Blood permeability of a novel ceramic scaffold for bone morphogenetic protein-2

A functionally graded apatite (fg-HAp) with body fluid permeability was developed from bovine bone. The tissue reaction of fg-HAp and its efficacy as a scaffold for recombinant human bone morphogenetic protein-2 (BMP-2) were evaluated histomorphometrically, and a component of permeable fluid into the fg-HAp was analyzed by immunoblotting assay. The fg-HAp block (27 mm(3)) combined with and without BMP-2 (5 microg) was implanted subcutaneously in 4-week-old Wistar rats. Histological examination showed that the surface and bulk degradations of the fg-HAp proceeded extensively and giant cells appeared on the fg-HAp at 2 weeks. Body fluid permeation was found inside the fg-HAp, and the fluid component was immunopositive for albumin. In addition, albumin was detected as a main component among proteins collected from the in vivo implanted fg-HAp. The bioabsorption of the fg-HAp was accelerated as BMP-2-induced bone matured. Histomorphometrical analysis at 4 weeks in the BMP-2/fg-HAp implant showed 59.0% in the total volume of bone and marrow. These results indicate that fg-HAp is an innovative, bioabsorbable bioceramic with fluid permeability characteristic, and may become a biointegrated scaffold for bone engineering.     

Porous titanium and silicon-substituted hydroxyapatite biomodification prepared by a biomimetic process: Characterization and in vivo evaluation

Porous titanium with a pore size of 150–600 μm and a porosity of 67% was prepared by fiber sintering. The porous titanium had a complete three-dimensional (3D) interconnected structure and a high yield strength of 100 MPa. Si-substituted hydroxyapatite (Si-HA) was coated on the surface by a biomimetic process to improve the surface bioactivity. X-ray diffraction results showed that Si-HA coating was not well crystallized. New bone tissue was found in the uncoated porous titanium after 2 weeks of implantation and a significant increase (p < 0.05) in the bone ingrowth rate (BIR) was found after 4 weeks of implantation, indicating the good osteoconductivity of the porous structure. The HA-coated and Si-HA-coated porous titanium exhibited a significantly higher BIR than the uncoated titanium at all intervals, highlighting the better surface bioactivity and osteoconductivity of the HA- and Si-HA coatings. Also, the Si-HA-coated porous titanium demonstrated a significantly higher BIR than the HA-coated porous titanium, showing that silicon plays an active role in the surface bioactivity. For Si-HA-coated porous titanium, up to 90% pore area was covered by new bone tissue after 4 weeks of implantation in cortical bone. In the bone marrow cavity, the pore spaces were filled with bone marrow, displaying that the interconnected pore structure could provide a channel for body fluid. It was concluded that both the 3D interconnected pore structure and the Si-HA coating contributed to the high BIR.     

Analysis of primary bone formation in porous alumina: a fluorescence and scanning electron microscopic study of marrow cell induced osteogenesis.

Porous alumina ceramics alone and combined with rat marrow cells were implanted subcutaneously in the back of syngeneic Fischer rats. Fluorochrome-labeling was performed post operatively and the ceramics were harvested 6 and 8 weeks after implantation. Undecalcified sections of the implants were observed under fluorescence microscopy and the de novo bone-ceramic interfacial areas were analyzed by a scanning electron microscope equipped with an electron probe microanalyzer. Alumina ceramics alone did not show any bone formation, while all marrow cell loaded ceramics showed new bone formation 6 and 8 weeks after implantation. Bone formation was first observed in the center of the pores and proceeded in a centrifugal direction, leading to contact with the ceramic. These results suggest that bone marrow cells have inherent osteogenic capacity and in the pore region of alumina ceramics progression of the osteogenesis causes the dissipation of intervening fibrous tissue between the de novo bone and alumina ceramic surface.     

煅烧与冻干制备去抗原异种骨支架材料的性能

背景：异种骨因其结构及生物特性与人体骨相似,经理化处理后抗原性降低,具有多孔结构,并且来源丰富、可以长期保存等特点,被认为是解决自体骨与异体骨来源不足的有效方法。 目的：比较2种不同处理方法处理后去抗原异种松质骨支架材料的各项理化特性。 方法：将经过化学方法处理的羊松质骨放入马弗炉中,在1 000 ℃高温下煅烧2 h制备成煅烧骨,将另一组经过化学处理的羊松质骨放入-80 ℃冰箱中冷冻4周,然后在真空仪器中干燥制备成冻干骨,将经过超纯水冲洗干净的羊松质骨当做对照组。 结果与结论：3组样品均保留与人体骨组织类似的多孔三维结构,通过微观观察,它们的框架保存完整,有较小的孔隙(55-650 μm)和较高的孔隙率(65%-80%),煅烧后的支架材料韧性降低而脆性明显增加,冷冻干燥处理后力学性能则下降不大,经过衍射分析仪测定它们的主要成分为羟基磷灰石,但煅烧组中还含有少量的β-磷酸三钙,而能谱分析则证实3组的钙磷含量比均接近人体的钙磷比。结果提示经过这两种方法处理的松质骨与人体骨组织结构相似,其中各主要元素的含量比也与人体接近,而且具有足够小的孔隙和较高的孔隙率,但是煅烧处理对支架材料的力学性能影响比较大,而经过冷冻干燥处理力学性能下降不大但无法完全去除材料中的抗原成分,它们基本能达到骨组织工程支架材料的基本要求。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Long-term implantation of zinc-releasing calcium phosphate ceramics in rabbit femora

Zinc is an essential trace element that has stimulatory effects on bone formation. Recently, we developed zinc-releasing calcium phosphate ceramics in order to add the pharmacologic effect of zinc to calcium phosphate ceramics. In our previous study, we showed that the optimum zinc content for promoting bone formation was 0.316 wt %. Therefore a zinc composite ceramic of zinc-containing beta-tricalcium phosphate and hydroxyapatite, with a zinc content of 0.316 wt %, was chosen for long-term implantation. Cylindrical rods of the zinc composite ceramic were implanted in rabbit femora for 2 to 60 weeks. Using computer-aided image analysis, a histomorphometric study was carried out to investigate bone formation and resorption around the implants. The control was a composite ceramic of beta-tricalcium phosphate and hydroxyapatite without zinc. The addition of zinc to the implant demonstrated both favorable and unfavorable effects on bone remodeling. The favorable effect was enhanced bone apposition to the implant surface, demonstrated by a significant increase in intramedullary bone apposition rate at 6 weeks and in cortical bone apposition rate at 24 and 60 weeks (p < 0.05). The unfavorable effect was increased bone resorption, demonstrated by a significant increase in medullary cavity area at 60 weeks (p < 0.05). In order to utilize the favorable effect and avoid the unfavorable effect of zinc, either a reduction in zinc content in the zinc composite ceramic or the selection of implantation sites that do not have excessive exposure to bone marrow are required.     

Bioactivity and mechanical properties of cellulose/carbonate hydroxyapatite composites prepared in situ through mechanochemical reaction

Organic-inorganic composites, prepared from bone-bonding bioactive ceramics and organic polymers, are useful for novel bone substitutes having mechanical properties analogous to natural bone. We synthesized composites from cellulose and carbonate hydroxyapatite (CHAp) in situ through mechanochemical reaction. They contained B-type CHAp analogous to bone apatite. They showed a bending strengths of 10-13 MPa and Young's modulus of 1.5-2.2 GPa. We predicted their microstructure by comparing the measured density with the theoretical one. Cellulose was assumed to be distributed in the pore of CHAp at low cellulose content, and in grain boundaries of CHAp at high cellulose content. The composites formed calcium phosphate on their surfaces in simulated body fluid, meaning that they have a potential to be bioactive.     

介孔-大孔生物活性玻璃的合成及其生物活性**◆

背景：多孔生物活性玻璃材料具有较大的比表面积、孔隙率以及贯通的孔道结构,可以加速羟基磷灰石沉积的动力学速率从而提高材料诱导形成新骨的能力。 目的：利用有机酸替代无机酸来合成新型介孔-大孔生物活性玻璃。 方法：将柠檬酸和P123加入到无水乙醇中,在室温下搅拌2 h至溶液澄清。依次加入正硅酸乙酯、四水硝酸钙和三磷酸乙酯,继续搅拌24 h。将所得溶胶倒入培养皿中,在室温下放置7 d,之后高温下烧结除去有机物模板。 结果与结论：①利用有机酸所制备的材料具有大孔结构,拥有较大的比表面积、孔隙率和孔径。②合成的介孔-大孔生物活性玻璃在人工模拟体液中可以诱导形成含碳羟基磷灰石,表现出了较好的生物活性,有望成为一种具有发展前景的骨缺损修复替代材料。     

Mediation of bone ingrowth in porous hydroxyapatite bone graft substitutes

Previous investigations have shown that both the early biological response and the mechanical properties of a porous hydroxyapatite bone graft substitute are highly sensitive to its pore structure. The objective of this study was to evaluate whether the pore structure continued to influence bone integration in the medium to long term. Two screened batches of porous hydroxyapatite (PHA) designated as batch A and batch B, with porosities of approximately 60 and 80%, respectively, were selected for this study and implanted for periods of 5, 13, and 26 weeks into the lower femur of New Zealand White rabbits. Histomorphometric analysis of the absolute volume of bone ingrowth within batch A and B implants from 5 to 26 weeks showed that the absolute volume of bone ingrowth was consistently lower in batch A (10-21%), compared to batch B implants (24-31%). However, when the volume of bone ingrowth was normalised for the available pore space, this difference was reduced (23-47% and 32-42% for batches A and B, respectively). These observations suggest that differences in the volume of bone ingrowth initially depended on pore interconnectivity rather than pore size, whereas the volume or morphology of the PHA influenced the volume and morphology of bone ingrowth at later time points. Compression testing showed that bone ingrowth had a strong reinforcing effect on PHA bone graft substitutes, and a strong correlation was identified between mechanical properties and the absolute volume of ingrowth for both batches A and B. Furthermore, at 13 and 26 weeks, there was no significant variation in the ultimate compressive strength of integrated batch A and B implants. This similarity in ultimate mechanical properties indicated that the absolute volume of ingrowth may be mediated by the PHA structure through its impact on the dynamics of the local biomechanical environment. The results of push-out testing showed that fixation of PHA bone graft substitutes was independent of density within the range studied, with no significant difference in the interfacial shear stress between batches A and B at each time point throughout the study.     

锶磷灰石多孔陶瓷复合成骨细胞的体内异位成骨研究

目的比较不同孔隙率的锶磷灰石（含5%锶的羟磷灰石,Sr-HA）陶瓷复合成骨细胞后体内异位成骨能力。方法实验试样分复合成骨细胞的不同孔隙率组和未复合成骨细胞的不同孔隙率组。先抽取健康兔的骨髓组织,体外培养,诱导分化为成骨细胞,分别与孔径约为450～700μm、孔隙率分别为50%、60%、70%的Sr-HA及孔隙率为70%的纯羟磷灰石陶瓷体复合,自体回植到兔左侧背脊肌内;未复合成骨细胞的不同孔隙率组试样则种植在兔右侧背脊肌内作为阴性对照。植入后4、12、24周取材,行大体观察、组织学检查。结果复合成骨细胞的各组陶瓷在植入兔背脊肌内4周后均有类骨质形成,随着植入时间的延长,骨组织的数量不断增加;孔隙率为70%的Sr-HA陶瓷和HA陶瓷的成骨性能明显优于50%、60%孔隙率的Sr-HA陶瓷。未复合细胞的陶瓷材料在组织学检查中未见有类骨质形成。结论Sr-HA多孔陶瓷是较理想的骨组织工程支架材料,成骨细胞复合Sr-HA陶瓷用于骨缺损的修复,具有广阔的临床应用前景。     

Oscillatory Pressurization of an Animal Cell as a Poroelastic Spherical Body

The analytical solution of a spherical poroelastic body under an oscillatory hydrostatic pressurization is obtained. This solution is then parameterized and interpreted in terms of a spherical animal cell with the cytoskeleton serving as the solid phase. It is found that for a cell with free or nearly free membrane leakage (such as in the case of an osteocytic cell body), the induced pore fluid pressure amplitude near the center of the cell exceeds the amplitude of the applied pressure by 50% if the loading frequency falls near that of normal human gait (1 Hz). A parametric analysis shows that the leakage coefficient is proportional to the intrinsic permeability ratio between the boundary and the bulk matrix. The physiological implication of the solution is further interpreted and discussed through an anatomical analysis of two representative cellular entities under compression: a chondrocyte and an osteocytic cell body. Finally, through a comparison between the characteristic time of gait and the characteristic pore fluid pressure relaxation times of various fluid-saturated entities in the body (such as a tumor, the brain, the cortical bone, the trabecular bone, and the cartilage, etc.), it is found that the gait-induced pore fluid transport seems to be important only in deeply buried cells and the mineralized cartilage.     

Mechanical properties and in vitro cell compatibility of hydroxyapatite ceramics with graded pore structure

In order to improve the mechanical strength of hydroxyapatite (HA) ceramics used as osteoimplants and to enhance cellular penetration functionally graded ceramics with a transition in porosity from the surface towards the centre were designed. The multilayer structures were prepared by multiple tape casting based on an aqueous HA slurry containing polybutylmethacrylate (PBMA) spheres with diameters ranging from 100 to 300 microns. After burning out the PBMA, pores of 70-200 microns were generated. The pore-graded laminates were sintered at temperatures between 1250 degrees C and 1450 degrees C. Bending strength of the pore-graded ceramics was approximately 50% higher as compared to HA of the same pore volume fraction but without gradient structure. The materials were tested in vitro for attachment and activity of osteoblast-like MC3T3-E1 cells over a period of 3 weeks. Cells formed confluent layers on the ceramic surface, penetrated into the graded porosity ranging from 100-150 microns to 250-300 microns in size and showed increasing alkaline phosphatase activity over 3 weeks. The results demonstrated initial in vitro cell compatibility of the functionally graded HA materials and their potential as osteoimplants.     

Bone formation after 4 weeks around blood-plasma-modified titanium implants with varying surface topographies: an in vivo study

The aim of the present study was to investigate and compare the stability and bone ingrowth capacity to screw-shaped titanium implants with five different surface treatments. The implants were: (1) standard turned with a thin blood plasma coat (TP), (2) NaOH-etched dito with pore size 0.2-0.3 microm (E), (3) NaOH-etched with pore size 0.2-0.3 microm and a thin blood plasma coat (EP), (4) electrochemically oxidised with pore size 1-2 microm (O), (5) electrochemically oxidised with pore size 1-2 microm and a thin blood plasma coat (OP). A total of 66 implants were divided into the above-described five groups and inserted for 4 weeks into tibia and femur of 11 rabbits. The implants were evaluated by resonance frequency (RF) measurements at the time of insertion and removal, and analysed histomorphometrically at removal. The RF measurements showed that the implant stability was lower in soft bone compared to dense and increased with time. No significant differences were observed between the different surface modifications. The histomorphometric analysis revealed no statistically significant differences between the implants regarding bone-to-metal contact (BMC) and bone area inside the threads (BA). The above results indicate that thin blood plasma-coated and non-coated screw-shaped titanium implants with turned, NaOH-etched and electrochemically etched surface profiles integrate similarly to bone at 1 month of implantation.     

Ca,P-rich layer formed on high-strength bioactive glass-ceramic A-W

Glass-ceramic A-W, containing crystalline apatite and wollastonite in a MgO-CaO-SiO2 glassy matrix shows high bioactivity as well as high mechanical strength, but other ceramics containing the same kinds of crystalline phases in different glassy matrices do not show the same bioactivity. In order to investigate the bone-bonding mechanism of this type of glass-ceramic, surface structural changes of the glass-ceramics after exposure to simulated body fluid were analyzed with various techniques. A solution with ion concentrations which are almost equal to those of the human blood plasma was used as the simulated body fluid, instead of Tris-buffer solution hitherto used. For analyzing the surface structural changes, thin-film x-ray diffraction was used in addition to conventional techniques. It was found that a bioactive glass-ceramic forms a Ca, P-rich layer on its surface in the fluid but nonbioactive ones do not, and that the Ca, P-rich layer consists of carbonate-containing hydroxyapatite of small crystallites and/or defective structure. These findings were common to those of Bioglass-type glasses. So, we conclude that the essential condition for glass and glass-ceramic to bond to bone is the formation of the surface apatite layer in the body environment but it is not essential to contain apatite within the material. Bioactivity of glass and glass-ceramic can be evaluated in vitro by examining the formation of the surface apatite layer in the simulated body fluid described above.     

Bioactive tantalum metal prepared by NaOH treatment

Untreated tantalum metal formed an apatite on its surface in simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma. However, it took an induction period as long as 4 weeks for apatite formation. The tantalum metal formed the apatite within 1 week when it was previously soaked in a 0.2 or 0.5M NaOH aqueous solution at 60 degrees C for 24 h to form a sodium tantalate hydrogel layer on its surface. The decrease in the induction period of apatite formation was attributed to the catalytic effect of the Ta-OH groups on the surface of the tantalum metal for apatite nucleation and acceleration of the apatite nucleation by an increased ionic activity product of the apatite in the fluid due to the release of Na(+) ions. The NaOH-treated tantalum metal can form apatite in a short period even in the living body and bond to the bone through this apatite layer. This indicates that a highly bioactive tantalum metal can be obtained by a simple chemical treatment.     

Fabrication methods of porous metals for use in orthopaedic applications

Implant stability is not only a function of strength but also depends on the fixation established with surrounding tissues [Robertson DM, Pierre L, Chahal R. Preliminary observations of bone ingrowth into porous materials. J Biomed Mater Res 1976;10:335-44]. In the past, such stability was primarily achieved using screws and bone cements. However, more recently, improved fixation can be achieved by bone tissue growing into and through a porous matrix of metal, bonding in this way the implant to the bone host. Another potentially valuable property of porous materials is their low elastic modulus. Depending on the porosity, moduli can even be tailored to match the modulus of bone closer than solid metals can, thus reducing the problems associated with stress shielding. Finally, extensive body fluid transport through the porous scaffold matrix is possible, which can trigger bone ingrowth, if substantial pore interconnectivity is established [Cameron HU, Macnab I, Pilliar RM. A porous metal system for joint replacement surgery. Int J Artif Organs 1978;1:104-9; Head WC, Bauk DJ, Emerson Jr RH. Titanium as the material of choice for cementless femoral components in total hip arthroplasty. Clin Orthop 1995;85-90]. Over the years, a variety of fabrication processes have been developed, resulting in porous implant substrates that can address unresolved clinical problems. The advantages of metals exhibiting surface or bulk porosity have led researchers to conduct systematic research aimed at clarifying the fundamental aspects of interactions between porous metals and hard tissue. This review summarises all known methods for fabricating such porous metallic scaffolds.     

Marrow cell induced osteogenesis in porous hydroxyapatite and tricalcium phosphate: a comparative histomorphometric study of ectopic bone formation

To investigate the bone formation ability of porous hydroxyapatite (HA) and tricalcium phosphate (TCP), ceramic discs were implanted with or without rat marrow cells into subcutaneous sites in syngeneic rats. The discs of HA and TCP had identical microstructures: pore size was 190-230 microns, porosity was 50-60%, and they were fully interconnected. Implants without marrow cells (discs themselves) did not show bone formation, whereas implants with marrow cells showed bone formation in the pores of the ceramics. The bone formation of both HA and TCP occurred initially on the surface of the ceramic and progressed towards the center of the pore. The de novo bone was quantitated from decalcified serial sections of the implants. One month after implantation with marrow cells, the percentage fractions of the pore area filled with bone for implanted HA and TCP were 16.9 and 15.1, respectively. At 2 months after implantation with marrow cells, the fractions of bone were 34.3 and 30.9, respectively. These results indicate that both HA and TCP ceramics can show comparable osteogenic ability in the presence of marrow cells.     

Effects of ceramic component on cephalexin release from bioactive bone cement consisting of Bis-GMA/TEGDMA resin and bioactive glass ceramics

The purpose of this study was to elucidate the effect of amount of ceramic cement powder on drug release from bioactive bone cement. The associated bone-bonding strength was also investigated. The bioactive bone cement under investigation consisted of bisphenol-alpha-glycidyl methacrylate (Bis-GMA), triethylene-glycol dimethacrylate (TEGDMA) resin and a combination of apatite- and wollastonite-containing glass-ceramic (A-W GC) powder. A-W GC powder (50%, 70% and 80% w/w) containing 5% cephalexin (CEX) powder hardened within 5 min after mixing with Bis-GMA/TEGDMA resin. The compressive strength of the cement with or without drug increased with increasing the amount of ceramic powder. The compressive strength of the 80% ceramic cement without the incorporation of cephalexin was 194 MPa. This compressive strength was about 3 times higher than that for polymethylmethacrylate cement. After the cement was implanted in the proximal metaphysis of the tibiae of male rabbits, the failure load for the cement was found to increase with increasing of the amount of ceramic powder. This finding suggested that the cement formed a bonding with bone. In vitro CEX release from bioactive bone cement pellets in a simulated body fluid at pH 7.25 and 37 degrees C continued for more than 2 weeks. Drug release profile followed the Higuchi equation initially, but not at later stages. The drug release rate increased with increasing amount of ceramic powder in the mixture. Since the pore volume of the cement increased with increasing of amount of ceramic powder, the drug diffused in the pores between the ceramics particle and polymer matrix. As hydroxyapatite precipitated on the cement surface, the drug release rate decreased, as observed at the later release stage. These results suggest that varying the amount of ceramic powder in the cement system could control the drug release rate from bioactive bone cement.     

新型羟基磷灰石多孔支架的制备与性状

背景：目前几种钙羟磷灰石作为骨的替代品已经在临床上使用,但是由于缺少内部交互的连通孔,使得骨形成过程中经常发生病理性的骨折。 目的：采用改良的技术制备互联多孔羟磷灰石陶瓷支架材料,并对其物理化学特征进行检测。 方法：将羟基磷灰石(质量分数60%)与聚乙烯亚胺质量分数40%混合,采用改良的泡沫凝胶技术技术(聚氧乙烯十二烷基醚质量分数1%)制备多孔羟磷灰石陶瓷。扫面电镜检测材料内部结构,将制备的材料移植动物体骨缺损区观察成骨情况。 结果与结论：多孔羟磷灰石陶瓷具有三维结构,内部充满大小较均一的球形孔隙,直径平均为150 μm,孔隙率75%,孔隙上充满窗孔样相互交通的小洞,平均40 μm, 具有足够的抗压度(10~12 MPa)。动物实验表明,多孔羟磷灰石陶瓷表现出较好的成骨诱导性,可见孔隙里形成了新生骨。结果提示改良的羟基磷灰石支架能够在骨组织工程上应用,有望成为新型的改良品。     

Water-based sol-gel synthesis of hydroxyapatite: process development

Hydroxyapatite (HA) ceramics were synthesized using a sol-gel route with triethyl phosphite and calcium nitrate as phosphorus and calcium precursors, respectively. Two solvents, water and anhydrous ethanol, were used as diluting media for HA sol preparation. The sols were stable and no gelling occurred in ambient environment for over 5 days. The sols became a white gel only after removal of the solvents at 60 degrees C. X-ray diffraction showed that apatitic structure first appeared at a temperature as low as 350 degrees C. The crystal size and the HA content in both gels increase with increasing calcination temperature. The type of initial diluting media (i.e., water vs. anhydrous ethanol) did not affect the microstructural evolution and crystallinity of the resulting HA ceramic. The ethanol-based sol dip-coated onto a Ti substrate, followed by calcination at 450 degrees C, was found to be porous with pore size ranging from 0.3 to 1 microm. This morphology is beneficial to the circulation of physiological fluid when the coating is used for biomedical applications. The satisfactory adhesion between the coating and substrate suggests its suitability for load-bearing uses.     

Bone formation process in porous calcium carbonate and hydroxyapatite.

This study determined the bone formation in porous calcium carbonate (CC) and porous hydroxyapatite (HA) in ectopic sites. The bone formation stimulus was derived from bone marrow cells. CC and HA in the shape of disks were implanted with or without rat marrow cells into subcutaneous sites of syngeneic rats. The CC and HA had identical microstructure: pore size was 190-230 microns, porosity was 50-60% and they were fully interconnected. Bone did not form in any implants without marrow cells (disks themselves), whereas bone consistently formed in the pores of all implants with marrow cells after 4 weeks. The bone formation of both CC and HA occurred initially on surface of the pore regions and progressed toward the center of the pore. Scanning electron microscopy and electron-probe microanalysis revealed a continuum of calcium at the interfaces of both bone/CC and bone/HA implants. These results indicate that the bone formation in calcium carbonate derived from marine corals is comparable to the bioactive hydroxyapatite.