Takuya Taniwaki

Characterization of an exchangeable gene trap using pU-17 carrying a stop codon-βgeo cassette

We have developed a new exchangeable gene trap vector, pU‐17, carrying the intron‐lox71‐splicing acceptor (SA)‐βgeo‐loxP‐pA‐lox2272‐pSP73‐lox511. The SA contains three stop codons in‐frame with the ATG of βgalactosidase/neomycin‐resistance fusion gene (βgeo) that can function in promoter trapping. We found that the trap vector was highly selective for integrations in the introns adjacent to the exon containing the start codon. Furthermore, by using the Cre‐mutant lox system, we successfully replaced the βgeo gene with the enhanced green fluorescent protein (EGFP) gene, established mouse lines with the replaced clones, removed the selection marker gene by mating with Flp‐deleter mice, and confirmed that the replaced EGFP gene was expressed in the same pattern as the βgeo gene. Thus, using this pU‐17 trap vector, we can initially carry out random mutagenesis, and then convert it to a gain‐of‐function mutation by replacing the βgeo gene with any gene of interest to be expressed under the control of the trapped promoter through Cre‐mediated recombination.

Intracellular accumulation of advanced glycation end products induces apoptosis via endoplasmic reticulum stress in chondrocytes

Mammalian cells attempt to maintain their homeostasis under endoplasmic reticulum (ER) stress. If the stress cannot be alleviated, cells are led to apoptosis through induction of C/EBP homologous protein (CHOP). ER stress is provoked in osteoarthritis chondrocytes, and intracellular accumulation of advanced glycation end products (AGEs) in chondrocytes is a possible cause. To clarify the role of intracellular AGE accumulation in chondrocytes, the present study investigated the effect of intracellular AGE accumulation on ER stress and apoptosis by in vitro and in vivo analysis. Intracellular AGE accumulation induced by AGE precursors caused apoptosis, induced expression of ER stress markers, and led to co‐localization of AGEs with glucose‐regulated protein 78, leading to formation of high‐molecular‐weight complexes in cultured chondrocytes. These reactions were inhibited by an AGE formation inhibitor. CHOP deletion inhibited apoptosis induced by intracellular AGE accumulation. In vivo intracellular AGE accumulation induced by intra‐articular injection of AGE precursors caused ER stress and apoptosis in chondrocytes and led to degradation of articular cartilage. Additionally, intracellular AGE accumulation increased the degree of cartilage degradation in an osteoarthritis model. These data indicate that intracellular accumulation of AGEs induces modification of unfolded protein response‐related protein by AGEs and apoptosis via ER stress in chondrocytes. Moreover, the in vivo study showed that intracellular AGE accumulation in chondrocytes is involved in the occurrence and progression of osteoarthritis through ER stress. Thus, research on mechanisms of apoptosis via ER stress induced by intracellular AGE accumulation in chondrocytes will lead to a new understanding of osteoarthritis pathology.

FGF-2 Stimulates the Growth of Tenogenic Progenitor Cells to Facilitate the Generation of Tenomodulin-Positive Tenocytes in a Rat Rotator Cuff Healing Model

Background: Fibroblast growth factor (FGF)–2 has the potential to enhance tendon-to-bone healing after rotator cuff (RC) injury. Hypothesis: FGF-2 stimulates tenogenic differentiation of progenitors to improve the biomechanical strength and histological appearance of repaired RCs in rats. Study Design: Controlled laboratory study. Methods: Adult male Sprague-Dawley rats (N = 156) underwent unilateral surgery to repair the supraspinatus tendon to insertion sites. The FGF-2-treated group (gelatin hydrogel containing 5 μg of FGF-2) and a control group (gelatin hydrogel only) were compared to investigate the effects of FGF-2 at 2, 4, 6, 8, and 12 weeks postoperatively. Biomechanical testing was performed at 6 and 12 weeks. Semiquantitative histological analysis and immunohistochemical analysis for the proliferating cell nuclear antigen (PCNA) were performed, and the expression of tendon-related markers, including Scleraxis (Scx) and Tenomodulin (Tnmd), was monitored by real-time reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. SRY-box containing gene 9 (Sox9) expression was monitored by RT-PCR and immunohistochemical analysis. At 2 and 4 weeks, immunohistochemical analysis for mesenchymal stem cell (MSC) markers was also performed. Results: The FGF-2-treated group demonstrated a significant improvement in mechanical strength at 6 and 12 weeks and significantly higher histological scores than the control group at ≥4 weeks. The average incidence of PCNA-positive cells was significantly higher at 2 and 4 weeks, and more cells expressing MSC markers were detected at the insertion site in the FGF-2-treated group. The expression level of Scx increased significantly in the FGF-2-treated group from 4 to 8 weeks, while the Tnmd level increased significantly from 4 to 12 weeks postoperatively. The localization of Tnmd overlapped with the locations of reparative tissues accompanying collagen fibers with an aligned orientation. Sox9 expression was significantly upregulated at 4 weeks in the FGF-2-treated group. Conclusion: FGF-2 promotes growth of the tenogenic progenitor cells, which participate in tendon-to-bone healing, resulting in biomechanical and histological improvement of the repaired RC. Clinical Relevance: These findings provide clues regarding the clinical development of regenerative repair strategies for RC injury.

Angiopoietin-like protein 2 induced by mechanical stress accelerates degeneration and hypertrophy of the ligamentum flavum in lumbar spinal canal stenosis

Chronic inflammation and subsequent fibrosis induced by mechanical stress play an important role in ligamentum flavum (LF) hypertrophy and degeneration in patients with lumbar spinal canal stenosis (LSCS). Angiopoietin-like protein 2 (Angptl2) is a chronic inflammatory mediator induced under various pathological conditions and increases the expression of TGF-β1, which is a well-characterized mediator in LF hypertrophy. We investigated whether Angptl2 is induced by mechanical stress, and whether it contributes to LF hypertrophy and degeneration by activating the TGF-β1 signaling cascade. In this study, we investigated human LF tissue and LF fibroblasts isolated from patients who underwent lumbar surgery. We found that Angptl2 was abundantly expressed in fibroblasts of hypertrophied LF tissues at both the mRNA and protein levels. This expression was not only positively correlated with LF thickness and degeneration but also positively correlated with lumbar segmental motion. Our in vitro experiments with fibroblasts from hypertrophied LF tissue revealed that mechanical stretching stress increases the expression and secretion of Angptl2 via activation of calcineurin/NFAT pathways. In hypertrophied LF tissue, expression of TGF-β1 mRNA was also increased and TGF-β1/Smad signaling was activated. Angptl2 expression in LF tissue was positively correlated with the expression of TGF-β1 mRNA, suggesting cooperation between Angptl2 and TGF-β1 in the pathogenesis of LF hypertrophy. In vitro experiments revealed that Angptl2 increased levels of TGF-β1 and its receptors, and also activated TGF-β1/Smad signaling. Mechanical stretching stress increased TGF-β1 mRNA expression, which was partially attenuated by treatment with a calcineurin/NFAT inhibitor or Angptl2 siRNA, indicating that induction of TGF-β1 expression by mechanical stretching stress is partially mediated by Angptl2. We conclude that expression of Angptl2 induced by mechanical stress in LF fibroblasts promotes LF tissue degeneration by activation of TGF-β1/Smad signaling, which results in LF hypertrophy in patients with LSCS.

Detecting ICRS grade 1 cartilage lesions in anterior cruciate ligament injury using T1ρ and T2 mapping

OBJECTIVE The purpose of this study was to clarify the detectability of the International Cartilage Repair Society (ICRS) grade 1 cartilage lesions in anterior cruciate ligament (ACL)-injured knees using T1ρ and T2 mapping. MATERIALS AND METHODS We performed preoperative T1ρ and T2 mapping and 3D gradient-echo with water-selective excitation (WATS) sequences on 37 subjects with ACL injuries. We determined the detectability on 3D WATS based on arthroscopic findings. The T1ρ and T2 values (ms) were measured in the regions of interest that were placed on the weight-bearing cartilage of the femoral condyle. The receiver operating characteristic (ROC) curve based on these values was constructed using the arthroscopic findings as a reference standard. The evaluation of cartilage was carried out only in the weight-bearing cartilage. The cut-off values for determining the presence of a cartilage injury were determined using each ROC curve, and the detectability was calculated for the T1ρ and T2 mapping. RESULTS The cut-off values for the T1ρ and T2 were 41.6 and 41.2, respectively. The sensitivity and specificity of T1ρ were 91.2% and 89.5%, respectively, while those of T2 were 76.5% and 81.6%, respectively. For the 3D WATS images, the same values were 58.8% and 78.9%, respectively. CONCLUSIONS Our study demonstrated that the T1ρ and T2 values were significantly higher for ICRS grade 1 cartilage lesions than for normal cartilage and that the two mappings were able to non-invasively detect ICRS grade 1 cartilage lesions in the ACL-injured knee with a higher detectability than were 3D WATS images.

L5 radiculopathy caused by a ganglion cyst of the posterior longitudinal ligament in a teenager

BACKGROUND CONTEXT There is no previous report on the intraspinal ganglion cyst of the posterior longitudinal ligament in a teenager. PURPOSE To report a case of radiculopathy caused by a ganglion cyst of the posterior longitudinal ligament in a teenager. STUDY DESIGN Case report. METHODS A 17-year-old male with a 4-month history of left L5 radicular pain was found to have an intraspinal cystic lesion causing radicular compression. Magnetic resonance imaging showed a cystic lesion located in the ventral side of the dura. The patient suffered from severe leg pain. As a result, a surgical operation was therefore performed. RESULTS The cyst containing jelly-like components and a hemorrhage was punctured and then extirpated. It originated from the posterior longitudinal ligament. A histological study revealed the cyst to be without any synovial layers. CONCLUSIONS This is the first report to describe a ganglion cyst originating from the posterior longitudinal ligament in a teenager. This possible etiology should be kept in mind for any other individuals displaying symptoms of spinal nerve root compression as well as disc herniation.

Effects of the water‐holding capability of polyvinyl formal sponges on osteogenic ability in in vivo experiments

In this study, dextran-coated polyvinyl formal (PVF) sponges with high water-holding capability were developed to increase the osteogenic response in the PVF sponge. The study aimed to estimate the effect of the increased water-holding capability of the sponges on osteogenic capacity at a bone defect site in the rabbit femur epiphysis. Bone formation was evaluated using radiography, microcomputed tomography (CT), and histological analysis at 2, 4, and 6 weeks after implantation. As shown by radiography and micro-CT findings, the dextran-coated PVF sponge without water-holding capability showed little bone formation at all evaluated time points. However, the dextran-coated PVF sponge with high water-holding capability showed increasing bone formation around the implant at 4 and 6 weeks after implantation. Furthermore, as shown by micro-CT quantitative analysis, the grafted PVF sponge with high water-holding capability showed significantly greater values for percentage of bone volume per total volume and mean bone mineral density compared with the grafted PVF sponge without water-holding capability at 4 and 6 weeks after implantation. These results suggest that the dextran-coated PVF sponge with high water-holding capability promoted osteogenesis in vivo. The PVF sponge might be a new biomaterial to be used as a fill material for bone defects.

Corresponding scapular pain with the nerve root involved in cervical radiculopathy.

Purpose. To correspond scapular pain with the nerve root involved in cervical radiculopathy. Methods. In the anatomic study, 11 Japanese adult cadavers were dissected to examine the numbers and courses of the cutaneous nerves from C3 to C8 dorsal rami. In the clinical study, 14 men and 11 women aged 34 to 77 years who presented with scapular pain as well as pain, numbness or motor weakness in the upper limbs secondary to cervical radiculopathy were assessed. The involved nerve roots were identified based on the symptoms and signs in the arm and/or fingers, the radiological diagnosis, and the pain response to cervical nerve root blocks. The sites and characteristics of radicular pain were assessed. Results. In the anatomic study of 22 cutaneous nerves from medial branches of dorsal rami, 18 involved the C5 nerve root, 0 the C6 root, one the C7 root, and 8 the C8 root. In the clinical study, the radicular pain often occurred in the suprascapular region involving the C5 root, in the suprascapular to posterior deltoid region involving the C6 root, in the interscapular region involving the C7 root, and in the interscapular and scapular regions involving the C8 root. All patients with C5 or C8 radiculopathy had both superficial and deep pain, whereas almost all patients with C6 or C7 radiculopathy had deep pain only. No patient had superficial pain only. Conclusion. Cervical radiculopathy can cause scapular pain. Pain sites and characteristics are related to the affected nerve root.

Gas-filled intradural cysts of the lumbar spine and the possible pathogenesis

BACKGROUND CONTEXT There have only been four reports of gas-filled intradural cysts, and the pathogenesis is unknown. PURPOSE To document the radiologic and histopathologic features of gas-filled intradural cysts and to discuss the pathogenesis with a review of the literature. STUDY DESIGN Case report. METHODS A 67-year-old woman, admitted to our institute, presented with severe right thigh pain. On admission to the institute, enhanced magnetic resonance imaging, showed a cystic lesion in the spinal canal at the L2-L3 level, with an intensity suggesting the presence of gas. An enhanced region around the cyst was noted. Computed tomography after discography also revealed a water-soluble contrast filled the subarachnoid space and area around the cyst, but not inside. RESULTS The cyst was surgically resected. One of the nerve roots was firmly adherent to the gas-filled cyst. The cyst wall comprised fibrous tissue, including small granulations and herniated disc material. CONCLUSION Gas-filled intradural cysts are rare. The pathogenesis appears to involve gas in a degenerated intervertebral disc, and spontaneous absorption of herniated disc material.

Evaluation of the relationship between T1ρ and T2 values and patella cartilage degeneration in patients of the same age group.

OBJECTIVE The aim of this study was to investigate the association between the T1ρ and T2 values and the progression of cartilage degeneration in patients of the same age group. MATERIALS AND METHODS Sagittal T1ρ and T2 mapping and three-dimensional (3D) gradient-echo images were obtained from 78 subjects with medial knee osteoarthritis (OA). The degree of patella cartilage degeneration was classified into four groups using MRI-based grading: apparently normal cartilage, mild OA, moderate OA, and severe OA group. We measured the T1ρ and T2 values (ms) in the regions of interest set on the full-thickness patella cartilage. Then, we analyzed the relationship between the T1ρ and T2 values and the degree of patella cartilage degeneration. RESULTS There were no significant differences in age among the four groups. Both the T1ρ and T2 values showed a positive correlation with the degree of OA progression (ρ=0.737 and ρ=0.632, respectively). By comparison between the apparently normal cartilage and the mild OA groups, there were significant differences in the T1ρ mapping, but not in the T2 mapping. CONCLUSIONS Our study confirmed that T1ρ and T2 mapping can quantitatively evaluate the degree of patella cartilage degeneration in patients within the same age group.