Assessment of Bitterness in Non-Charged Pharmaceuticals with a Taste Sensor: A Study on Substances with Xanthine Scaffold and Allopurinol.

Taste sensors with an allostery approach have been studied to detect non-charged bitter substances, such as xanthine derivatives, used in foods (e.g., caffeine) or pharmaceuticals (e.g., etofylline). In this study, the authors modified a taste sensor with 3-bromo-2,6-dihydroxybenzoic acid and used it in conjunction with sensory tests to assess the bitterness of non-charged pharmaceuticals with xanthine scaffolds (i.e., acefylline and doxofylline), as well as allopurinol, an analogue of hypoxanthine. The results show that the sensor was able to differentiate between different levels of sample bitterness. For instance, when assessing a 30 mM sample solution, the sensor response to acefylline was 34.24 mV, which corresponded to the highest level of bitterness (τ = 3.50), while the response to allopurinol was lowest at 2.72 mV, corresponding to relatively weaker bitterness (τ = 0.50). Additionally, this study extended the application of the sensor to detect pentoxifylline, an active pharmaceutical ingredient in pediatric medicines. These results underscore the taste sensor's value as an additional tool for early-stage assessment and prediction of bitterness in non-charged pharmaceuticals.

Effect of Hydroxybenzoic Acids on Caffeine Detection Using Taste Sensor with Lipid/Polymer Membranes.

A taste sensor with lipid/polymer membranes can objectively evaluate taste. As previously reported, caffeine can be detected electrically using lipid/polymer membranes modified with hydroxybenzoic acids (HBAs). However, a systematic understanding of how HBAs contribute to caffeine detection is still lacking. In this study, we used various HBAs such as 2,6-dihydroxybenzoic acid (2,6-DHBA) to modify lipid/polymer membranes, and we detected caffeine using a taste sensor with the modified membranes. The effect of the concentrations of the HBAs on caffeine detection was also discussed. The results of the caffeine detection indicated that the response to caffeine and the reference potential measured in a reference solution were affected by the log P and pKa of HBAs. Furthermore, the taste sensor displayed high sensitivity to caffeine when the reference potential was adjusted to an appropriate range by modification with 2,6-DHBA, where the slope of the change in reference potential with increasing 2,6-DHBA concentration was steep. This is helpful in order to improve the sensitivity of taste sensors to other taste substances, such as theophylline and theobromine, in the future.

An SPR Sensor Chip Based on Peptide-Modified Single-Walled Carbon Nanotubes with Enhanced Sensitivity and Selectivity in the Detection of 2,4,6-Trinitrotoluene Explosives.

In this study, we developed a surface plasmon resonance (SPR) sensor chip based on 2,4,6-trinitrotoluene (TNT) recognition peptide-modified single-walled carbon nanotubes (SWCNTs). The carboxylic acid-functionalized SWCNTs were immobilized on a 3-aminopropyltriethoxysilane (APTES)-modified SPR Au chip surface. Through π-stacking between the aromatic amino acids and SWCNTs, the TNT recognition peptide TNTHCDR3 was immobilized onto the surface of the SWCNTs. The peptide⁻SWCNTs-modified sensor surface was confirmed and evaluated by atomic force microscope (AFM) observation. The peptide⁻SWCNTs hybrid SPR sensor chip exhibited enhanced sensitivity with a limit of detection (LOD) of 772 ppb and highly selective detection compared with commercialized carboxymethylated dextran matrix sensor chips.

[Research of the Cancer-Odor].

Early detection and resection of cancer is the most effective in the treatment of solid cancer. Development of a new cancer detection method is expected to become a breakthrough to solve various problems for early detection. It has been reported that there is the specific odors of cancer by using bio olfaction such as dogs, and it has been recognized that there is the odors of cancer. Cancer cells acquire malignant traits as a result of metabolic changes originating from genetic mutation. The cancer specific odorous substances may be considered to be the end products of their metabolic changes. Omics researches such as genomics, proteomics, and metabolomics have been extensively performed to comprehensively analyze changes in DNA, RNA, protein, metabolism and its products specific to cancer for the purpose of developing a new cancer detection marker. It is thought that the research on the odor of cancer is also on the line of omics research. It is difficult to identify cancer-specific odorants buried in various environmental substances. However, it is expected that human will be able to acquire the technology, from the fact that they can be recognized by biological olfaction. We are continuing the research with the dream that identification of the odorous substances as a new cancer detection marker and sensor development for it will lead to the happiness of colleagues in the world.

Rational Design of Peptide-Functionalized Surface Plasmon Resonance Sensor for Specific Detection of TNT Explosive.

In this study, a rationally-designed 2,4,6-trinitrotoluene (TNT) binding peptide derived from an amino acid sequence of the complementarity-determining region (CDR) of an anti-TNT monoclonal antibody was used for TNT detection based on a maleimide-functionalized surface plasmon resonance (SPR) sensor. By antigen-docking simulation and screening, the TNT binding candidate peptides were obtained as TNTHCDR1 derived from the heavy chain of CDR1, TNTHCDR2 derived from CDR2, and TNTHCDR3 from CDR3 of an anti-TNT antibody. The binding events between candidate peptides and TNT were evaluated using the SPR sensor by direct determination based on the 3-aminopropyltriethoxysilane (APTES) surface. The TNT binding peptide was directly immobilized on the maleimide-functionalized sensor chip surface from N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). The results demonstrated that peptide TNTHCDR3 was identified and selected as a TNT binding peptide among the other two candidate peptides. Five kinds of TNT analogues were also investigated to testify the selectivity of TNT binding peptide TNTHCDR3. Furthermore, the results indicated that the APTES-GMBS-based SPR sensor chip procedure featured a great potential application for the direct detection of TNT.

Towards an electronic dog nose: surface plasmon resonance immunosensor for security and safety.

This review describes an "electronic dog nose" based on a surface plasmon resonance (SPR) sensor and an antigen-antibody interaction for security and safety. We have concentrated on developing appropriate sensor surfaces for the SPR sensor for practical use. The review covers different surface fabrications, which all include variations of a self-assembled monolayer containing oligo(ethylene glycol), dendrimer, and hydrophilic polymer. We have carried out detection of explosives using the sensor surfaces. For the SPR sensor to detect explosives, the vapor or particles of the target substances have to be dissolved in a liquid. Therefore, we also review the development of sampling processes for explosives, and a protocol for the measurement of explosives on the SPR sensor in the field. Additionally, sensing elements, which have the potential to be applied for the electronic dog nose, are described.

Fabrication of an SPR sensor surface with antifouling properties for highly sensitive detection of 2,4,6-trinitrotoluene using surface-initiated atom transfer polymerization.

In this study, we modified a surface plasmon resonance immunosensor chip with a polymer using surface-initiated atom transfer polymerization (SI-ATRP) for the highly sensitive detection of 2,4,6-trinitrotoluene (TNT). To immobilize a TNT analogue on the polymer, mono-2-(methacryloyloxy)ethylsuccinate (MES), which has a carboxyl group, was used in this study. However, the anti-TNT antibody may adsorb non-specifically on the polymer surface by an electrostatic interaction because MES is negatively charged. Therefore, a mixed monomer with MES and diethylaminoethylmethacrylate (DEAEM), which has a tertiary amino group and is positively charged, was prepared to obtain electroneutrality for suppressing the nonspecific adsorption. The detection of TNT was performed by inhibition assay using the polymer surface. To ensure high sensitivity to TNT, the affinity between the surface and the antibody was optimized by controlling the density of the initiator for ATRP by mixing two types of self-assembled monolayer reagents. As a result, a limit of detection of 5.7 pg/mL (ppt) for TNT was achieved using the optimized surface.

Development of two-dimensional qualitative visualization method for isoflavones secreted from soybean roots using sheets with immobilized bovine serum albumin.

A visualization method for the qualitative evaluation of soybean isoflavones secreted from soybean roots by transferring them onto a sheet with immobilized bovine serum albumin (BSA) was developed. BSA was chemically bonded onto a glass microfiber filter. The fluorescence quenching resulting from the interaction of BSA with soybean isoflavones such as daidzein and daidzin was utilized. Fluorescence images before and after soybean roots were placed in contact with the sheets with immobilized BSA were taken with an electron-multiplying charge-coupled device camera. The fluorescence quenching in the images was visualized and analyzed. Soybean isoflavones were extracted from the sheets for quantitative analysis, and the correlation coefficient between the quenched fluorescence intensity per sheet and the total amount of soybean isoflavones was 0.78 (p < 0.01), indicating a high correlation. The quenched fluorescence intensity was lower in pumpkin roots, which do not secrete soybean isoflavone. It was found from analyzed images that soybean isoflavone is secreted in larger amounts from the basal region of the taproot and the tips of the lateral roots of soybean.

A bioinspired peptide matrix for the detection of 2,4,6-trinitrotoluene (TNT).

A novel peptide-based three-dimensional probe called "peptide matrix," inspired by the antibody paratope region, was fabricated on a surface plasmon resonance (SPR) sensor chip to enhance the sensitivity of detecting the explosive 2,4,6-trinitrotoluene (TNT). Although peptide aptamer is an attractive candidate for a molecular recognition probe because of its ease of synthesis and chemical stability, it still has difficulty in applying to highly sensitive (i.e. parts-per-billion (ppb) or sub-ppb level) detections. Thus, we developed the concept of peptide matrix structure, which is constructed by consecutive disulfide bond formation between a large number of peptide fragments. This robust three-dimensional structure displays multiple binding sites which can efficiently associate with each TNT molecule. The peptide matrix lowered the dissociation constant (KD) by two orders of magnitude compared to the linear peptide aptamer, estimating KD as 10.1 nM, which is the lowest concentration reported by using peptide-based TNT probe. Furthermore, the concentration limit of detection of peptide matrix modified SPR sensor was 0.62 ppb, and hence comparable to single-chain variable fragment (scFv)-based TNT sensors. To our knowledge, this is the first report demonstrating peptide matrix fabrication and its application for small explosive molecule detection. This peptide matrix-based approach, which has the advantage of simple synthesis and high sensitivity, will be applicable to many other small-molecule label-free detections.

Identification of characteristic compounds of moderate volatility in breast cancer cell lines.

In this study, we were challenging to identify characteristic compounds in breast cancer cell lines. GC analysis of extracts from the culture media of breast cancer cell lines (MCF-7, SK-BR-3, and YMB-1) using a solid-phase Porapak Q extraction revealed that two compounds of moderate volatility, 1-hexadecanol and 5-(Z)-dodecenoic acid, were detected with markedly higher amount than those in the medium of fibroblast cell line (KMST-6). Furthermore, LC-TOF/MS analysis of the extracts clarified that in addition to the above two fatty acids, the amounts of five unsaturated fatty acids [decenoic acid (C10:1), decadienoic acid (C10:2), 5-(Z)-dodecenoic acid (C12:1), 5-(Z)-tetradecenoic acid (C14:1), and tetradecadienoic acid (C14:2)] in MCF-7 medium were higher than those in medium of KMST-6. Interestingly, H2O2-oxidation of 5-(Z)-dodecenoic acid and 5-(Z)-tetradecenoic acid produced volatile aldehydes that were reported as specific volatiles in breath from various cancer patients, such as heptanal, octanal, nonanal, decanal, 2-(E)-nonenal, and 2-(E)-octenal. Thus, we concluded that these identified compounds over-produced in breast cancer cells in this study could serve as potential precursors producing reported cancer-specific volatiles.

Metabolome Analysis Identified Okaramines in the Soybean Rhizosphere as a Legacy of Hairy Vetch.

Inter-organismal communications below ground, such as plant-microbe interactions in the rhizosphere, affect plant growth. Metabolites are shown to play important roles in biological communication, but there still remain a large number of metabolites in soil to be uncovered. Metabolomics, a technique for the comprehensive analysis of metabolites in samples, may uncover the molecules that intermediate these interactions. We conducted a multivariate analysis using liquid chromatography (LC)-mass spectrometry (MS)-based untargeted metabolomics in several soil samples and also targeted metabolome analysis for the identification of the candidate compounds in soil. We identified okaramine A, B, and C in the rhizosphere soil of hairy vetch. Okaramines are indole alkaloids first identified in soybean pulp (okara) inoculated with Penicillium simplicissimum AK-40 and are insecticidal. Okaramine B was detected in the rhizosphere from an open field growing hairy vetch. Okaramine B was also detected in both bulk and rhizosphere soils of soybean grown following hairy vetch, but not detected in soils of soybean without hairy vetch growth. These results suggested that okaramines might be involved in indirect defense of plants against insects. To our knowledge, this is the first report of okaramines in the natural environment. Untargeted and targeted metabolomics would be useful to uncover the chemistry of the rhizosphere.

Development of an oligo(ethylene glycol)-based SPR immunosensor for TNT detection.

This paper describes the development of novel biosensor surfaces supported by robust self-assembled monolayers (SAMs) of aromatic alkanedithiol and oligo(ethylene glycol) (OEG) linker for highly sensitive surface plasmon resonance (SPR) detection of 2,4,6-trinitrotoluene (TNT). Aromatic alkanedithiol SAMs were firstly formed on Au sensor surface and TNT analogues were immobilized on it through OEG chain. Two kinds of OEG containing amine compounds, where H(2)N(C(2)H(4)O)(11)C(2)H(4)NHCOOC(CH(3))(3) served as a linker to react with carboxyl groups of TNT analogues while H(2)N(C(2)H(4)O)(3)C(2)H(4)OH served as a protein non-fouling background, were covalently bound to carboxyl terminal groups of SAMs with a certain ratio. Optimal ratio of them was also examined. Three kinds of TNT analogues, namely TNP-glycine, DNP-glycine, and DNP-acetic acid were used as immobilized ligands. Highly sensitive TNT detection by indirect competitive assay was conducted on the fabricated sensor surfaces; we examined how structural variations of them affect sensitivity in order to choose optimal hapten as well to improve sensitivity. The DNP-acetic acid immobilized surface, which had the lowest affinity to the TNT antibody among the three, showed the best limit of detection (LOD) value (ca. 80 ppt (pg ml(-1))). On the other hand, the TNP-glycine immobilized surface, which had the highest affinity, showed the worst LOD value (ca. 220 ppt). The LOD got lower to ca. 50 ppt by the use of the secondary antibody on the DNP-acetic acid immobilized surface. The sensor surfaces are durable for more than 100 times repeated use without any noticeable deterioration by their chemical stability and rather mild regeneration condition.

Effects of Manufacturing Processing Conditions on Retronasal-Aroma Odorants from a Milk Coffee Drink.

To develop a ready-to-drink (RTD) milk coffee that retains the original coffee flavor, the effects of manufacturing processing conditions on retronasal-aroma (RA) odorants were investigated by gas chromatography-olfactometry (CharmAnalysis™) using an RA simulator (RAS). Twenty-nine of 33 odorants detected in the RAS effluent (RAS odorants) were identified. The detected odorants were classified into 19 odor-description groups. The total odor intensity (charm value, CMV) of all coffee RAS odorants decreased approximately 68% following pH adjustment, whereas the total CMV increased 6% to 7% following ultra-high-temperature sterilization. The total CMV ratio (about 83%) of the milk coffee produced using a new blending-after-sterilization (BAS) process without pH adjustment of the coffee was greater than that (approximately 56%) prepared using a conventional blending-before-sterilization (BBS) process with pH adjustment. In BAS-processed milk coffees, the total CMV ratio (91%) with infusion (INF)-sterilized reconstituted milk (r-milk) was greater than that (83%) of plate (PLT)-sterilized r-milk. Principal component analysis of odor-description CMVs indicated that the effect of coffee pH adjustment on odor characteristics was greater than that of sterilization, that BAS and BBS samples differed, and that BAS milk coffee prepared using INF sterilization was more similar to homemade milk coffee (blending unsterilized coffee without pH adjustment with PLT-sterilized milk) than milk coffee prepared using PLT sterilization. In conclusion, the BAS process using INF sterilization is superior for manufacturing RTD milk coffee that retains odor characteristics similar to targeted homemade milk coffee. PRACTICAL APPLICATION: Ready-to-drink milk coffee beverages produced using conventional blending-before-sterilization methods do not retain their original coffee flavor following adjustment of the pH of the coffee during manufacturing. The use of newly developed blending-after-sterilization methods, by contrast, produces ready-to-drink milk coffee with an aroma more similar to that of homemade milk coffee, as demonstrated using an analytical system for characterizing food product aromas. The blending-after-sterilization process is now being used in Japan to produce ready-to-drink milk coffee beverages.

Effects of Processing Conditions During Manufacture on Retronasal-Aroma Compounds from a Milk Coffee Drink.

To develop a ready-to-drink (RTD) milk coffee retaining the original coffee flavor, the effects of processing conditions during manufacture on retronasal-arma (RA) compounds from the milk coffee were investigated by gas chromatography-mass spectrometry using an RA simulator (RAS). Thirteen of 46 detected compounds in the RAS effluent (RAS compounds) decreased significantly following pH adjustment of coffee (from pH 5.1 to 6.8) and 5 compounds increased. RAS compounds from coffee tended to decrease through the pH adjustment and subsequent sterilization. Significantly higher amounts of 13 RAS compounds were released from the milk coffee produced using a blending-after-sterilization (BAS) process without the pH adjustment than from that using a blending-before-sterilization (BBS) process with the pH adjustment. In BAS-processed milk coffee, significantly lower amounts of 8 high-volatility compounds and 1H-pyrrole were released from coffee containing infusion-sterilized (INF) milk than from coffee containing plate-sterilized (PLT) milk, whereas 3 low-volatility compounds were released significantly more from coffee using PLT milk. Principal component analysis revealed that the effect of the manufacturing process (BAS, BBS, or homemade (blending unsterilized coffee without pH adjustment with sterilized milk)) on milk coffee volatiles was larger than that of the sterilization method (INF or PLT) for milk, and that the sterilization method could result in different RAS volatile characteristics in BAS and homemade processes. In conclusion, a BAS process was found to be superior to a BBS process for the manufacture of an RTD milk coffee that retains volatile characteristics similar to that of a homemade milk coffee. PRACTICAL APPLICATION: Ready-to-drink (RTD) milk coffee manufactured using the conventional blending-before-sterilization process does not retain its original coffee flavor due to pH adjustment of the coffee during the process. The new blending-after-sterilization (BAS) process enabled the production of RTD milk coffee whose volatiles are closer to that of homemade milk coffee, as demonstrated by the results of RAS-GC-MS analysis. The BAS process has already been applied to the manufacture of RTD milk coffees in Japan.

Array-Based Rational Design of Short Peptide Probe-Derived from an Anti-TNT Monoclonal Antibody.

Complementarity-determining regions (CDRs) are sites on the variable chains of antibodies responsible for binding to specific antigens. In this study, a short peptide probe for recognition of 2,4,6-trinitrotoluene (TNT), was identified by testing sequences derived from the CDRs of an anti-TNT monoclonal antibody. The major TNT-binding site in this antibody was identified in the heavy chain CDR3 by antigen docking simulation and confirmed by an immunoassay using a spot-synthesis based peptide array comprising amino acid sequences of six CDRs in the variable region. A peptide derived from heavy chain CDR3 (RGYSSFIYWF) bound to TNT with a dissociation constant of 1.3 µM measured by surface plasmon resonance. Substitution of selected amino acids with basic residues increased TNT binding while substitution with acidic amino acids decreased affinity, an isoleucine to arginine change showed the greatest improvement of 1.8-fold. The ability to create simple peptide binders of volatile organic compounds from sequence information provided by the immune system in the creation of an immune response will be beneficial for sensor developments in the future.

Fabrication of surface plasmon resonance sensor surface with control of the non-specific adsorption and affinity for the detection of 2,4,6-trinitrotoluene using an antifouling copolymer.

We fabricated a surface plasmon resonance (SPR) sensor using a hydrophilic polymer for the highly sensitive detection of 2,4,6-trinitrotoluene (TNT). The hydrophilic polymer was made from mono-2-(methacryloyloxy)ethylsuccinate (MES) and 2-hydroxyethylmethacrylate (HEMA) by surface-initiated atom transfer radical polymerization. The detection of TNT was carried out by displacement assay with the SPR measurement. In displacement assay, the affinity between anti-TNT antibody and the sensor surface, affects to the sensitivity. In the SPR measurement, non-specific adsorption should be controlled because SPR sensor cannot discriminate between specific and non-specific adsorption. Therefore, the affinity and non-specific adsorption were controlled by changing the ratio of HEMA to MES. A detection limit of 0.4 ng/ml (ppb) for TNT was achieved using a sensor surface with the lowest affinity without non-specific adsorption.

Development of Indirect Competitive Immuno-Assay Method Using SPR Detection for Rapid and Highly Sensitive Measurement of Salivary Cortisol Levels.

The monitoring of salivary cortisol as a key biomarker of an individual's stress response has been increasingly focused on. This paper describes the development of a novel cortisol immuno-assay method based on an indirect competitive method using a commercially available surface plasmon resonance instrument. The surface of an Au chip was modified with PEG6-COOH aromatic dialkanethiol self-assembled monolayers and hydrocortisone 3-(O-carboxymethyl) oxime (hydrocortisone 3-CMO) as a cortisol analog. A detection limit of 38 ppt range with a measurement range of 10 ppt-100 ppb was accomplished without the incubation of a mixing solution consisting of standard cortisol and an anti-cortisol antibody, and the time for quantification of cortisol concentration was 8 min from the sample injection. We experimentally compared our immuno-assay with a commercialized salivary cortisol enzyme-linked immunosorbent assay (ELISA) kit using human saliva samples. It was found that the results obtained by the cortisol immuno-assay had a good correlation with those obtained by ELISA assay (R = 0.96). Our findings indicate the potential utility of the cortisol immuno-assay for measurements of human salivary cortisol levels.

Sensitive detection of capsaicinoids using a surface plasmon resonance sensor with anti-homovanillic Acid polyclonal antibodies.

Recently, highly functional biosensors have been developed in preparation for possible large-scale terrorist attacks using chemical warfare agents. Practically applicable sensors are required to have various abilities, such as high portability and operability, the capability of performing rapid and continuous measurement, as well as high sensitivity and selectivity. We developed the detection method of capsaicinoids, the main component of some lachrymators, using a surface plasmon resonance (SPR) immunosensor as an on-site detection sensor. Homovanillic acid, which has a vanillyl group similar to capsaicinoids such as capsaicin and dihydrocapsaicin, was bound to Concholepas concholepas hemocyanin (CCH) for use as an immunogen to generate polyclonal antibodies. An indirect competitive assay was carried out to detect capsaicinoids using SPR sensor chips on which different capsaicin analogues were immobilized. For the sensor chip on which 4-hydroxy-3-methoxybenzylamine hydrochloride was immobilized, a detection limit of 150 ppb was achieved. We found that the incubation time was not required and the detection can be completed in five minutes.

Noncompetitive immunodetection of benzaldehyde by open sandwich ELISA.

Benzaldehyde (Bz) is a typical fragrant compound for peach-flavored beverages. In the food and beverage industries there is great demand for a sensitive and easy detection system of Bz in order to ensure product quality control and to avoid contamination. For the noncompetitive detection of Bz, we applied an open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) utilizing an antigen-dependent reassociation of antibody variable region fragments, VH and VL. We cloned the VH and VL genes of an anti-Bz monoclonal antibody, and the fragments were individually expressed and purified as a bacterial alkaline phosphatase (BAP)-conjugated form for VH and as a thioredoxine (Trx)-fused form for VL, respectively. Using these VH and VL fragments, we successfully constructed the OS-ELISA system for Bz detection. The Bz-induced formation of a trimolecular complex composed of VH-BAP/Bz/Trx-VL was readily detected by a dose-dependent increase in the BAP activity of the VH-fusion protein.

Effects of anterior lumbar spinal fusion on the distribution of nerve endings and mechanoreceptors in the rabbit facet joint: quantitative histological analysis.

The purpose of this study was to investigate the density and distribution of neural endings in rabbit lumbar facet joints after anterior spinal fusion and to evaluate the effects of intervertebral immobilization. An extraperitoneal approach was applied, and L5/6 was fixed with a plate and screws. Bilateral L4/5, L5/6, and L6/7 facet joint capsules were harvested from the rabbits 4, 8, and 16 weeks postoperatively. Capsular tissues were processed using a modified gold chloride staining method, and the specimens were sliced into 15-micro m sections. All sections were analyzed microscopically, and neural ending numbers per unit volume were calculated. Three types of neural ending were identified in each specimen: Pacinian corpuscles, Ruffini corpuscles, and free nerve endings. In the L5/6 fusion segment there was a significant decrease in the number of Pacinian corpuscles at 4 weeks and of Ruffini corpuscles at 4, 8, and 16 weeks after the fusion compared with the control; and in the L4/5 upper adjacent segment there was a significant increase in the number of free nerve endings. The number of Ruffini endings for the L6/7 lower adjacent segment was significantly lower more than 8 weeks after the fusion. These results suggest that immobilization of the intervertebral segment causes a reduction in the number of mechanoreceptors in the facet joint capsules because of the reduction in mechanical stimulation. Moreover, in the upper adjacent facet joint there may be neural sprouting caused by nociceptive stimulation.