Kouki Hatori

Appositional Bone Formation by OCP-Collagen Composite

Synthetic octacalcium phosphate (OCP) has been shown to enhance bone formation and to biodegrade if implanted into bone defects. Here, we hypothesized that an OCP-atelocollagen complex (OCP/Col) is biodegradable and can induce bone formation in a thickness-dependent manner when implanted into the calvaria. OCP/Col disks (diameter, 9 mm; thickness, 1 or 3 mm) were implanted into a subperiosteal pocket in the calvaria of 12-week-old Wistar rats for 4, 8, and 12 weeks and subsequent bone formation was monitored. X-ray diffraction analysis and Fourier transform infrared spectroscopy showed that OCP in the OCP/Col implants was converted into a carbonate-rich apatite after 4 weeks. Although thinner disks tended to be replaced by new bone, thicker disks were progressively resorbed by osteoclast-like cells until 12 weeks, possibly via the increased mechanical load in the subperiosteal pocket. Therefore, OCP/Col can increase appositional intra-membranous bone formation if the appropriate size of the implant is applied.

Joint Immobilization Induced Hypoxic and Inflammatory Conditions in Rat Knee Joints

The purpose of this study was to examine the hypoxic and inflammatory conditions after immobilization in the joint capsule of rat knees. The unilateral knee joints of adult male rats were immobilized with an internal fixator (Im group) for 1 day, 3 days, and 1, 2, 4, 8, and 16 weeks. Sham-operated animals had holes drilled in the femur and tibia and screws inserted without a plate (control group). The number of cells and blood vessels in the capsule were histologically examined. The hypoxic condition in the capsule was histologically examined with a Hypoxyprobe™-1. The gene expressions related to the hypoxic (hypoxia inducible factor-1α, vascular endothelial growth factor, and fibroblast growth factor 2) and inflammatory conditions [interleukin-6 (IL-6), IL-1α, IL-1β, tumor necrosis factor-α, and tumor necrosis factor-β] were evaluated by quantitative reverse transcription polymerase chain reaction. The number of cells was unchanged at 1 day in the two groups; however, the number significantly increased at 3 days in the Im group. The number of blood vessels in the Im group gradually decreased. Strong immunostaining of Hypoxyprobe™-1 around the blood vessels was observed in the Im group. The gene expressions of hypoxia inducible factor-1α and fibroblast growth factor 2 were significantly higher in the Im group compared with those in the control group. The gene expressions of IL-6, IL-1α, IL-1β, and tumor necrosis factor-β were significantly higher in the Im group compared with those in the control group. These data indicated that joint immobilization induced hypoxic and inflammatory conditions in the joint capsule, which might be an initiating factor for joint contracture.

Chondrogenic and fibrotic process in the ligamentum flavum of patients with lumbar spinal canal stenosis.

Study Design. A histological, biological, and immunohisto-chemical study of human lumbar ligamentum flavum. Objective. To analyze changes in the hypertrophied ligamentum flavum and clarify their etiology. Summary of Background Data. Hypertrophy of the ligamentum flavum has been considered a major contributor to the development of lumbar spinal canal stenosis (LSCS). Although previous studies have reported some factors related to ligamentum flavum hypertrophy, its etiology is still unclear. Methods. Ligamentum flavum samples were collected from 20 patients with LSCS (LSCS group) and 10 patients with lumbar disc herniation (LDH group) as a control. The thickness of the ligamentum flavum was measured histologically. The amounts of elastic fibers and proteoglycans were assessed by Elastica-Masson staining and alcian blue staining, respectively. Gene and protein expressions related to fibrosis, inflammation, and chondrogenesis were analyzed by quantitative reverse transcription–polymerase chain reaction and immunohistochemistry. The total genes of the 2 groups were compared by DNA microarray analysis. Results. The ligamentum flavum was significantly thicker in the LSCS group, which had a smaller amount of elastic fibers and a larger amount of proteoglycans. The gene expression related to fibrosis was significantly higher in the LSCS group; however, the immunoreactivities of collagen types I and III were weaker on the dorsal side of the ligamentum flavum in the LSCS group. The gene expression related to chondrogenesis and proteoglycan synthesis was significantly higher in the LSCS group. There was no significant difference in the gene expression related to inflammation between the 2 groups. Conclusion. Synthesis of the collagenous fibers and degradation of the elastic and collagenous fibers are both accelerated in the ligamentum flavum of patient with LSCS, which may be the reason for hypertrophy of the tissue. In addition, chondrogenesis and proteoglycan synthesis may have critical roles in the pathogenesis of the ligamentum flavum hypertrophy. Level of Evidence: 5

Expression of type I collagen in the capsule of a contracture knee in a rat model

Contracture is a very common complication in daily examination and a fibrotic change of a capsule is suggested to be a one of the main causes. Type I collagen is a major component of a synovial capsule and also has been implicated in tissue elasticity of other organs. We immobilized the knee joints of 66 rats in 150 degrees of flexion using a plastic plate and metal screws. Sham operated knee joints had holes drilled and screws inserted but none of them were plated. The expression patterns of type I collagen were characterized using in situ hybridization and immunohistochemistry. The in situ hybridization demonstrated that the mRNA of type I collagen decreased rapidly after immobilization. However, the immunoreactivity of the capsule was not changed in the immobilized and the control groups at any time points. Other processes might be considered to evaluate the contracture capsule.

Comparison of articular cartilage images assessed by high-frequency ultrasound microscope and scanning acoustic microscope

PurposeThe purpose of this study was to compare images of a newly developed high-frequency ultrasound imaging system (HFUIS) and scanning acoustic microscope (SAM) and to calculate their Pearson product moment correlations with a view to applying HFUIS for clinical use.MethodsCylindrical cartilage–bone complexes from adult male Sprague-Dawley rats were obtained. The specimens were immersed in normal saline and scanned by HFUIS. Intensity by HFUIS was normalised by reflection from a steel plate at the same distance. After the scanning, specimens were fixed with paraformaldehyde, decalcified and embedded in paraffin. Thinly sliced tissues were prepared for SAM evaluation. After the scanning, three layers of articular cartilage (superficial, middle and deep) were independently evaluated and their relationships calculated.ResultsThe superficial and deep layers indicated high relative intensity, whereas the middle layer showed nonhomogeneous relative intensity by HFUIS. A high relative intensity by HFUIS and high sound speed area by SAM had strong correlations (Pearson product moment correlation, superficial layer 0.704, middle layer 0.731).ConclusionsHFUIS produced high-resolution images of the articular cartilage and its intensity was strongly correlated with sound speed by SAM.

Decreased elastic fibers and increased proteoglycans in the ligamentum flavum of patients with lumbar spinal canal stenosis.

Elastic fibers and proteoglycans are major components of the extracellular matrix and their changes have been reported in some pathological conditions. Further, recent studies have indicated that some glycosaminoglycans and proteoglycans inhibit elastic fiber assembly. The purpose of this study was to investigate changes of the elastic fibers and proteoglycans in the ligamentum flavum and analyze their relationships to thickening of the ligamentum flavum from lumbar spinal canal stenosis (LSCS). Ligamentum flavum samples were collected from 20 patients with LSCS (thickened flavum group) and 10 patients with lumbar disc herniation (non‐thickened flavum group) as a control. Elastica–Masson staining and alcian blue staining were used to compare the relationship between the changes in the elastic fibers and proteoglycans. Gene and protein expressions of the elastic fibers and proteoglycans were analyzed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. Histological changes indicated that proteoglycans mainly increased on the dorsal side of the ligamentum flavum in accordance with the decreased elastic fibers in the thickened flavum group. The gene and protein expressions of fibrillin‐2 and DANCE were significantly lower and decorin, lumican, osteoglycin, and versican were significantly higher in the thickened flavum group. Our study shows that elastic fibers decrease and proteoglycans increase in the thickened ligamentum flavum. Decreased gene expression of elastogenesis and disrupted elastic fiber assembly caused by increased proteoglycans may lead to a loss of elasticity in the thickened ligamentum flavum. Decreased elasticity may cause buckling of the tissue, which leads to thickening of the ligamentum flavum.

Root development of rat tooth germs implanted in the tooth socket and in the subcutaneous tissue.

OBJECTIVE This study was designed to investigate root development of a rat tooth germ implanted in a tooth socket or in a subcutaneous region. MATERIALS AND METHODS Tooth germs of the upper left first molars in 2-week-old rats were extracted and implanted in the original tooth socket or in the subcutaneous region of the back. The upper right first molar was used as a control. The rats were fixed in weeks 1, 2, 4, 8 and 12. The root development was examined quantitatively with X-ray radiographic morphometry. The cellular activity of producing matrix proteins was assessed using in situ hybridization for type I collagen. RESULTS Root development was observed in the implanted teeth in the tooth socket as also in the control teeth. In contrast, roots hardly developed in subcutaneously implanted teeth. Histology showed that periodontal ligaments were arranged around roots of implanted teeth in the tooth socket as around control teeth, but few periodontal ligaments were identified in the subcutaneous implantation. Dentin and cementum formed in both the implanted teeth as also in the control teeth and odontoblasts, cementoblasts and cementocytes expressed type I collagen. CONCLUSION Tooth sockets may possess specific environments that allow root development of a tooth germ.

Acoustic Diagnosis Device for Dentistry

There are a lot of diseases which show the abnormal elastic property. Although many medical doctors and dentists have noticed the change of tissue elasticity due to the disease, the diagnostic device to examine the tissue elastic property objectively has not well developed.

Changes of masseter muscle in a rat unilateral occlusal model assessed by photoacoustic imaging system

Biomedical photoacoustic (PA) imaging system has the unique capability of combining high optical contrast with high ultrasound (US) resolution in a single modality. PA imaging system with 532 nm laser and 50 MHz US transducer has been developed. When the laser with the wavelength of 532 nm is used, PA signals are reflected from blood vessels. Patients with unilateral occlusion due to the extraction of unilateral molars are often seen in dental practice. Although the period of the unilateral occlusion induces the changes of masseter muscle (MM), it is not known that how blood vessels and tissue elasticity of MM are affected by the unilateral occlusion. In this study, we aimed to evaluate the sequential changes of MM due to the unilateral occlusion in rats with PA imaging system. PA signals from the MM of the non-occlusal side were significantly lower than that of the occlusal side, and US signals from the MM of the occlusal side were significantly higher than that of the non-occlusal side depending on the ...

High Frequency Ultrasound Imaging of Cartilage-Bone Complex

High frequency ultrasound microscope with central frequency of 100 MHz was developed. The system was capable of (1) conventional C-mode acoustic microscope imaging of thinly sliced tissue, (2) ultrasound impedance imaging of the surface of in vitro thick tissue and (3) 3D ultrasound imaging of inside of the in vitro tissue. In the present study, cylindrical cartilage-bone unit specimens were removed from rat knee joints and evaluated with the equipment. The resolution was enough to visualize the articular cartilage surface morphology and the subchondral bone. Compared with histological sections observed by optical microscope, it can also differentiate the non-calcified zone and calcified zone of the articular cartilage. High frequency ultrasound microscope will provide important information of the structural changes of the articular cartilage.