Evaluation of bone ingrowth into porous titanium implant : histomorphometric analysis in rabbits

A porous material for bone ingrowth with adequate pore structure and appropriate mechanical properties has long been sought as the ideal bone-implant interface. This study aimed to assess in vivo the influence of three types of porous titanium implant on the new bone ingrowth. The implants were produced by means of a powder metallurgy technique with different porosities and pore sizes: Group 1 = 30% and 180 μm; Group 2 = 30% and 300 μm; and Group 3 = 40% and 180 μm. Six rabbits received one implant of each type in the right and left tibiae and were sacrificed 8 weeks after surgery for histological and histomorphometric analyses. Histological analysis confirmed new bone in contact with the implant, formed in direction of pores. Histomorphometric evaluation demonstrated that the new bone formation was statistically significantly lower in the group G1 than in group G3, (P = 0.023). Based on these results, increased porosity and pore size were concluded to have a positive effect on the amount of bone ingrowth. Descriptors: Titanium; Osseointegration; Porosity; Topography, Medical. Introduction Titanium (Ti) and its alloys are the most frequently used materials for endosseous implants in dentistry and orthopedics due to their high degree of biocompatibility and good mechanical properties.1 Successful integration of an implant is generally accepted to rely on its surface characteristics such as chemical composition, morphology, and energy.2 Surface morphology is an important factor determining the long-term implant stability, especially when bone quality is poor.3 Porous metals have been exploited for several decades to increase friction force between the implant and bone and promote the initial and long-term stability through bone ingrowth.3,4,5,6 The advantage of porous materials is their ability to provide biological anchorage for the surrounding bone tissue via the ingrowth of mineralized tissue into the pore spaces.7 The architecture of a porous implant has been shown to substantially affect the bone ingrowth into pore space.5,8,9 Optimal pore size for bone ingrowth ranges from 100 μm to 600 μm, and the pores must be interconnected to maintain the vascular system required for continuing bone development.8,9 Although great progress has been made in the manufacture of porous Evaluation of bone ingrowth into porous titanium implant: histomorphometric analysis in rabbits Braz Oral Res. 2010 Oct-Dec;24(4):399-405 400 structures with various available fabrication processes, some limitations continue to exist.10 Several pore-generation fabrication methods have been proposed, and the technique chosen is usually dependent on the properties of the material being used.11 Current fabrication methods include the use of TiH2 suspension for reactive sintering12 or organic additives for powder metallurgy (PM),4,9,13,14,15 multiple coating,16 the tape casting17 and environmental electro-discharge sintering process.18 Based on the potential for a porous surface to enhance implant-bone contact, the purpose of this study was to evaluate the influence of pore size and porosity on bone neoformation in the use of titanium implants produced by PM technique. Material and methods Porous-surface titanium implant fabrication Pure Ti powder developed in the General Command of Aerospace Technology (CTA), Institute of Air and Space (IAE), Division of Materials (AMR), Brazil (purity ≥  99.5%, particle size ≤  8 μm), was used as starting material in the present study. Urea particles (Synth, Diadema, SP, Brazil) with particle sizes from 177 to 350 μm were used as spacer material. The selection of particle sizes was obtained by a screening technique. Three types of interconnected porous-surface titanium scaffolds were produced by PM. Initially, Ti powder and urea particles were mixed. The mixture was uniaxially pressed at 100 MPa into a stainless steel mold and then pressed isostatically at 200 MPa. Next, the green specimens were heat treated at 200°C for 2 h in air to burn out the spacer particles (EDG 1800 furnace, Sonora, Campo Grande, MS, Brazil). Finally, the specimens were sintered at high temperature, 1200°C, for 1 h in a vacuum (10-7 torr) furnace (Astro, São Paulo, SP, Brazil). The implants were fabricated with different porosities and pore sizes by control of the quantity and size of the spacer particles added to the Ti powder: • Group 1 (G1), porosity =  30%, spacer particle size = 180 μm; • Group 2 (G2), porosity =  30%, spacer particle size = 300 μm; and • Group 3 (G3), porosity =  40%, spacer particle size = 180 μm. After sintering, the porous-surface titanium implants had an average diameter and length of 4 mm with pores located only on the specimen surface and a solid core aimed to optimize the mechanical properties of the specimens (Figure 1). Animal experiments Six New Zealand albino rabbits aged 4-6 months and with a mean weight of 4.0 kg were used in this study. The rabbits were provided by the Animal Center of the São José dos Campos School of Dentistry, kept in individual cages and fed with commercial food (Coelhil R Socil, Belo Horizonte, MG, Brazil) and water ad libitum. The rabbits received one specimen of each type (G1, G2, G3) in the left and right tibiae. This study was approved by the Ethics in Research Committee of the Graduate School of Dentistry of São José dos Campos, UNESP (021/2005). Before surgery, the rabbits were weighed and intramuscularly anesthetized with a mixture of 13 mg/ kg aqueous solution of 2% 2-(2,6-xylidine)-5,6dihydro-4H-1,3-thiazine hydrochloride (Rompun Bayer, São Paulo, SP, Brazil), an analgesic, sedative, and muscle relaxant substance, and 33 mg/kg ketamine (Dopalen, Agibrands do Brazil Ltda., São Paulo, SP, Brazil), a general anesthetic. A local anesthetic composed of 3% octapressin combined with Figure 1 SEM micrograph of a longitudinal section: porous-surface () titanium implant with solid core () fabricated by a powder metallurgy technique, with original magnification of 20X. Vasconcellos LMR, Leite DO, Oliveira FN, Carvalho YR, Cairo CAA Braz Oral Res. 2010 Oct-Dec;24(4):399-405 401 prilocaine hydrochloride and felypressin (3% Citanest, Dentsply, Petrópolis, RJ, Brazil) was also used. The procedures were performed under standard sterile conditions. After hair removal, shaving, disinfection, and draping, a straight 3-cm skin incision was made over the medial portion of both tibiae. Three perforations of 4 mm in diameter were made bilaterally at 0.5 cm intervals using an electric surgical drill (AEU707Av2, Aseptico, Washington USA). During drilling, the drill was continuously cooled with saline. Just before insertion of the implants, the hole was irrigated with saline to remove any bone shards. One specimen of each type was placed into one perforation on each tibia and pressed into the surgical cavity until it was fixed to the cortical bone. The muscle tissue and skin were sutured with mononylon 4-0 surgical thread (Johnson & Johnson, São José dos Campos, SP, Brazil). The rabbits received one injection of Pentabiotico (Fort Dodge Saúde Animal, São Paulo, SP, Brazil) after the surgery and were then inspected daily for clinical signs of complications or adverse reactions. The rabbits were sacrificed 8 weeks after surgery by intramuscular injection of a high dose of the anesthetic solution. Histological and histomorphometric examination Six animals were prepared for histological analysis. Implant-containing bone fragments were fixed in 10% formalin for 48  h and subjected to serial alcohol dehydration for 24  h each. The fragments were then embedded in xylol (Synth, Diadema, SP, Brazil) for 3 days, followed by methyl methacrylate resin (Synth, Diadema, SP, Brazil). Next, they were sectioned longitudinally with a low speed diamond saw (Labcut 1010, Extec Corp, Enfield, CT, USA) in serial sections of 400 μm and ground to a thickness of 50 μm in a polishing machine (Labpol 8-12, Extec Corp, Enfield, CT, USA). Microscopic analyses were performed using an optical microscope (Axion Vision 3.1, MO, Carl Zeiss, Germany) attached to a digital camera (DSC-S85 Cybershot, Sony Brasil Ltda., Manaus, AM, Brazil) and with scanning electron microscopy (Leo, SEM, Leo Ldta., Cambridge, UK). In the histomorphometric analysis, the percentage of new bone at the bone-implant interface was evaluated in two sections of each implant. Two fields of each section were digitized (100x), representing the medial and distal interface of the implant. For standardization, the images for histomorphometric analysis were taken in the same position, previously standardized on a television monitor (TC-L32C Viera, Panasonic Brasil Ltda., Manaus, AM, Brazil), attached to the equipment. Thus, 48 sections per region were evaluated in the histomorphometric analysis. The amount of new bone and bone ingrowth to the interior of the pores was calculated using image processing software (Image J, Microsoft Java1.4, Maryland, USA) as a percentage of total bone-implant interface area. Statistical analyses were performed on the histomorphometric results using one-way ANOVA followed by a post hoc test (Tukey multiple comparison test) with P < 0.05 to determine differences between implant conditions. Results Histological examination All the rabbits presented satisfactory postoperative results, with no evidence of inflammation or infection at the surgical site. No adverse reaction was observed during the procedure. Regardless of the type of specimen, new bone was observed at the implant-bone interface, leading to osseointegration, and new bone was also observed growing into the pores (Figures 2 and 3). This new bone consisted of mature bone trabeculae that presented lamellar arrangement and different-sized medullar spaces. Bone ingrowth into the pores was observed in all rabbits, even deep inside the more internal pores (Figure 3). In general, smaller pores were totally filled with bone, whereas bigger pores were partially filled (


Introduction
Titanium (Ti) and its alloys are the most frequently used materials for endosseous implants in dentistry and orthopedics due to their high degree of biocompatibility and good mechanical properties. 1 Successful integration of an implant is generally accepted to rely on its surface characteristics such as chemical composition, morphology, and energy. 2 Surface morphology is an important factor determining the long-term implant stability, especially when bone quality is poor. 3Porous metals have been exploited for several decades to increase friction force between the implant and bone and promote the initial and long-term stability through bone ingrowth. 3,4,5,6he advantage of porous materials is their ability to provide biological anchorage for the surrounding bone tissue via the ingrowth of mineralized tissue into the pore spaces. 7The architecture of a porous implant has been shown to substantially affect the bone ingrowth into pore space. 5,8,9Optimal pore size for bone ingrowth ranges from 100 µm to 600 µm, and the pores must be interconnected to maintain the vascular system required for continuing bone development. 8,9lthough great progress has been made in the manufacture of porous structures with various available fabrication processes, some limitations continue to exist. 10Several pore-generation fabrication methods have been proposed, and the technique chosen is usually dependent on the properties of the material being used. 11urrent fabrication methods include the use of TiH 2 suspension for reactive sintering 12 or organic additives for powder metallurgy (PM), 4,9,13,14,15 multiple coating, 16 the tape casting 17 and environmental electro-discharge sintering process. 18ased on the potential for a porous surface to enhance implant-bone contact, the purpose of this study was to evaluate the influence of pore size and porosity on bone neoformation in the use of titanium implants produced by PM technique.

Porous-surface titanium implant fabrication
Pure Ti powder developed in the General Command of Aerospace Technology (CTA), Institute of Air and Space (IAE), Division of Materials (AMR), Brazil (purity ≥ 99.5%, particle size ≤ 8 µm), was used as starting material in the present study.Urea particles (Synth  , Diadema, SP, Brazil) with particle sizes from 177 to 350 µm were used as spacer material.The selection of particle sizes was obtained by a screening technique.Three types of interconnected porous-surface titanium scaffolds were produced by PM.Initially, Ti powder and urea particles were mixed.The mixture was uniaxially pressed at 100 MPa into a stainless steel mold and then pressed isostatically at 200 MPa.Next, the green specimens were heat treated at 200°C for 2 h in air to burn out the spacer particles (EDG 1800 furnace, Sonora  , Campo Grande, MS, Brazil).Finally, the specimens were sintered at high temperature, 1200°C, for 1 h in a vacuum (10 -7 torr) furnace (Astro  , São Paulo, SP, Brazil).
The implants were fabricated with different porosities and pore sizes by control of the quantity and size of the spacer particles added to the Ti powder: • Group 1 (G1), porosity = 30%, spacer particle size = 180 µm; • Group 2 (G2), porosity = 30%, spacer particle size = 300 µm; and • Group 3 (G3), porosity = 40%, spacer particle size = 180 µm.After sintering, the porous-surface titanium implants had an average diameter and length of 4 mm with pores located only on the specimen surface and a solid core aimed to optimize the mechanical properties of the specimens (Figure 1).prilocaine hydrochloride and felypressin (3% Citanest, Dentsply  , Petrópolis, RJ, Brazil) was also used.

Animal experiments
The procedures were performed under standard sterile conditions.After hair removal, shaving, disinfection, and draping, a straight 3-cm skin incision was made over the medial portion of both tibiae.Three perforations of 4 mm in diameter were made bilaterally at 0.5 cm intervals using an electric surgical drill (AEU707Av2, Aseptico, Washington USA).During drilling, the drill was continuously cooled with saline.Just before insertion of the implants, the hole was irrigated with saline to remove any bone shards.
One specimen of each type was placed into one perforation on each tibia and pressed into the surgical cavity until it was fixed to the cortical bone.The muscle tissue and skin were sutured with mononylon 4-0 surgical thread (Johnson & Johnson, São José dos Campos, SP, Brazil).The rabbits received one injection of Pentabiotico  (Fort Dodge Saúde Animal, São Paulo, SP, Brazil) after the surgery and were then inspected daily for clinical signs of complications or adverse reactions.The rabbits were sacrificed 8 weeks after surgery by intramuscular injection of a high dose of the anesthetic solution.

Histological and histomorphometric examination
Six animals were prepared for histological analysis.Implant-containing bone fragments were fixed in 10% formalin for 48 h and subjected to serial alcohol dehydration for 24 h each.The fragments were then embedded in xylol (Synth  , Diadema, SP, Brazil) for 3 days, followed by methyl methacrylate resin (Synth  , Diadema, SP, Brazil).Next, they were sectioned longitudinally with a low speed diamond saw (Labcut 1010, Extec Corp  , Enfield, CT, USA) in serial sections of 400 µm and ground to a thickness of 50 µm in a polishing machine (Labpol 8-12, Extec Corp  , Enfield, CT, USA).Microscopic analyses were performed using an optical microscope (Axion Vision 3.1, MO, Carl Zeiss, Germany) attached to a digital camera (DSC-S85 Cybershot, Sony Brasil Ltda., Manaus, AM, Brazil) and with scanning electron microscopy (Leo, SEM, Leo Ldta., Cambridge, UK).
In the histomorphometric analysis, the percentage of new bone at the bone-implant interface was evaluated in two sections of each implant.Two fields of each section were digitized (100x), representing the medial and distal interface of the implant.For standardization, the images for histomorphometric analysis were taken in the same position, previously standardized on a television monitor (TC-L32C Viera, Panasonic Brasil Ltda., Manaus, AM, Brazil), attached to the equipment.Thus, 48 sections per region were evaluated in the histomorphometric analysis.The amount of new bone and bone ingrowth to the interior of the pores was calculated using image processing software (Image J, Microsoft Java1.4,Maryland, USA) as a percentage of total bone-implant interface area.Statistical analyses were performed on the histomorphometric results using one-way ANOVA followed by a post hoc test (Tukey multiple comparison test) with P < 0.05 to determine differences between implant conditions.

Histological examination
All the rabbits presented satisfactory postoperative results, with no evidence of inflammation or infection at the surgical site.No adverse reaction was observed during the procedure.
Regardless of the type of specimen, new bone was observed at the implant-bone interface, leading to osseointegration, and new bone was also observed growing into the pores (Figures 2 and 3).This new bone consisted of mature bone trabeculae that presented lamellar arrangement and different-sized medullar spaces.Bone ingrowth into the pores was observed in all rabbits, even deep inside the more internal pores (Figure 3).In general, smaller pores were totally filled with bone, whereas bigger pores were partially filled (Figure 4).A distinct border occurred between newly formed bone and preexisting bone (Figure 5).New bone was also observed both above (Figure 2) and below the implants, and the pores in these areas also presented new bone.

Histomorphometric examination
The bone ingrowth rates are presented in table 1. Group 1 (25.64%)presented the lowest quantity of new bone and Group 3 (30.31%)showed the greatest quantity of new bone, with a statistically significant difference observed between the two implant types (P = 0.023).No difference between Groups 2 and 3 was observed.

Discussion
Implant surface properties have a direct role in osteogenesis at the bone-implant interface, influencing a series of coordinated events including protein adsorption, cell proliferation, and bone tissue depo-sition. 4Therefore, many efforts to modify the titanium implant surface for improved tissue response have been reported. 19,20,21The advantage of porous materials is their ability to provide biological anchorage for surrounding bone tissues 18,22 and greater contact area at the implant-bone interface. 4,9Additionally, the bone-filled porous structure is a composite, an important area of transition between the titanium core and bone. 8he influence of porosity, pore size, and spatial arrangement on the biological behavior of bone has    been investigated in several studies. 8,9,14,15Changes in pore shape can also radically affect the success of bone development; the optimal morphogenetic geometry for bone ingrowth has a concave shape with specific dimensions. 23,24New bone growth into porous metal implants also depends on factors, including interconnecting pore size, 7,25 pore throats open to the surface, 8 and the presence of gaps between the implant and bone surface. 7However, no consensus has yet been reached regarding optimal pore structure.
The optimal pore size for bone ingrowth has been reported to range from 150 to 600 µm.In the present study, the pore sizes of implants evaluated were between the lower and upper limits of this optimal range for porous materials.Data indicate that scaffolds with the smallest pore size and lowest porosity had significantly less bone ingrowth.Thus, pore size, porosity, and interconnected pore size can all affect osteoconductivity. 5,7,26Although increased porosity and pore size are clearly preferential for new bone growth facilitation in Ti implants, it should be kept in mind that another consequence of increased porosity and pore size is a reduction in the mechanical properties of the implant.Thus, depending on the intended application, a balance between mechanical properties and biological performance should be established.
The pores must be connected to maintain the vascular system required for continuing bone development.A three-dimensional open porous structure is particularly suitable for implant fixation by tissue ingrowth. 26Pore interconnection is believed to be critical for the proliferation of bone tissue, since different networks can affect bone ingrowth or tissue differentiation in each pore, but its influence is not yet well understood.All porosity parameters will affect or compromise cell proliferation and differentiation within the pores to different degrees, and, since bone ingrowth invades the porous structure from the periphery towards the center, short routes should be considered important. 8In this study, implants were developed with solid cores and porous surfaces; therefore the pores were restricted to the surface of the implant, a characteristic that should help to fill the entire pore by bone tissue since the routes were all short.
Traditionally, two-dimensional image analyses have been used to quantify the amount of bone in pores and evaluate bone-implant contact by measuring contact between the two phases (i.e., Ti and bone) with optical or radiographic techniques or SEM. 15 In this study, microscopic analysis was performed using optical microscopy and SEM.Histomorphometric analysis revealed that Group 1 obtained significantly less bone ingrowth.This occurred as a consequence of the implant structure, which presented smaller pores and lower porosity, thereby decreasing the space for bone growth in this group.In contrast, Group 2 and 3 implants exhibited an increased area of porous Ti compared with Group 1 implants.These data suggest that, for the implants investigated in the present study, bone conduction is definitely influenced by pores and porosity.
The use of Ti associated with different concentrations and sizes of urea granules permitted the fabrication of implants with well-controlled porous structures, i.e., control of porosity, pore size, and spatial arrangement.Such control over specimen architecture permits the investigation of how geometric parameters influence biological performance of biomaterials, such as osseointegration.In this study, the occurrence of a distinct border was noted between newly formed and preexisting bone, emphasizing the biocompatibility of the material as an appropriate surface for new bone proliferation.
Porous titanium implants have not yet been applied in dentistry.Indeed, few technologies allow the fabrication of this surface in implants as small as dental implants. 15However, a smaller segment of porous implant allows for more effective contact with the bone, due to the relatively greater contact area determined by its topography. 9,25his study sought to demonstrate the capacity for the production of porous-surface titanium while controlling pore size, percentage, and distribution using a powder metallurgy technique.The versatility of manufacturing implants with a solid core and different types of porous surface permits the development of implants that produce distinct rates of bone tissue proliferation.Given the positive results obtained, future studies can focus on determining the specific parameters required to develop implants for use in clinical applications.

Conclusion
According to the specific model used in this study, it can be concluded that the bone ingrowth into porous titanium implants demonstrated distinct biological behaviors varying with pore size and porosity.Increased porosity and pore size showed a direct positive effect on the amount of new bone growth.

SixFigure 1 -
Figure 1 -SEM micrograph of a longitudinal section: porous-surface () titanium implant with solid core () fabricated by a powder metallurgy technique, with original magnification of 20X.

Figure 5 -
Figure 5 -SEM micrograph of the bone-implant interface: delimitation between newly formed bone and preexisting bone (), with original magnification of 400X.

Figure 4 -
Figure 4 -SEM micrograph of the smaller pores totally filled with bone (), whereas bigger pores were partially filled with bone and resin (), with original magnification of 200X.

Figure 3 -
Figure 3 -SEM micrograph of bone ingrowth into the pores (), even deep inside the more internal pores () and near the solid core (), with original magnification of 150X.

Table 1 -
Histomorphometry: percentage of new bone formation at the bone-implant interface.