-3 lm and larger pits of approximately 6-10 lm) that appears to enhance early endosseous integration and the stability of the implant. 35This may be related to the changes in surface roughness and chemical composition.Studies by Wennerberg et al. 36,37 demonstrated an optimal blasting particles of 75 lm, which made implant more resistant to torque and gave it greater bone-to-metal contact than did small (25 lm) or coarse (250 lm) particles.The optimal surface had an average height deviation of approximately 1.5 lm, resulting in a surface enlargement of 50%.

Plasma-coated spray
A coating produces a rough implant surface that significantly improves the anchorage of the implant in bone.This process can be used for both metal and ceramic implants.Plasma coating works by blowing an inert gas through an intense electric arc.Down the arc, the coating material is introduced in the form of an extremely hot gas.The inert gas is broken into ions and electrons in the arc.This state is known as plasma.The titanium hydride (coating material) decomposes in the gas stream and forms droplets of molten metal that are projected onto the implant surface to build up a coating.The layer is typically 20-30 lm thick with a roughness of approximately 15 lm.Gases in titanium harden the metal, which is an advantageous enhancement for the surface of an implant. 38The bond strength between the porous plasma layer and the substrate is limited, but excessive treatment, such as exposure to an ultrasound source, is needed to cause this bond to fail.Titanium implants with this type of coating have an average BIC in cancellous bone of nearly 40%, which is significantly higher than for smoothly polished or finely structured titanium implants, which have values of slightly over 20%. 39Plasma sprayed coatings using antibacterial agents, such as ionic silver, can be used as an alternative to classical antibiotics due to its broad activity against Gram-positive and Gram-negative bacterial strains. 40

Sand blasting
Sand blasting roughens the surface of the implant and achieves both microretentive topography and increased surface area. 41,42A sand-blasting treatment consists of the mechanical abrasion of surfaces using particles shot against the implant.The treatment produces a surface with a roughness that depends on the size, shape, and kinetic energy of the particles.However, this increased roughness may reduce the endurance properties of metals. 43Healing around titanium sand-blasted implants is similar to that observed around plasma-spray surfaces.

D. Biocompatibility and osseointegration
Nanostructuring a material changes its biological properties compared with material of the same chemical composition, but the mechanism of this phenomenon has not yet been clarified. 44The first evidence of such an effect was provided by Webster et al. in 1999, 45 who found significantly increased osteoblast adhesion and bone formation on a nanostructured titanium surface compared with conventional titanium.][51][52] The implantation of metal materials often induces an adverse response in the bone, such as implant rejection, reaction to wear debris, or fibrosis of the adjacent tissues.Similarly, leaking contact between the implant and bone, which is filled with biological active body fluids, may cause metal surface corrosion. 53In addition, an important component of long-term integrity success is improving the osseointegration of an implant with its surrounding natural bone tissue. 24,54One factor that can improve and accelerate the osseointegration of an implant is surface nanotopography. 55,56

III. METHODS OF IMPLANTATION A. Animal species and bones used in experimental dental implantology
Animals of various species, size, and ages are used for the experimental evaluation of osseointegration.No species fulfils all requirements of an ideal model to determine an optimal interface between bone and dental implants.However, dog, sheep, goat, pig, and rabbit models are commonly used to evaluate bone-implant interactions. 57,58he dog seems to be more promising as animal model for testing bone implant materials, but there are increasing ethical issues relating to the use of dogs in medical research due to their status as companion animals. 59ith regard to bone anatomy, morphology, healing, and remodeling, the pig demonstrates a good likeness to human bone.However, challenges may be encountered in relation to its large size and difficulty in handling.][65][66] The rabbit is the most commonly used animal model.][69][70][71][72] The most common bones for implantation are a miniature pig maxilla [63][64][65][66] and a rabbit femur. 67,69,70,72The most frequently used experimental animals, their age/weight (according to the primary literature), the bones selected for implantation, and healing time are shown in Table I.

B. Implanting into compact and spongy bone
Selecting the target bone is an important step of the study design.For example, implants intended to be used in human maxilla may be tested in porcine maxilla. 64The maxilla has a variable layer of compact cortical bone on the surface, below which mostly spongy bone is found.However, both porcine and human mandibles have a substantially greater proportion of very thick compact bone.According to Katranji et al., 61 the average buccal cortical thicknesses are 1.69 mm (molar region), 1.43 mm (premolar region), and This will inevitably result in lower BIC parameter values.To prevent any significant bias, we suggest that studies on implant osseointegration should be accompanied by details on the ratio between compact and spongy bone at the site of implantation.Unfortunately, these values are rarely found in the literature.The cortical thickness of rabbit femoral proximal diaphysis is 9.5 6 0.4 mm. 74

C. ISO standards in implant testing
The results of an experimental evaluation of osseointegration should be compatible with ISO Standards (ISO 10993-6 Biological evaluation of medical devices-Part 6: Tests for local effects after implantation), and several recommendations should be considered (ISO 10993-6, 2014) during the design of the experiment, as follows: (1) the diameter of the dental implant should be between 2 and 4.5 mm; (2) when using rabbit as an animal model, the maximum number of implants per animal is 6; and (3) when using a pig as a model, the number of implants per animal must not exceed 12.

D. In vivo evaluation
In an in vivo evaluation, animals are used to test the effect of various biological entities as well as to observe the overall effect of an experiment on a living subject.Under general anesthesia and standard disinfection and aseptic conditions, skin incision and soft tissue preparation will be performed, and a mucoperiosteal flap will be incised and raised to expose the entire extent of the bone where the implants are to be placed.After bone exposure and surgical preparation of the bone, the implant will be inserted [Fig.2(a)].It is essential to use copious amounts of sterile saline irrigation and sharp drills to prevent heating the bone above 47 C, which could result in bone cell death and osseointegration failure.The insertion technique depends largely on the implant system being used.Wounds will be closed primarily layer by layer from deep to superficial tissue with resorbable suture material after thorough cleaning and irrigation.
Following the surgical insertion of an implant in the animal and postoperative care, the animal is allowed to mature and will then be sacrificed.During the period of healing and osseointegration, it is possible to perform radiographic examinations, such as x-ray, CT, or densitometry analysis, to track the healing progress and determine the exact position of the implants [Fig.2(b)].After sacrificing the animal and separating the bone containing the implants from the soft tissue, other radiographic images will be taken for analysis [Fig.2(c)].
In general, handling wild animals during experiments is difficult, and complications may occur at any time.At the time of surgery, the animal can be lost due to the introduction of intratracheal intubation during general anesthesia.Infection of the wound, which is a common postoperative complication, may develop because maintaining animals in a hygienic environment is extremely challenging [Figs.At the end of the experiment, tissue samples are harvested from the animals and processed.The anatomical position and orientation of implants in bone should be verified using x-rays.This is also very helpful for subsequent cutting of bones into smaller tissue blocks of bone with implants.A suitable tissue-block size for histological processing depends on the size of the implants or the size of formed defects during the evaluation of degradable osteoinductive biomaterial.The tissue block should be as small as possible for better penetration of fixation fluid.However, at least two millimetres of bone tissue surrounding the implant should be preserved to allow for a reliable evaluation of osseointegration.Therefore, tissue blocks exceeding 2 Â 2 Â 2 cm are cut using either a diamond band saw [Fig.4(a)] or a special low speed diamond wheel saw.The orientation of the tissue blocks relative to the original anatomical positions should be marked and carefully maintained during all processing steps.Further processing might differ in samples of surrounding bone without implants (in this case, the samples may be decalcified), or in samples with implants (undemineralized samples).

B. Undemineralized ground sections
There are methods for processing hard tissue, and for the evaluation of osseointegration of titanium implants, a resin and cutting-grinding system is suitable.This method provides information on the formation and mineralization of a new bone matrix adjacent to the implant.

Fixation and dehydration
The time of fixation in a 10% formaldehyde solution depends on the sample thickness.Tissue blocks 2-4 mm thick require approximately 12 h for penetration. 72,75lightly thicker samples require approximately 24 h, 71 but some researchers recommend up to 5 days for 16 mm thick samples. 63An important factor affecting the fixation time is the ratio of spongy bone (with relatively easy fixation) to compact bone (which requires a longer fixation time).The samples are then dehydrated in ascending grades of alcohol of 70%, 80%, 96%, and 100% (two days required per grade of alcohol).After the 100% alcohol treatment, the samples are transferred to a 1:1 mixture of acetone-alcohol for approximately 12 h for better permeabilization of the tissue, and they are then put back into 100% alcohol.

Embedding
Methacrylate-based resins are currently the most commonly used embedding media. 68,71,72,75The time required for dehydration and saturation by resin can be shortened using an automatic embedding device with an agitation and vacuum system. 75The samples are put in methyl methacrylate (MMA) without an initiator of polymerization for approximately four days.The samples are then put in a mold with resin, and the polymerization of resin is initiated either by blue/ultraviolet light 63,67,72,76 or peroxide. 66,77A plastic block is formed [Fig.4(b)].Another resin frequently used for embedding is epoxy resin (e.g., Epon).When using epoxy-based embedding resins, after dehydration, the samples are put into 100% propylene oxide twice for 15 min, followed by a 1:1 mixture of propylene oxide and Epon overnight and then into clean Epon for two days at 37 C. Verifying the position of an implant in a plastic block with an x-ray might be necessary in nontransparent resins.

Sectioning
The blocks of samples are sectioned along the long axis in the middle of the implants to form two blocks.The sample can be cut with a diamond saw band (Exact Apparatebau, Norderstedt, Germany), 68,72 a diamond disc [Fig.4(c)] (Struers, Ballerup, Hovedstaden, Denmark), 71 or a special saw microtome, 69 The cutting area is ground by P1200 grit sandpaper until the all tissue segments for observation are exposed to the surface.After grinding, the cutting area is polished with P4000 grit sandpaper.A clean slide is glued to the polished side.The second incision is made approximately 100-300 lm from and parallel to the slide.

Grinding and polishing
The slide with the section is ground using a sequence of abrasive papers (P320, P1200, and P2500 grit) and polished using P4000 grit sandpaper under water irrigation to a final thickness of approximately 50-30 lm.For the final polishing of the surface paper coated with a fine cloth and diamond paste with grain roughness 3 lm are used.The papers for grinding and polishing are on a rotating desk, and the samples are pressed toward the desk [Fig.4(d)].

Staining
The most commonly used staining method is toluidine blue 67,71,[76][77][78][79][80] [Fig.5(a)].Toluidine blue can easily differentiate unmineralized osteoid from fully mineralized bone tissue and provides sufficient preservation of morphological details.Basic fuchsine can be used alone 81 [Fig.5(c)] for staining microporous bone or can be combined with toluidine blue.Other staining methods for MMA-embedded samples are shown in Table II.
MMA-embedded sections can also be stained by immunohistochemistry as described in decalcified sections.The sections have to undergo dissolution of the embedding resin using 2-methoxy ethyl acetate for 24 h and are then cleaned with ethanol, rehydrated using a descending ethanol series, and transferred into distilled water. 80,86MMA-embedded nondecalcified bone yields a stronger immunostaining reaction compared to decalcified bone embedded in paraffin.There is also better preservation of the trabecular bone morphology. 87s demonstrated in this section, the methods used for histological processing of undemineralized sections are rather general and not entirely specific for the nanostructured implants.Cutting, grinding, polishing, and histological staining of hard tissues, such as bone and teeth, are widely used in general for evaluating metal implants, 88,89 but also in other research fields, such as osteology and archeology. 90espite playing an important biological and physiological role, the dimensions fine nanostructural and surface modifications of the nanoimplants are beyond the resolution limits used in histology and light microscopy.However, histology is the level most decisive for evaluating the osseointegration.The advantage of using the same processing in nanoimplants as in conventional implants without nanostructural modifications is that the results of both types of implant can be directly compared in experiments.

C. Demineralized (decalcified) histological sections
Alternatively, bone samples without metal implants, such as biopsies of bone tissue or bone from the regions between the implantation sites, may undergo demineralization.This technique may be beneficial for the evaluation of degradable osteoinductive biomaterials because it demonstrates organic components of bone, such as osteocalcin 82,91,92 [Figs.5(b) and 5(e)]; osteopontin; and type I collagen [Fig.5(d)].After using a decalcification solution, [91][92][93] the samples may be processed using a routine histological paraffin-embedding technique.][93] Decalcification time depends on the sample size.Because the demineralization in EDTA requires approximately 8-10 weeks, alternative solutions of acids may be used to accelerate the process. 94However, some of the more aggressive decalcifying solutions, such as formic acid, may damage the structure of the tissue, thereby limiting its further evaluation. 95Moreover, some biomaterials are harder than bone and have to remain in a decalcification solution for a longer period of time.This can affect the morphology of bone, its dyeability, and the next evaluation.Unfortunately, osteoinductive materials, such as hydroxyapatite granules, may affect the cutting of samples because the material can be harder than bone and needs to remain in the decalcification solution for a longer period of time.Some hard particles of biomaterial can fall out during sectioning.

Qualitative evaluation
Using the staining methods shown in Table II, qualitative findings, such as the formation of unmineralized osteoid and mineralized tissue, loose connective tissue surrounding the implant surface, or the formation of granuloma with excessive vascularization but poor mechanical retention, can be obtained.

Histological quantification of bone-implant contact
Quantitative histological techniques are frequently used to demonstrate which part of the bone directly binds to fully differentiated bone tissue.The most commonly used quantitative parameter for the evaluation of osseointegration 96,97 is the BIC, which is expressed as follows: where bone contact (BC) refers to the length of direct contact between the implant profile and bone tissue, and interface length (IL) indicates the total length of the implant surface projected to the section plane.The BIC is a continuous variable expressed as a dimensionless ratio of h0;1i, where BIC ¼ 0 denotes no direct contact between implant and bone, and BIC ¼ 1 theoretically means that the entire implant is fully embedded within the bone tissue.Alternatively, BIC is often expressed as a percentage value.When quantifying objects of macroscopic size (such as the implant-bone interface) on a microscopic scale, an appropriate and unbiased sampling of the microscopic fields of view from the entire object has to be performed.Briefly, all parts of the specimen should be given the same chance to be sampled with the microscopic fields of view and included in the quantification.Selecting a lower objective magnification results in a larger field of view but a poor differentiation of the bone-implant interface details.Therefore, the magnification should be adjusted according to the amount of detail in a particular study so that an unambiguous identification of that detail is possible.Either all the adjacent microscopic fields of view are included [Fig.6(a)] or for larger implants, a systematic uniform sampling 98 of the objective image fields is performed by selecting, for example, every second field of view [Fig.6(b)].
Delineating the BC and IL lines could be done manually. 96,97Alternatively, stereological grids 99 and principles of stochastic geometry may be used for this task as follows: (1) a two-dimensional system of lines with known geometrical properties is randomly positioned over the series of calibrated micrographs sampled from the bone-implant interface [Fig.6(b)].The line system consisting of perpendicular equidistant lines with randomized orientation is isotropic, i.e., it has the same geometrical properties in all directions and does not favor any direction (for alternative stereological line probes, see Mouton et al. 99 ).( 2) The number of intersections between the line system and the profile of the implant surface is counted manually.According to the modified stereological Buffon's method, 99 the total number of intersections {orange points in [Figs.6(c) and 6(d)]} is directly proportional to the IL, i.e., implants with more structured and greater surface will have more intersections with the testing grid.(3) In contrast, the number of intersections hitting the profile of the implant surface attached to the bone is counted.Again, the total number of these intersections {light blue points in [Figs.6(c) and 6(d)]} is proportional to the BC.After summing all BC and IL points from all microscopic image fields from the same specimen, an average BIC value may be calculated as the BC/IL ratio.The total number of points hitting the profile of the implant surface should be a minimum of 150. 98Based on our experience, this stereological method is highly reproducible, gives consistent results, does not require calibration, is easy to learn, and is robust to any possible histological artifacts.
Next to stereology, also image processing techniques may be used for quantification of osseointegration.These are mainly based on automatic image segmentation of the boneimplant interface, which has usually sufficient contrast to be traced and thresholded automatically.Various general or specialized software packages are available for this purpose, such as NIS Elements D3.2 (Nikon Instruments, Inc., Melville, NY, USA), 100 Bio Quant Osteo 7.10.10(BIOQUANT Image Analysis Corporation, Nashville, TN, USA), 101 Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA), 102 or LAS-4.1.0(Version-Image processing and analysis system, Leica-Wetzlar, GmbH, Germany). 103The advantage of these automatic or semiautomatic image processing methods is that high throughput batch processing of multiple photomicrographs can be done at once.Once established automatic image processing can be coupled with whole slide imaging, thus evaluating large images with detailed resolution acquired using histological scanners.However, automatic image processing requires highly uniform staining of the slides and excellent technical quality of the ground sections, because these methods may be more sensitive to any processing artifacts than interactive stereological methods.

Histological quantification of other bone characteristics
Vascularization of the bone adjacent to the implant and the numerical density of osteocyte lacunae within the bone may be assessed using the stereological point-counting technique reported in the human mandible by Tonar et al. 81 The lacunar microporosities (density of osteocytes) may be statistically independent from the vascular microporosities (bone FIG. 6. Stereological quantification of the bone-implant contact.(a) Sampling of image fields (red squares) using a low microscope magnification; all adjacent fields showing the implant surface (black) and bone (purple) are taken, (b) alternatively, every second image filed is sampled; especially used when higher magnification with small field of view is needed, (c) stereological grid randomly positioned over the micrograph; intersections between the test lines and the implant surface (orange points) and intersections between the implant-bone interface and test lines (light blue points), and (d) the same way evaluation, but using a higher magnification; details of BIC and the testing grid is shown in embedded rectangle (the bone has a blue color).vascularization), and therefore, both parameters should be regarded as complementary characteristics of bone quality.Alternatively, the amount of bone tissue formed within a certain time interval in the region used for implantation may be quantified using the tetracycline labeling test. 104,105riefly, a tetracycline antibiotic is administered at two time points.Due to its relatively short biological half-time but high affinity to the bone tissue, tetracycline incorporates into bone tissue formed within a specific period, i.e., 1-2 days after administration.Using the autofluorescence of the tetracycline (for details on excitation and emission wavelengths of different types of tetracyclines, see Pautke et al. 106 ), the bone formed between the two administrations of tetracycline can be visualized using fluorescence microscopy of the ground sections [Fig.5(f)].The width of the bone region between the two lines marked with tetracycline is proportional to the bone that forms between the two administrations of tetracycline.

V. REMARKS AND RECOMMENDATIONS
Based on the results of our experiments, we can conclude that the surface of the implant has a very important role in osseointegration and that mechanically added surface roughness significantly increases the contact area between the implant surface and the peri-implant bone.
It is important to note that there are many physiological and pathological influences that can affect the results, and it is vital to repeat the method several times to increase the accuracy of the results.
Implant technology is a rapidly progressing science with frequent production of new designs, materials, shapes, and surface treatments.Therefore, based on the success of our method, we could in the future utilize this method to analyze these new modifications.The current method that was used made the evaluation of osseointegration more efficient, more accurate, and faster.An important area of research involves the creation of a "biomimetic surface," or a surface that closely resembles that of real tissue, which would assist in the stimulation and proliferation of bone tissue because it stimulates the regularity and dimensions of the bone tissue itself without altering the properties of titanium.
To perform a similar experiment, older animals or miniature pigs that have a slower rate of growth are recommended.A histological evaluation of experimental osseointegration of nanostructured titanium implants requires a careful choice of experimental animals, bones, and implantation sites.Bones with a similar ratio of compact to spongy bone, such as the human maxilla and mandible, are preferred.Quantification of the BIC contact may be performed using an unbiased stereological method, but an appropriate and fair sampling of the microscopic image fields is an absolute prerequisite of the quantification procedure.
In conclusion, this paper reviews current methods used in the experimental evaluation of the osseointegration of nanostructured titanium implants.These methods can be used for both dental and orthopedic implants.Practical recommendations for the experiment, harvesting of samples, tissue processing, and quantitative histological evaluation are provided.
3(a)-3(c)].Based on our experience, the utilization of young animals for experiments may bring another complication.Radiographic images have revealed the occurrence of opposition of bone above the implant head due to osteoblastic activity, and the largest part of the implant shifted inside the bone marrow cavity due to the osteoclastic activity of osteoclasts.This led to resorption of bone at the distal part of implant because of the rapid growth of the animal.

FIG. 2 .
FIG. 2. (a) Implants inserted into the pig mandible, (b) x-ray of rabbit femur 2 weeks after surgery, and (c) x-ray of rabbit femur 12 weeks after surgery (shortly after the animal has been euthanized).

FIG. 5 .
FIG. 5.Staining methods for bone samples evaluation.(a) Sky blue osteoid on the surface of bone trabeculae adjacent to titanium implant (toluidine blue stain), (b) brown osteocalcin in osteoblasts and extracellular matrix on decalcified section (antiosteocalcin antibody OCG3 visualized by diaminobenzidine), (c) pink blood vessels and osteocyte lacunes in bone (basic fuchsine stain), (d) red or yellow type I collagen in extracellular matrix and green type III collagen (picrosirius red stain, polarized light), (e) details of osteocalcin staining, and (f) tetracycline incorporated into newly formed bone visualized as a strong green fluorescence signal; the distance between the two outlines of fluorescence is proportional to the bone newly built between the two administrations of tetracycline (double tetracycline labeling, confocal fluorescence microscope).Scale bars 50 lm (a)-(e) and 100 lm (f).

TABLE I .
62imals, bones, and healing time in recent studies on osseointegration of implants.As some of the studies do not provide details on animal age and size consistently, both values are included in the second column.mm(anteriorregion) in the edentulous maxilla and 2.06 mm (molar region), 1.78 mm (premolar region), and 1.36 mm (anterior region) in the edentulous mandible.Changes in the relative composition between trabecular and cortical bone in atrophic edentulous mandibles are due to loss of height and total area, mainly at the expense of trabecular bone, but not to changes in the cortical bone.62Due to a different spatial organization of the compact and spongy bone, the choice of implant location might result in various values of the BIC parameter, which is the most widely used quantitative parameter for evaluating osseointegration (see below for details on BIC quantification).Moreover, different proximal versus distal segments of long bones, such as the femur, tibia, and humerus (TableI), may vary in the proportions of compact and spongy bones.Again, variable proportions of compact and spongy bone tissue within the same anatomical bone could represent a possible source of bias.While implants (or their parts) fully embedded within the compact bone could theoretically reach maximum values of BIC, parts of implants surrounded by spongy tissue will always be partially touching other types of tissue between the bone trabeculae, such as connective tissue or bone marrow.

TABLE II .
Staining methods and their results in ground sections.