Association of Ligamentum Flavum Hypertrophy with Adolescent Idiopathic Scoliosis Progression-Comparative Microarray Gene Expression Analysis.

The role of the ligamentum flavum (LF) in the pathogenesis of adolescent idiopathic scoliosis (AIS) is not well understood. Using magnetic resonance imaging (MRI), we investigated the degrees of LF hypertrophy in 18 patients without scoliosis and on the convex and concave sides of the apex of the curvature in 22 patients with AIS. Next, gene expression was compared among neutral vertebral LF and LF on the convex and concave sides of the apex of the curvature in patients with AIS. Histological and microarray analyses of the LF were compared among neutral vertebrae (control) and the LF on the apex of the curvatures. The mean area of LF in the without scoliosis, apical concave, and convex with scoliosis groups was 10.5, 13.5, and 20.3 mm2, respectively. There were significant differences among the three groups (p < 0.05). Histological analysis showed that the ratio of fibers (Collagen/Elastic) was significantly increased on the convex side compared to the concave side (p < 0.05). Microarray analysis showed that ERC2 and MAFB showed significantly increased gene expression on the convex side compared with those of the concave side and the neutral vertebral LF cells. These genes were significantly associated with increased expression of collagen by LF cells (p < 0.05). LF hypertrophy was identified in scoliosis patients, and the convex side was significantly more hypertrophic than that of the concave side. ERC2 and MAFB genes were associated with LF hypertrophy in patients with AIS. These phenomena are likely to be associated with the progression of scoliosis.

Direct Reprogramming and Induction of Human Dermal Fibroblasts to Differentiate into iPS-Derived Nucleus Pulposus-like Cells in 3D Culture.

Intervertebral disc (IVD) diseases are common spinal disorders that cause neck or back pain in the presence or absence of an underlying neurological disorder. IVD diseases develop on the basis of degeneration, and there are no established treatments for degeneration. IVD diseases may therefore represent a candidate for the application of regenerative medicine, potentially employing normal human dermal fibroblasts (NHDFs) induced to differentiate into nucleus pulposus (NP) cells. Here, we used a three-dimensional culture system to demonstrate that ectopic expression of MYC, KLF4, NOTO, SOX5, SOX6, and SOX9 in NHDFs generated NP-like cells, detected using Safranin-O staining. Quantitative PCR, microarray analysis, and fluorescence-activated cell sorting revealed that the induced NP cells exhibited a fully differentiated phenotype. These findings may significantly contribute to the development of effective strategies for treating IVD diseases.

Effect of hyperdry amniotic membrane in preventing tendon adhesion in a rabbit model.

BACKGROUND: No anti-adhesive materials are currently in clinical use for orthopaedic surgery. We developed a hyperdry amniotic membrane (HD-AM) for easy storage and transplantation as amniotic membrane. The purpose of this study was to examine the application of HD-AM to reduce peritendinous adhesions without impairing tendon healing. METHODS: We randomly divided 3 digits (2nd, 3rd, and 4th digits) from each rabbit into three groups: a tendon repair group; a tendon repair with HD-AM group (HD-AM group); and a control group (cast only). The effects of HD-AM on peritendinous adhesions and tendon healing were examined using microscopic, histological, and mechanical analyses in a rabbit flexor digitorum profundus tendon model. RESULTS: Adhesions on macroscopic evaluation of the tendon repair site were significantly smaller in the HD-AM group than in the tendon repair group. Little adhesion formation or foreign body reactions were seen by on histologic evaluation in the HD-AM group. Range of motion following tendon repair was significantly better in the HD-AM group than in the tendon repair group. Maximal tensile strength required to pull the tendon from the site of adhesion was significantly smaller in the HD-AM group than in the tendon repair group. As for tendon repair site, no significant difference was seen between the tendon repair and HD-AM groups. CONCLUSIONS: HD-AM prevented peritendinous adhesion macroscopically, pathologically, and mechanically without impairing the sutured tendon. HD-AM has already been clinically applied in neurosurgery, ophthalmology, and otolaryngology, and clinical application as an anti-adhesive materials may be achieved in the future.

Evaluation of complete and incomplete subscapularis tendon tear on preoperative magnetic resonance imaging.

BACKGROUND: This study aims to evaluate the significance of preoperative magnetic resonance imaging findings to predict subscapularis tear confirmed at arthroscopic surgery. METHODS: Sixty-four consecutive shoulders that underwent preoperative magnetic resonance imaging examination and arthroscopic shoulder operations were retrospectively reviewed. Under arthroscopic examination, complete subscapularis tear was defined as a full-thickness tear and incomplete subscapularis tear as tendon detachment larger than 5 mm from the insertion on the joint side. RESULTS: In arthroscopic findings, they were included 11 shoulders with complete subscapularis tear, 13 with incomplete subscapularis tear, and the remaining 28 shoulders without subscapularis tear. Subscapularis discontinuity by axial magnetic resonance imaging had the highest sensitivity and specificity in detecting complete subscapularis tear compared with other magnetic resonance imaging findings. Long head biceps subluxation or dislocation showed significantly higher prevalence in the complete and incomplete subscapularis tear groups than in the group with no tear. Incomplete subscapularis tear groups had a higher incidence of superior subscapularis recess fluid, and this fluid was present in all the shoulders with incomplete subscapularis tear. CONCLUSIONS: The presence of subscapularis discontinuity is useful for diagnosis of complete subscapularis tear. In addition, in cases of incomplete subscapularis tear, the presence of superior subscapularis recess fluid had 100% sensitivity. Thus, this finding may be a characteristic diagnosis of subscapularis tear including incomplete tear.

Distribution of amniotic stem cells in human term amnion membrane.

Amnion membrane studies related to miscarriage have been conducted in the field of obstetrics and gynecology. However, the distribution of stem cells within the amnion and the differences in the properties of each type of stem cells are still not well understood. We address this gap in knowledge in the present study where we morphologically classified the amnion membrane, and we clarified the distribution of stem cells here to identify functionally different amniotic membrane-derived stem cells. The amnion can be divided into a site that is continuous with the umbilical cord (region A), a site that adheres to the placenta (region B), and a site that is located opposite the placenta (region C). We found that human amnion epithelial stem cells (HAECs) that strongly express stem cell markers were abundant in area A. HAEC not only expressesed stem cell-specific surface markers TRA-1-60, Tra-1-81, SSEA4, SSEA3, but was also OCT-3/4 positive and had alkaline phosphatase activity. Human amniotic mesenchymal stem cells expressed KLF-A, OCTA, Oct3/4, c-MYC and Sox2 which is transcription factor. Especially, in regions A and B they have expressed CD73, and the higher expression of BCRP which is drug excretion transporter protein than the other parts. These data suggest that different types of stem cells may have existed in different area. The understanding the relation with characteristics of the stem cells in each area and function would allow for the efficient harvest of suitable HAE and HAM stem cells as using tool for regenerative medicine.

Is it possible to predict the final component gap in flexion before femoral posterior condylar osteotomy in cruciate-retaining and posterior-stabilized total knee arthroplasty?

BACKGROUND: In total knee arthroplasty (TKA) with posterior condylar osteotomy using anatomical landmarks, predicting the final flexion gap is impossible, as it differs with the presence or absence of the posterior cruciate ligament. We compared the predicted flexion gap, based on pre-femoral posterior condylar osteotomy measurements, with the postsurgical final flexion gap in cruciate-retaining (CR) and posterior-stabilized (PS) TKA. METHODS: One hundred knees of patients with osteoarthritis were included: 35 underwent CR, and 65 PS TKA. Distal femoral and proximal tibial osteotomy using the measured resection technique was performed. An anterior and posterior femoral osteotomy guide was set parallel to the surgical epicondylar axis, and the predicted flexion gap was measured using a seesaw tensor attached to the guide. After all procedures, the final component gap in flexion was measured using a similar seesaw tensor at the patella reduction position and was compared with the predicted gap. RESULTS: The correlation coefficients for predicted vs. final component gap were 0.45 (P < 0.05) in CR and 0.82 (P < 0.001) in PS. The mean differences between predicted and final gaps were 1.8 mm for CR and 1.0 mm for PS. In 34.3% of CR cases, the gap difference was more than 2 mm. CONCLUSION: It is possible to predict the final flexion gap before femoral posterior condylar osteotomy, with a strong correlation observed between predicted and final component gaps in PS TKA. However, in CR, more than 30% of the cases showed unexpectedly large final flexion gaps.

Differential proteome analysis in adolescent idiopathic scoliosis patients with thoracolumbar/lumbar curvatures.

BACKGROUND: Although the pathogenesis of adolescent idiopathic scoliosis (AIS) remains unclear, there are little evidences of the pathogenesis in patients with thoracolumbar/lumbar AIS. The purpose of this study was to identify proteins or proteomes that may be causally related to the pathogenesis of AIS with structured thoracolumbar/lumbar curvature using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). METHODS: A total of 20 control volunteers and 61 AIS in patients with thoracolumbar/lumbar curvature were included. First, the plasma samples of each five AIS with pure thoracolumbar/lumbar curvature and control samples were subjected to 2D-DIGE analysis. Protein spots that were expressed differently by the AIS and control groups were selected and identified by nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis. To characterize the differently-expressed proteins in AIS patients, we performed functional pathway analysis using the Protein ANalysis THrough Evolutionary Relationships (PANTHER) system. Additionally, the proteins were compared between control and AIS using western blotting. Lastly, prospectively collected 15 control and 41 AIS with thoracolumbar/lumbar curvature samples were compared to the differentially expressed proteins. RESULTS: A total of 3862 ± 137 spots were detected, of which 11 spots met the criteria when compared with controls. Nine proteins were identified by nanoLC-MS/MS. Functional analysis showed the association of the proteins in AIS patients with blood coagulation using the PANTHER system. Of the proteins, vitamin D binding protein (DBP) significantly correlated with Cobb angle in thoracolumbar/lumbar curvatures. DBP expression of the prospectively collected AIS samples were significantly higher than those of controls (P < 0.05). CONCLUSIONS: This study suggests that DBP and several coagulation-related proteins may play a role in the pathogenesis of AIS. DBP appears to be a marker of severity of AIS with thoracolumbar/lumbar curvature.

A selective c-Fos/AP-1 inhibitor prevents cartilage destruction and subsequent osteophyte formation.

The objective of the present study is to demonstrate that a newly developed selective c-Fos/activator protein (AP)-1 inhibitor, T-5224, inhibits the expression of matrix metalloproteinases (MMPs) in human articular chondrocytes, and prevents cartilage destruction in an osteoarthritis (OA)-induced mouse model. First, we examined the effect of T-5224 on MMP and inflammatory cytokine expression by real-time polymerase chain reaction in human articular chondrocytes. We created an OA model by destabilization of the medial meniscus (DMM) in mice. T-5224 was orally administered once a day and the OA pathology was assessed by histological, immunohistochemical, and micro-computed tomography (CT) analyses. T-5224 inhibited the mRNA expression levels of MMP-1, 3, and 13, and interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and IL-6 in IL-1-stimulated human chondrocytes. Oral administration of T-5224 to OA-induced mice prevented cartilage destruction. The histological scores for OA were significantly better in the T-5224-treated group than the vehicle-treated group. Type X collagen and MMP-13 were not increased in the T-5224-treated group by immunohistochemical staining. Micro-CT analysis showed mild but apparent osteophyte development in the femoral condyle and antero-medial aspect of the tibia in the vehicle-treated group but not in the T-5224-treated group. Taken together, specific inhibition of c-Fos/AP-1 and the resulting inhibition of the transactivation of a broad spectrum of downstream MMPs, along with inflammatory cytokines, effectively prevented cartilage destruction and osteophyte formation.

A selective inhibition of c-Fos/activator protein-1 as a potential therapeutic target for intervertebral disc degeneration and associated pain.

Intervertebral disc (IVD) degeneration is a major cause of low back pain. The transcription factor c-Fos/Activator Protein-1 (AP-1) controls the expression of inflammatory cytokines and matrix metalloproteinases (MMPs) that contribute to the pathogenesis IVD degeneration. We investigated the effects of inhibition of c-Fos/AP-1 on IVD degeneration and associated pain. A selective inhibitor, T-5224, significantly suppressed the interleukin-1ß-induced up-regulation of Mmp-3, Mmp-13 and Adamts-5 transcription in human nucleus pulposus cells and in a mouse explant culture model of IVD degeneration. We used a tail disc percutaneous needle puncture method to further assess the effects of oral administration of T-5224 on IVD degeneration. Analysis of disc height, T2-magnetic resonance imaging (MRI) findings, and histology revealed that IVD degeneration was significantly mitigated by T-5224. Further, oral administration of T-5224 ameliorated pain as indicated by the extended tail-flick latency in response to heat stimulation of rats with needle-puncture-induced IVD degeneration. These findings suggest that the inhibition of c-Fos/AP-1 prevents disc degeneration and its associated pain and that T-5224 may serve as a drug for the prevention of IVD degeneration.

A Human Amnion-Derived Extracellular Matrix-Coated Cell-Free Scaffold for Cartilage Repair: In Vitro and In Vivo Studies.

OBJECTIVE: Extracellular matrix (ECM) derived from human amniotic mesenchymal cells (HAMs) has various biological activities. In this study, we developed a novel HAM-derived ECM-coated polylactic-co-glycolic acid (ECM-PLGA) scaffold, examined its property on mesenchymal cells, and investigated its potential as a cell-free scaffold for cartilage repair. MATERIALS AND METHODS: ECM-PLGA scaffolds were developed by inoculating HAM on a PLGA. After decellularization by irradiation, accumulated ECM was examined. Exogenous cell growth and differentiation of rat mesenchymal stem cells (MSCs) on the ECM-PLGA were analyzed in vitro by cell attachment/proliferation assay and reverse transcription-polymerase chain reaction. The cell-free ECM-PLGA scaffolds were implanted into osteochondral defects in the trochlear groove of rat knees. After 4, 12, or 24 weeks, the animals were sacrificed and the harvested tissues were examined histologically. RESULTS: The ECM-PLGA contained ECM that mimicked natural amniotic stroma that contains type I collagen, fibronectin, hyaluronic acid, and chondroitin sulfates. The ECM-PLGA showed excellent properties of cell attachment and proliferation. MSCs inoculated on the ECM-PLGA scaffold showed accelerated type II collagen mRNA expression after 3 weeks in culture. The ECM-PLGA implanted into an osteochondral defect in rat knees induced gradual tissue regeneration and resulted in hyaline cartilage repair, which was better than that in the empty control group. CONCLUSION: These in vitro and in vivo experiments show that the cell-free scaffold composed of HAM-derived ECM and PLGA provides a favorable growth environment for MSCs and facilitates the cartilage repair process. The ECM-PLGA may become a "ready-made" biomaterial for cartilage repair therapy.

Successful treatment of methicillin-resistant Staphylococcus aureus osteomyelitis with combination therapy using linezolid and rifampicin under therapeutic drug monitoring.

Linezolid is an effective antibiotic against most gram-positive bacteria including drug-resistant strains such as methicillin-resistant Staphylococcus aureus. Although linezolid therapy is known to result in thrombocytopenia, dosage adjustment or therapeutic drug monitoring of linezolid is not generally necessary. In this report, however, we describe the case of a 79-year-old woman with recurrent methicillin-resistant S. aureus osteomyelitis that was successfully treated via surgery and combination therapy using linezolid and rifampicin under therapeutic drug monitoring for maintaining an appropriate serum linezolid concentration. The patient underwent surgery for the removal of the artificial left knee joint and placement of vancomycin-impregnated bone cement beads against methicillin-resistant S. aureus after total left knee implant arthroplasty for osteoarthritis. We also initiated linezolid administration at a conventional dose of 600 mg/h at 12-h intervals, but reduced it to 300 mg/h at 12-h intervals on day 9 because of a decrease in platelet count and an increase in serum linezolid trough concentration. However, when the infection exacerbated, we again increased the linezolid dose to 600 mg/h at 12-h intervals and performed combination therapy with rifampicin, considering their synergistic effects and the control of serum linezolid trough concentration via drug interaction. Methicillin-resistant S. aureus infection improved without reducing the dose of or discontinuing linezolid. The findings in the present case suggest that therapeutic drug monitoring could be useful for ensuring the therapeutic efficacy and safety of combination therapy even in patients with osteomyelitis who require long-term antibiotic administration.

Cartilage intermediate layer protein promotes lumbar disc degeneration.

Lumbar disc disease (LDD) is one of the most common musculoskeletal disorders, and accompanies intervertebral disc degeneration. CILP encodes cartilage intermediate layer protein, which is highly associated with LDD. Moreover, CILP inhibits transcriptional activation of cartilage matrix genes in nucleus pulposus (NP) cells in vitro by binding to TGF-ß1 and inhibiting the phosphorylation of Smads. However, the aetiology and mechanism of pathogenesis of LDD in vivo are unknown. To demonstrate the role of CILP in LDD in vivo, we generated transgenic mice that express CILP specifically in the intervertebral disc tissues and assessed whether CILP exacerbates disc degeneration. Degeneration of the intervertebral discs was assessed using magnetic resonance imaging (MRI) and histology. The level of phosphorylation of Smad2/3 in intervertebral discs was measured to determine whether overexpressed CILP suppressed TGF-beta signalling. Although the macroscopic skeletal phenotype of transgenic mice appeared normal, histological findings revealed significant degeneration of lumbar discs. MRI analysis of the lumbar intervertebral discs indicated a significantly lower signal intensity of the nucleus pulposus where CILP was overexpressed. Intervertebral disc degeneration was also observed. The number of phosphorylation of Smad2/3 immuno-positive cells in the NP significantly was decreased in CILP transgenic mice compared with normal mice. In summary, overexpression of CILP in the NP promotes disc degeneration, indicating that CILP plays a direct role in the pathogenesis of LDD.

Establishment of immortalized human amniotic mesenchymal stem cells.

Human amniotic mesenchymal cells (HAM cells) are known to contain somatic stem cells possessing the characteristics of pluripotency. However, little is known about the biology of these somatic cells because isolated HAM cells from amniotic membrane have a limited lifespan. To overcome this problem, we attempted to prolong the lifespan of HAM cells by infecting retrovirus encoding human papillomavirus type16E6 and E7 (HPV16E6E7), bmi-1, and/or human telomerase reverse transcriptase (hTERT) genes and investigated their characteristics as stem cells. We confirmed the immortalization of the four lines of cultured HAM cells for about 1 year. Immortalized human amnion mesenchymal cells (iHAM cells) have continued to proliferate over 200 population doublings (PDs). iHAM cells were positive for CD73, CD90, CD105, and CD44 and negative for CD34, CD14, CD45, and HLA-DR. They expressed stem cell markers such as Oct3/4, Sox2, Nanog, Klf4, SSEA4, c-myc, vimentin, and nestin. They showed adipogenic, osteogenic, and chondrogenic differentiation abilities after induction. These results suggested that immortalized cell lines with characteristics of stem cells can be established. iHAM cells with an extended lifespan can be used to produce good experimental models both in vitro and in vivo.

Isolation and characterization of human amniotic mesenchymal stem cells and their chondrogenic differentiation.

BACKGROUND: Freshly isolated human amniotic mesenchymal (fHAM) cells contain somatic stem cells possessing proliferative ability and pluripotency, including a chondrogenic lineage. However, little is known about the biology of amnion-derived mesenchymal stem cells (MSCs) because fHAM cells can barely survive to expand under culture conditions in vitro for a long time. METHODS: In this study, we separated fHAM cells and seeded them to isolate MSCs and analyze its character. In addition, suitable chondrogenic growth factor was determined by pellet culture, and their viability under xenogenic environment was examined by transplantation into rabbit knee joints. RESULTS: We succeeded in purifying proliferative subpopulations of fHAM cells, which could continue to proliferate more than 50 cumulative population doubling levels, and designated them as HAMα cells. Flow cytometry analysis revealed that they were positive for MSC markers (CD44, CD73, CD90, and CD105) and negative for hematopoietic cell markers (CD34, CD14, and CD45) and major histocompatibility complex class II antigen (human leukocyte antigen-DR). The expression of various stem-cell markers such as OCT3/4, C-MYC, SOX2, NANOG, CD44, SSEA-3, and SSEA-4 was also proved by immunocytochemical staining. Pellet culture using chondrogenic medium supplemented with transforming growth factor ß3, transforming growth factor ß3 plus bone morphogenetic protein (BMP)-2, or BMP-2 implied that supplementation of BMP-2 alone most effectively induced chondrogenesis in vitro. Xenotransplantation of HAMα cells achieved 8-week survival in vivo. CONCLUSIONS: These results suggest that HAMα cells correspond to MSCs that are highly proliferative and multipotent. Their chondrogenic potential and low immunogenicity indicate that HAMα cells could be an allotransplantable cell resource for cartilage repair.