Hydroxyapatite and titania sol-gel composite coatings on titanium for hard tissue implants; mechanical and in vitro biological performance

Hydroxyapatite (HA) composites with titania (TiO2) up to 30 mol % were coated on a titanium (Ti) substrate by a sol-gel route, and the mechanical and biological properties of the coating systems were evaluated. Using polymeric precursors, highly stable HA and TiO2 sols were prepared prior to making composite sols and coatings. Coatings were produced under a controlled spinning and heat treatment process. Pure phases of HA and TiO2 were well developed on the composites after heat treatment above 450 degrees C. The HA-TiO2 composite coating layers were homogeneous and highly dense with a thickness of about 800-900 nm. The adhesion strength of the coating layers with respect to Ti substrate increased with increasing the TiO2 addition. The highest strength obtained was as high as 56 MPa, with an improvement of about 50% when compared to pure HA (37 MPa). The osteoblast-like cells grew and spread actively on all the composite coatings. The proliferation and alkaline phosphatase (ALP) activity of the cells grown on the composite coatings were much higher than those on bare Ti, and even comparable to those on pure HA coating. Notably, the HA-20% TiO2 composite coating showed a significantly higher proliferation and ALP expression compared to bare Ti (p < 0.05). These findings suggest that the sol-gel-derived HA-TiO2 composite coatings possess excellent properties for hard tissue applications from the mechanical and biological perspective.     

Hydroxyapatite coating on titanium substrate with titania buffer layer processed by sol-gel method

Hydroxyapatite (HA) was coated onto a titanium (Ti) substrate with the insertion of a titania (TiO2) buffer layer by the sol-gel method. The HA layer was employed to enhance the bioactivity and osteoconductivity of the Ti substrate, and the TiO2 buffer layer was inserted to improve the bonding strength between the HA layer and Ti substrate, as well as to prevent the corrosion of the Ti substrate. The HA layer coated over the TiO2 showed a typical apatite phase at 400 degrees C and the phase intensity increased above 450 degrees C. The sol-gel derived HA and TiO2 films, with thicknesses of approximately 800 and 200 nm, respectively, adhered tightly to each other and to the Ti substrate. The bonding strength of the HA/TiO2 double layer coating on Ti was markedly improved when compared to that of the HA single coating on Ti. The highest strength of the double layer coating was 55 MPa after heat treatment at 500 degrees C. The improvement in bonding strength with the insertion of TiO2 was attributed to the resulting enhanced chemical affinity of TiO2 toward the HA layer, as well as toward the Ti substrate. Human osteoblast-like cells, cultured on the HA/TiO2 coating surface, proliferated in a similar manner to those on the TiO2 single coating and on the pure Ti surfaces. However, the alkaline phosphatase activity of the cells on the HA/TiO2 double layer was expressed to a higher degree than that on the TiO2 single coating and pure Ti surfaces. The corrosion resistance of Ti was improved by the presence of the TiO2 coating, as confirmed by a potentiodynamic polarization test.     

Mesoporous titanium dioxide coating for metallic implants

A bioactive mesoporous titanium dioxide (MT) coating for surface drug delivery has been investigated to develop a multifunctional implant coating, offering quick bone bonding and biological stability. An evaporation induced self-assembly (EISA) method was used to prepare a mesoporous titanium dioxide coating of the anatase phase with BET surface area of 172 m(2)/g and average pore diameter of 4.3 nm. Adhesion tests using the scratch method and an in situ screw-in/screw-out technique confirm that the MT coating bonds tightly with the metallic substrate, even after removal from bone. Because of its high surface area, the bioactivity of the MT coating is much better than that of a dense TiO(2) coating of the same composition. Quick formation of hydroxyapatite (HA) in vitro can be related to enhance bonding with bone. The uptake of antibiotics by the MT coating reached 13.4 mg/cm(3) within a 24 h loading process. A sustained release behavior has been obtained with a weak initial burst. By using Cephalothin as a model drug, drug loaded MT coating exhibits a sufficient antibacterial effect on the material surface, and within millimeters from material surface, against E.coli. Additionally, the coated and drug loaded surfaces showed no cytotoxic effect on cell cultures of the osteoblastic cell line MG-63. In conclusion, this study describes a novel, biocompatiblemesoporous implant coating, which has the ability to induce HA formation and could be used as a surface drug-delivery system.     

Laser processing of bioactive tricalcium phosphate coating on titanium for load-bearing implants

Laser-engineered net shaping (LENS), a commercial rapid prototyping (RP) process, was used to coat titanium with tricalcium phosphate (TCP) ceramics to improve bone cell-materials interactions. During LENS coating process, the Nd:YAG laser melts the top surface of Ti substrate in which calcium phosphate powder is fed to create a TCP-Ti composite layer. It was found that an increase in laser power and/or powder feed rate increases the thickness of the coating. However, coating thickness decreased with increasing laser scan speed. TCP coating showed columnar titanium grains at the substrate side of the coating and transitioned to equiaxed titanium grains at the outside. When the scan speed was reduced from 15 to 10mms(-1), coating hardness increased from 882+/-67 to 1049+/-112Hv due to an increase in the volume fraction of TCP in the coating. Coated surfaces showed uniformly distributed TCP particles and X-ray diffraction data confirmed the absence of any undesirable phases, while maintaining a high level of crystallinity. The effect of TCP coating on cell-material interaction was examined by culturing osteoprecursor cells (OPC1) on coated surfaces. The results indicated that TCP coating had good biocompatibility where OPC1 cells attached and proliferated on the coating surface. The coating also initiated cell differentiation, ECM formation and biomineralization.     

Adherent apatite coating on titanium substrate using chemical deposition

Plasma-sprayed "HA" coatings on commercial orthopedic and dental implants consist of mixtures of calcium phosphate phases, predominantly a crystalline calcium phosphate phase, hydroxyapatite (HA) and an amorphous calcium phosphate (ACP) with varying HA/ACP ratios. Alternatives to the plasma-spray method are being explored because of some of its disadvantages. The purpose of this study was to deposit an adherent apatite coating on titanium substrate using a two-step method. First, titanium substrates were immersed in acidic solution of calcium phosphate resulting in the deposition of a monetite (CaHPO4) coating. Second, the monetite crystals were transformed to apatite by hydrolysis in NaOH solution. Composition and morphology of the initial and final coatings were identified using X-ray diffraction (XRD), Scanning Electron Microscopy, and Energy Dispersive Spectroscopy (EDS). The final coating was porous and the apatite crystals were agglomerated and followed the outline of the large monetite crystals. EDS revealed the presence of calcium and phosphorous elements on the titanium substrate after removing the coating using tensile or scratching tests. The average tensile bond of the coating was 5.2 MPa and cohesion failures were observed more frequently than adhesion failures. The coating adhesion measured using scratch test with a 200-microm-radius stylus was 13.1N. Images from the scratch tracks demonstrated that the coating materials were squashed without fracturing inside and/or at the border of the tracks until the failure point of the coating. In conclusion, this study showed the potential of a chemical deposition method for depositing a coating consisting of either monetite or apatite. This method has the advantage of producing a coating with homogenous composition on even implants of complex geometry or porosity. This method involves low temperatures and, therefore, can allow the incorporation of growth factors or biogenic molecules.     

Electrolytic deposition of calcium etidronate drug coating on titanium substrate

Wear debris-induced osteolysis is the major cause of aseptic loosening and failure of hip implants. One of the promising therapeutic interventions to improve the longevity of hip implants is to administrate bisphosphonate drug to inhibit osteoclastic bone resorption. This study aimed at developing new techniques of directly combining bisphosphonate with implants to achieve local delivery and controlled release of the drug. Instead of using soluble sodium salt, we proposed to apply sparingly soluble calcium salt of bisphosphonate as a potential long-term antiosteolysis coating on hip implants. Calcium salt of etidronate, a member of the bisphosphonate family of potent osteoclast inhibitors, was used in this pilot study. By adopting the electrolytic deposition (ELD) technique, which was developed for ceramic coatings including calcium phosphates, we demonstrated that a thin layer of calcium bisphosphonate could be deposited onto titanium surface. The drug coating is amorphous as characterized with X-ray diffraction, and has globular morphology under the scanning electron microscope. Electrospray-ionization mass-spectrometry (ESI-MS) and Fourier-transformed infrared spectroscopy confirmed that the molecular structure of the etidronate (m/z 205, H3L-, the single dissociated form of parent etidronic acid, denoted as H4L) was preserved after the ELD process. In vitro release into a "physiological" buffer solution confirmed that the etidronate concentration was limited by its low solubility. The etidronate concentration was 8 x 10(-5) M at day 1 and kept relatively stable at approximately 6 x 10(-5) M from day 2 to day 8. The deposition mechanisms of the drug coating and its potential efficacy as an antiosteolytic release source were discussed.     

Hydroxyapatite and fluor-hydroxyapatite layered film on titanium processed by a sol-gel route for hard-tissue implants

A double-layered coating, consisting of a hydroxyapatite (HA) outer film and a fluor-hydroxyapatite (FHA) inner film, was produced on a Ti substrate by a sol-gel route to improve the biocompatibility and functionality of the system. Dissolution behavior of and in vitro cellular responses to the layered film were investigated. Calcium nitrate and triethyl phosphite were used for calcium and phosphate precursors, respectively, and ammonium fluoride was added as a fluorine-ion source for FHA. The FHA layer was deposited on Ti by spin coating and subsequent heat treatment at 550 degrees C for 30 min in air, and then the HA layer was laid down over the FHA-coated Ti under the same conditions. After heat treatment, characteristic apatite structures and phases were developed on both FHA and HA films. The cross-section view of the HA/FHA film clearly showed a double-layered structure on Ti with each layer approximately 0.6-0.8-microm thickness. The coating layer was highly uniform and dense, and adhered to Ti substrate strongly with an adhesion strength of about 40 MPa. The in vitro solubility of the HA/FHA layered film in a physiological solution was between that of HA and FHA pure film, and the dissolution profile was quite biphasic, that is, an initial rapid period and a slowdown with increasing time, reflecting the gradient solubility of the fast HA outer structure/slow FHA inner structure. The human osteoblast-like HOS TE85 cells cultured on the HA/FHA layered film attached, spread, and grew favorably. The proliferation rate of the cells on the layered film was significantly higher (considered at p < 0.05 for n = 6) than that on Ti substrate and was similar to that on pure HA film. The alkaline phosphatase (ALP) activity and osteocalcin (OC) produced by the cells on the layered film were significantly higher (considered at p < 0.05 for n = 6) than those on Ti substrate. Moreover, the ALP and OC levels of cells on the layered film showed the trends of HA outer/FHA inner structure with respect to culture period, that is, HA initially and FHA later. These observations suggest that the HA/FHA layered film on Ti obtained by a sol-gel route possesses gradient functionality in terms of solubility and cellular responses, and find that those parameters can be tailored for specific use in hard-tissue implants.     

Plasma-sprayed hydroxyapatite+titania composite bond coat for hydroxyapatite coating on titanium substrate

A two-layer hydroxyapatite (HA)/HA+TiO(2) bond coat composite coating (HTH coating) on titanium was fabricated by plasma spraying. The HA+TiO(2) bond coat (HTBC) consists of 50 vol% HA and 50 vol% TiO(2) (HT). The microstructural characterization of the HTH coatings before and after heat treatment was conducted by using scanning electron microscopy (SEM), electron probe microanalyser (EPMA), X-ray diffractometer (XRD) and transmission electron microscopy (TEM), in comparison with that of HT coating and pure HA coating. The results revealed that HA and TiO(2) phases layered in an alternating pattern within the HTBC, and the HTBC bonded well to HA top coating (HAT coating) and Ti substrate. The as-sprayed HT coating consists mainly of crystalline HA, rutile TiO(2) and amorphous Ca-P phase. The post-spray heat treatment at 650 degrees C for 120 min effectively restores the structural integrity of HA by transforming non-HA phases into HA. It was found that there exists interdiffusion of the elements within the HTBC, but no chemical product between HA and TiO(2), such as CaTiO(3) was formed. The cross-sectional morphologies confirmed that there is a shift towards a relatively tighter bonding from the HAT coating/HTBC interface in the as-sprayed HTH coating to the HTBC/Ti substrate interface in the heat-treated HTH coating. On quenching the coatings into water, the surface cracking indicates more apparently the positive effect of the HTBC on the decrease of residual stress in HAT coating. The in situ surface cracking also suggests that the stress on the surface of the HTH coating is stable under subjection to a repetitious heat treatment. The toughening and strengthening of HTBC is thought to be mainly due to TiO(2) as obstacles embarrassing cracking, the reduction of the near-tip stresses resulting from stress-induced microcracking and the decrease of CTE mismatch. In the HTH composite coating, the HAT coating is toughened by the decreased CTE mismatch with Ti through the addition of HTBC, which bonds well to the Ti substrate via its TiO(2) hobnobbing with the Ti oxides formed on Ti substrate.     

A novel ordered nano hydroxyapatite coating electrochemically deposited on titanium substrate

A novel porous nano hydroxyapatite (HA) coating has been prepared on commercially pure titanium substrate by a modified electrochemical deposition method. The physico-chemical and biological properties of the coating were characterized by SEM, XRD, FTIR, Raman, and in vitro cell culture test respectively. The SEM patterns show a uniform microporous morphology consisting of wirelike crystals at nanometer scale. It is suggested that under controlled deposition conditions, the primary HA nanowires grow and self-assemble to construct an ordered microporous nest-like morphology, thus to form a nano-micro two-level structure. The XRD results demonstrate that the HA nanowires are orderly arranged with their c-axis preferentially perpendicular to the substrate surface. The Raman and IR spectra affirm that the main component of the coating is well crystallized HA. An interdigitation phenomenon of the MG63 human osteosarcoma cells with the HA nanowires is observed in the in vitro test, indicating excellent biocompatibility and bioactivity for the prepared coating.     

The effect of hydroxyapatite coating on bony ingrowth into grooved titanium implants

This study was undertaken to investigate the relative importance of a hydroxyapatite (HA) coating and the macrotexture of titanium implants to the quality of bony ingrowth and fixation. Various types of titanium cylinders were implanted into the cancellous bone of the intercondylar region of the distal femur of the dog. The animals were sacrificed at intervals post-implantation and the implants were evaluated histologically for amount of bony ingrowth and mechanically by the means of a push-out test. Our results demonstrated that when grooved titanium implants are used, the addition of HA coating significantly improved the biologic fixation. In addition, a groove depth of 1 mm was found to give significantly better fixation than 2 mm. When compared to implants with traditional, beads-coated porous surfaces, HA-coated grooved titanium implants were found to show better fixation at 4 weeks after implantation, but, significantly inferior fixation at 12 weeks after implantation. We concluded that while a groove depth of 1 mm was optimal in HA-coated, grooved titanium implants, they remain inferior to beads-coated titanium implants with respect to longer-term fixation. More research needs to be addressed at improving the macrotexture environment of grooved implants to enhance long-term bony ingrowth.     

Soft and hard tissue response to endosseous dental implants

The last two decades have seen a remarkable growth in the development of dental implants and their incorporation into the practice of dentistry. This turn of events was made possible by an improved understanding of the biological response of living tissues to implants as well as clinical trials that validated the long-term success of these implants. Despite major structural differences between teeth and implants, such as the absence of a periodontal ligament around implants, the latter appear to provide a reliable functional replacement for their natural counterparts. This review briefly summarizes the major structural differences of the interfacial region of teeth and dental implants and their supporting tissues. It focuses on our current understanding of the soft and hard tissue responses to submerged and nonsubmerged root-form dental implants. The influence of a number of factors that affect the tissue response is reviewed, including biomaterials, implant design, surgical technique, and the local microbiota. Our recently acquired ability to modulate wound healing with guided tissue regeneration and growth factors will undoubtedly play an important role in the future utilization and success rates of dental implants. © 1996 Wiley-Liss, Inc.     

Hydroxyapatite coating on titanium by thermal substrate method in aqueous solution.

A new hydrocoating method (the thermal substrate method) is proposed for coating calcium phosphates such as hydroxyapatite (HA), on titanium substrates in an aqueous solution. Several factors (e.g., the type of ion source, the heating time and temperature, and the surface roughness of the substrate) affected the characteristics of the precipitate formed by this method. The solution used included 3 mmol dm(-3) Ca(H(2)PO(4))(2) and 7 mmol dm(-3) CaCl(2), and its pH was adjusted to 6.5. The experimental studies were conducted under the following conditions: temperature 45-160 degrees C, heating time 10-20 min, and surface roughness of substrate #120-#2000 grid ground using energy paper. A high quality of precipitate, whose predominant component was HA, was obtained on titanium substrates by the thermal substrate method in an aqueous solution. No significant difference in the precipitates was found with the type of ion source. The amount of HA precipitate increased with increasing temperature and with increasing heating time. The features of the precipitate were different, depending on the surface roughtness of the substrate: HA regularly nucleated along the grooves of the rough surface (#120 and #400 grid), and in the case of the fine surface (#1200-#2000 grid), a uniform precipitation occurred.     

Biocompatibility of titanium implants modified by microarc oxidation and hydroxyapatite coating

A thin hydroxyapatite (HA) layer was coated on a microarc oxidized titanium (MAO-Ti) substrate by means of the sol-gel method. The microarc oxidation (anodizing) enhanced the biocompatibility of the Ti, and the bioactivity was improved further by the sol-gel HA coating on the anodized Ti. The HA sol was aged fully to obtain a stable and phase-pure HA, and the sol concentration was varied to alter the coating thickness. Through the sol-gel HA coating, the Ca and P concentrations in the coating layer increased significantly. However, the porous morphology and roughness of the MAO-Ti was altered very little by the sol-gel treatment. The proliferation and alkaline phosphatase (ALP) activity of the osteoblast-like cells on the MAO/HA sol-gel-treated Ti were significantly higher than those on the MAO-Ti without the HA sol-gel treatment.     

Effect of hydroxyapatite/tricalcium-phosphate coating on osseointegration of plasma-sprayed titanium alloy implants

This study determined the effects of a plasma-sprayed hydroxyapatite/tricalcium phosphate (HA/TCP) coating on osseointegration of plasma-sprayed titanium alloy implants in a lapine, distal femoral intramedullary model. The effects of the HA/TCP coating were assessed at 1, 3, and 6 months after implant placement. The HA/TCP coating significantly increased new bone apposition onto the implant surfaces at all time points. The ceramic coating also stimulated intramedullary bone formation at the middle and distal levels of the implants. Fluorescent bone labeling indicated that new bone formation occurred primarily during the first 3 months after implantation, with comparatively little activity detected in the latter stages of the study. There was no associated increase in pullout strength at either 3 or 6 months; however, post-pullout evaluation of the implants indicated that the HA/TCP coating itself was not the primary site of construct failure. Rather, failure was most commonly observed through the periprosthetic osseous struts that bridged the medullary cavity. The demonstrated osteoconductive activity of HA/TCP coating on plasma-sprayed titanium alloy implant surfaces may have considerable clinical relevance to early host-implant interactions, by accelerating the establishment of a stable prosthesis-bone interface.     

Soft tissue response to titanium dioxide nanotube modified implants

Titanium is widely used clinically, yet little is known regarding the effects of modifying its three-dimensional surface geometry at the nanoscale level. In this project we have explored the in vivo response in terms of nitric oxide scavenging and fibrotic capsule formation to nano-modified titanium implant surfaces. We compared titanium dioxide (TiO(2)) nanotubes with 100 nm diameters fabricated by electrochemical anodization with TiO(2) control surfaces. Significantly lower nitric oxide was observed for the nanostructured surface in solution, suggesting that nanotubes break down nitric oxide. To evaluate the soft tissue response in vivo TiO(2) nanotube and TiO(2) control implants were placed in the rat abdominal wall for 1 and 6 weeks. A reduced fibrotic capsule thickness was observed for the nanotube surfaces for both time points. Significantly lower nitric oxide activity, measured as the presence of nitrotyrosine (P<0.05), was observed on the nanotube surface after 1 week, indicating that the reactive nitrogen species interaction is of importance. The differences observed between the titanium surfaces may be due to the catalytic properties of TiO(2), which are increased by the nanotube structure. These findings may be significant for the interaction between titanium implants in soft tissue as well as bone tissue and provide a mechanism by which to improve future clinical implants.     

Interface between bone tissue and implants of solid hydroxyapatite or hydroxyapatite-coated titanium implants

Loaded prestressed implants of dense hydroxyapatite and non-loaded hydroxyapatite-coated titanium implants were placed in edentulous regions of the lower jaw of dogs. After 6 month the jaw specimens were fixed and embedded in methyl-methacrylate. Thin non-decalcified ground sections were made for histology. Although the hydroxyapatite showed histological differences between the coated implants and the prestressed solid ones, both had an extensive apposition of normal lamellar bone on the whole surface of the bone-buried part of the implant. The bone contact was very intimate and without any visible intermediate tissue layer. The tissue response observed forms a good biological base for the clinical application of hydroxyapatite-coated titanium implants.     

Nano-scale study of the nucleation and growth of calcium phosphate coating on titanium implants

The nucleation and growth of a calcium phosphate (Ca-P) coating deposited on titanium implants from simulated body fluid was investigated by using atomic force microscopy (AFM) and environmental scanning electron microscopy (ESEM). Forty titanium alloy plates were assigned into two groups. One group with a smooth surface having a maximum roughness R(max) < 0.10 microm (s-Ti6Al4V) and a group with a rough surface with an R(max) < 0.25 microm (r-Ti6Al4V) were used. Titanium samples were immersed in SBF concentrated by five (SBF x 5) from 10 min to 5 h and examined by AFM and ESEM. Scattered Ca-P deposits of approximately 15 nm in diameter appeared after only 10 min of immersion in SBF x 5. These Ca-P deposits grew up to 60-100 nm after 4 h on both s- and r-Ti6Al4V substrates. With increasing immersion time, the packing of Ca-P deposits with size of tens of nanometers in diameter formed larger globules and then a continuous Ca-P film on titanium substrates. A direct contact between the Ca-P coating and the Ti6Al4V surface was observed. The Ca-P coating was composed of nanosized deposits and of an interfacial glassy matrix. This interfacial glassy matrix might ensure the adhesion between the Ca-P coating and the Ti6Al4V substrate. In the case of s-Ti6Al4V substrate, failures within this interfacial glassy matrix were observed overtime. Part of the glassy matrix remained on s-Ti6Al4V while part detached with the Ca-P film. The Ca-P coating detached from the smooth substrate, whereas the Ca-P film extended onto the whole rough titanium surface over time. In the case of r-Ti6Al4V, the Ca-P coating covered evenly the substrate after immersion in SBF x 5 for 5 h. The present study suggested that the heterogeneous nucleation of Ca-P on titanium was immediate and did not depend on the Ti6Al4V surface topography. The further growth and mechanical attachment of the final Ca-P coating strongly depended on the surface, for which a rough topography was beneficial.     

CaTiO(3) coating on titanium for biomaterial application--optimum thickness and tissue response

The objectives of this study were to determine the optimum thickness of a CaTiO(3) film for biomaterial applications and to investigate the biocompatibility and bone formation of titanium with a CaTiO(3) film. First, CaTiO(3) films of 10, 20, 30, and 50 nm in thickness were deposited on titanium substrates using radiofrequency magnetron sputtering followed by annealing at 873 K in air for 7.2 ks. The optimum thickness of the CaTiO(3) film for bone formation was determined by comparison with its performance regarding calcium phosphate formation in Hanks' balanced saline solution (HBSS). Regarding calcium phosphate formation, the performance of the specimen with a 50-nm-thick CaTiO(3) film was superior to those of specimens with other thicknesses. A titanium prism with a CaTiO(3) film of 50-nm in thickness was surgically inserted in both soft and hard rat tissues. The biocompatibility of CaTiO(3)-deposited titanium and bone formation on it was investigated by histological observations. A slight inflammatory reaction was observed around the titanium with the 50-nm-thick CaTiO(3) film, while no severe response, such as degeneration and necrosis, was observed in either soft or hard rat tissue. New bone formation on the titanium plate with the CaTiO(3) film was more active than that without the film. The 50-nm-thick CaTiO(3) film has biocompatibility and can facilitate new bone formation in vivo, and, consequently, it is an excellent surface modification method for biomaterial applications.     

Polymers for healing caps on titanium implants

Healing caps are used during the healing period after abutment connection on titanium implants of the Branemark type. These healing caps consist of a screw embedded in polyamide-6 (PA-6) which is considerably weakened during this healing period. The properties of PA-6 and poly(4-methyl-1-pentene) (PMP) were studied. Storage in water at 37 degrees C led to a marked reduction in the flexural modulus of PA-6, but only a minor decrease for PMP. PA-6 showed a marked increase in volume due to water uptake, whereas PMP showed no significant change in volume. The improved properties of PMP compared to PA-6 when used for healing caps were demonstrated. The results of an agar overlay cytotoxicity test indicated that both PMP and PA-6 were nontoxic under laboratory controlled conditions.