Measurement of diffusion in articular cartilage using fluorescence correlation spectroscopy

<p>Abstract</p> <p>Background</p> <p>Fluorescence correlation spectroscopy (FCS) provides information about translational diffusion of fluorescent molecules in tiny detection volumes at the single-molecule level. In normal states, cartilage tissue lacks vascularity, so chondrocyte metabolism depends on diffusion for molecular exchanges. The abundant extracellular matrix (ECM) of cartilage is maintained by a limited number of chondrocytes. ECM plays an important role in the regulation of chondrocyte functions. In this study, FCS was used to measure diffusion behaviors of albumin, the major protein of the intra-articular space, using normal and degenerated cartilage. Preliminary investigation of fluorescence dyes including Alexa 488, Rhodamine 6G and Rhodamine 123 was conducted to evaluate their properties in cartilage.</p> <p>Results</p> <p>The results indicate that the diffusion behaviors of fluorescently lableded albumin can be observed using FCS in both normal and chemically degenerated cartilage.</p> <p>Conclusions</p> <p>This work demonstrates the capability of FCS for direct measurement of diffusion in cartilaginous ECM. When the diffusion characteristics of fluorescent probes in ECM are clarified using FCS evaluation, FCS will be applicable as a method for early diagnosis of osteoarthritis, which is accompanied by increased abnormalities of ECM and also as tool for evaluating bio-engineered artificial cartilage for autologous chondrocyte implantation.</p

Circadian factors BMAL1 and RORα control HIF-1α transcriptional activity in nucleus pulposus cells: implications in maintenance of intervertebral disc health.

BMAL1 and RORα are major regulators of the circadian molecular oscillator. Since previous work in other cell types has shown cross talk between circadian rhythm genes and hypoxic signaling, we investigated the role of BMAL1 and RORα in controlling HIF-1-dependent transcriptional responses in NP cells that exist in the physiologically hypoxic intervertebral disc. HIF-1-dependent HRE reporter activity was further promoted by co-transfection with either BMAL1 or RORα. In addition, stable silencing of BMAL1 or inhibition of RORα activity resulted in decreased HRE activation. Inhibition of RORα also modulated HIF1α-TAD activity. Interestingly, immunoprecipitation studies showed no evidence of BMAL1, CLOCK or RORα binding to HIF-1α in NP cells. Noteworthy, stable silencing of BMAL1 as well as inhibition of RORα decreased expression of select HIF-1 target genes including VEGF, PFKFB3 and Eno1. To delineate if BMAL1 plays a role in maintenance of disc health, we studied the spinal phenotype of BMAL1-null mice. The lumbar discs of null mice evidenced decreased height, and several parameters associated with vertebral trabecular bone quality were also affected in nulls. In addition, null animals showed a higher ratio of cells to matrix in NP tissue and hyperplasia of the annulus fibrosus. Taken together, our results indicate that BMAL1 and RORα form a regulatory loop in the NP and control HIF-1 activity without direct interaction. Importantly, activities of these circadian rhythm molecules may play a role in the adaptation of NP cells to their unique niche

Jellyfish mucin may have potential disease-modifying effects on osteoarthritis

<p>Abstract</p> <p>Background</p> <p>We aimed to study the effects of intra-articular injection of jellyfish mucin (qniumucin) on articular cartilage degeneration in a model of osteoarthritis (OA) created in rabbit knees by resection of the anterior cruciate ligament. Qniumucin was extracted from <it>Aurelia aurita </it>(moon jellyfish) and <it>Stomolophus nomurai </it>(Nomura's jellyfish) and purified by ion exchange chromatography. The OA model used 36 knees in 18 Japanese white rabbits. Purified qniumucin extracts from <it>S. nomurai </it>or <it>A. aurita </it>were used at 1 mg/ml. Rabbits were divided into four groups: a control (C) group injected with saline; a hyaluronic acid (HA)-only group (H group); two qniumucin-only groups (M groups); and two qniumucin + HA groups (MH groups). One milligram of each solution was injected intra-articularly once a week for 5 consecutive weeks, starting from 4 weeks after surgery. Ten weeks after surgery, the articular cartilage was evaluated macroscopically and histologically.</p> <p>Results</p> <p>In the C and M groups, macroscopic cartilage defects extended to the subchondral bone medially and laterally. When the H and both MH groups were compared, only minor cartilage degeneration was observed in groups treated with qniumucin in contrast to the group without qniumucin. Histologically, densely safranin-O-stained cartilage layers were observed in the H and two MH groups, but cartilage was strongly maintained in both MH groups.</p> <p>Conclusion</p> <p>At the concentrations of qniumucin used in this study, injection together with HA inhibited articular cartilage degeneration in this model of OA.</p

The properties of bioengineered chondrocyte sheets for cartilage regeneration

<p>Abstract</p> <p>Background</p> <p>Although the clinical results of autologous chondrocyte implantation for articular cartilage defects have recently improved as a result of advanced techniques based on tissue engineering procedures, problems with cell handling and scaffold imperfections remain to be solved. A new cell-sheet technique has been developed, and is potentially able to overcome these obstacles. Chondrocyte sheets applicable to cartilage regeneration can be prepared with this cell-sheet technique using temperature-responsive culture dishes. However, for clinical application, it is necessary to evaluate the characteristics of the cells in these sheets and to identify their similarities to naive cartilage.</p> <p>Results</p> <p>The expression of SOX 9, collagen type 2, 27, integrin α10, and fibronectin genes in triple-layered chondrocyte sheets was significantly increased in comparison to those in conventional monolayer culture and in a single chondrocyte sheet, implying a nature similar to ordinary cartilage. In addition, immunohistochemistry demonstrated that collagen type II, fibronectin, and integrin α10 were present in the triple-layered chondrocyte sheets.</p> <p>Conclusion</p> <p>The results of this study indicate that these chondrocyte sheets with a consistent cartilaginous phenotype and adhesive properties may lead to a new strategy for cartilage regeneration.</p

Characterization of chondrocyte sheets prepared using a co-culture method with temperature- responsive culture inserts

Abstract Conventional culture methods using temperature-responsive culture dishes require 4-5 weeks to prepare layered chondrocyte sheets that can be used in articular cartilage repair and regeneration. This study investigated whether the use of synovial tissue obtained from the same joint as the chondrocyte nutritive supply source could more quickly facilitate the preparation of chondrocyte sheets. After culturing derived synoviocytes and chondrocytes together (i.e. combined culture or co-culture) on temperature-responsive inserts, chondrocyte growth was assessed and a molecular analysis of the chondrocyte sheets was performed. Transplantable tissue could be obtained more quickly using this method (average 10.5 days). Real-time polymerase chain reaction and immunostaining of the three-layer chondrocyte sheets confirmed the significant expression of genes critical to cartilage maintenance, including type II collagen (COL2), aggrecan-1 and tissue metallopeptidase inhibitor 1. However, the expression of COL1, matrix metalloproteinase 3 (MMP3), MMP13 and A-disintegrin and metalloproteinase with thrombospondin motifs 5 was suppressed. The adhesive factor fibronectin-1 (FN1) was observed in all sheet layers, whereas in sheets generated using conventional preparation methods positive FN1 immunostaining was observed only on the surface of the sheets. The results indicate that synoviocyte co-cultures provide an optimal environment for the preparation of chondrocyte sheets for tissue transplantation and are particularly beneficial for shortening the required culture period

Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc.

Despite the high prevalence of intervertebral disc disease, little is known about changes in intervertebral disc cells and their regenerative potential with ageing and intervertebral disc degeneration. Here we identify populations of progenitor cells that are Tie2 positive (Tie2+) and disialoganglioside 2 positive (GD2+), in the nucleus pulposus from mice and humans. These cells form spheroid colonies that express type II collagen and aggrecan. They are clonally multipotent and differentiated into mesenchymal lineages and induced reorganization of nucleus pulposus tissue when transplanted into non-obese diabetic/severe combined immunodeficient mice. The frequency of Tie2+ cells in tissues from patients decreases markedly with age and degeneration of the intervertebral disc, suggesting exhaustion of their capacity for regeneration. However, progenitor cells (Tie2+GD2+) can be induced from their precursor cells (Tie2+GD2-) under simple culture conditions. Moreover, angiopoietin-1, a ligand of Tie2, is crucial for the survival of nucleus pulposus cells. Our results offer insights for regenerative therapy and a new diagnostic standard

Implementasi manajemen sarana dan prasarana dalam meningkatkan mutu pendidikan pada madrasah tsanawiyah negeri (MTSN) Rantauprapat Kabupaten Labuhanbatu

Penelitian ini bertujuan untuk mengetahui perencanaan, pengorganisasian, pelaksanaan, pengawasan manajemen sarana dan prasarana dalam meningkatkan mutu pendidikan pada Madrasah Tsanawiyah Negeri (MTsN) Rantauprapat Kabupaten Labuhanbatu. Penelitian ini dilakukan dengan menggunakan pendekatan kualitatif, Teknik pengumpulan data dalam penelitian ini menggunakan observasi, wawancara dan dokumen. Data yang didapat kemudian dianalisis dengan menggunakan analisis data kualitatif yang terdiri dari: (a).reduksi data, (b).penyajian data, dan (c) penarikan kesimpulan. Temuan penelitian: (1) perencanaan manajemen sarana dan prasarana di Madrasah Tsanawiyah Negeri (MTsN) Rantauprapat Kabupaten Labuhanbatu terlebih dahulu dilakukan analisis kebutuhan riil baik yang menyangkut kebutuhan administrasi maupun pendukung kegiatan proses pembelajaran, seperti ruang kelas, moubilair, dan lain sebagainya. Yang melibatkan: Kepala Madrasah, KTU, bendahara, PKM, dan bahkan utusan dari komite sekolah. (2).Pengorganisasian manajemen sarana dan prasarana pada Madrasah Tsanawiyah Negeri (MTsN) Rantauprapat Kabupaten Labuhanbatu dilakukan berdasarkan rumpun (kelompok) dari setiap jenis sarana itu sendiri, misalnya: bangunan fisik, moubilair, ATK, lingkungan, dan lain sebagainya yang kesemuanya itu di arsiparis berdasarkan ketentuan yang berlaku. (3) Pelaksanaan manajemen sarana dan prasarana di Madrasah Tsanawiyah Negeri (MTsN) Rantauprapat Kabupaten Labuhanbatu berjalan baik dan lancar. Pelaksanaannya masing-masing pihak bekerja sesuai job/pekerjaan masing-masing dan sesuai kepentingannya, sehingga sistem kerja tidak ada tumpang tindih antara satu sama lain. Dan pertanggung jawabannya langsung kepada Kepala madrasah MTsN Rantauprapat walaupun tetap di bawah koordinasi PKM sarana dan prasarana. (4).Pengawasan manajemen sarana dan prasarana pada Madrasah Tsanawiyah Negeri (MTsN) Rantauprapat Kabupaten Labuhanbatu dilakukan dengan cara: a) Pengawasan rutin setiap harinya yang dilakukan oleh PKM sarana jika menyangkut persoalan sarana pendukung pembelajaran, sedangkan yang menyangkut administrasi dilakukan oleh KTU. b) Secara berkala yakni setiap 6 (enam) bulan sekali diadakan rapat evaluasi tentang keadaan sarana dan prasarana. (5) Terkait dengan evaluasi diketahui bahwa sarana dan prasarana di Madrasah Tsanawiyah Negeri (MTsN) Rantauprapat Kabupaten Labuhanbatu sudah terpenuhi dan sesuai dengan standar pendidikan nasional.