miR-92a-3p-inspired shRNA exhibits pro-chondrogenic and chondrocyte protective effects in osteoarthritis treatment through targeting SMAD6/7.

INTRODUCTION: Osteoarthritis (OA) compromises patients' quality of life and requires further study. Although miR-92a-3p was reported to possess chondroprotective effects, the underlying mechanism requires further clarification. The objectives of this study were to elucidate the mechanism by which miR-92a-3p alleviates OA and to examine the efficacy of shRNA-92a-3p, which was designed based on mature miR-92a-3p. MATERIALS AND METHODS: TargetScan and luciferase reporter assay were used to predict the target of miR-92a-3p. Adipose-derived stem cells (ADSCs) were transfected with miR-92a-3p/miR-NC mimic for the analysis of chondrogenic biomarkers and SMAD proteins. ADSCs and osteoarthritic chondrocytes were transduced with shRNA-92a-3p for the analysis of chondrogenic biomarkers and SMAD proteins. OA was surgically induced in C57BL/6JJcl mice, and ADSCs with/without shRNA-92a-3p transduction were intra-articularly injected for the assessment of cartilage damage. RESULTS: SMAD6 and SMAD7 were predicted as direct targets of miR-92a-3p by TargetScan and luciferase reporter assay. Transfection of the miR-92a-3p mimic resulted in a decrease in SMAD6 and SMAD7 levels and an increase in phospho-SMAD2/3, phospho-SMAD1/5/9, SOX9, collagen type II, and aggrecan levels in ADSCs. Furthermore, shRNA-92a-3p decreased SMAD6 and SMAD7 levels, and increased phospho-SMAD2/3, phospho-SMAD1/5/9, SOX9, collagen type II, and aggrecan levels in ADSCs and osteoarthritic chondrocytes. Additionally, ADSC-shRNA-92a-3p-EVs reduced the rate of decrease of SOX9, collagen type II, and aggrecan in osteoarthritic chondrocytes. In mice with surgically induced OA, shRNA-92a-3p-treated ADSCs alleviated cartilage damage more effectively than nontreated ADSCs. CONCLUSIONS: miR-92a-3p and shRNA-92a-3p exhibit therapeutic effects in treating OA by targeting SMAD6 and SMAD7, thereby enhancing TGF-ß signaling.

Inhibition of cysteine protease disturbs the topological relationship between bone resorption and formation in vitro.

INTRODUCTION: Osteoporosis is a global health issue. Bisphosphonates that are commonly used to treat osteoporosis suppress both bone resorption and subsequent bone formation. Inhibition of cathepsin K, a cysteine proteinase secreted by osteoclasts, was reported to suppress bone resorption while preserving or increasing bone formation. Analyses of the different effects of antiresorptive reagents such as bisphosphonates and cysteine proteinase inhibitors will contribute to the understanding of the mechanisms underlying bone remodeling. MATERIALS AND METHODS: Our team has developed an in vitro system in which bone remodeling can be temporally observed at the cellular level by 2-photon microscopy. We used this system in the present study to examine the effects of the cysteine proteinase inhibitor E-64 and those of zoledronic acid on bone remodeling. RESULTS: In the control group, the amount of the reduction and the increase in the matrix were correlated in each region of interest, indicating the topological and quantitative coordination of bone resorption and formation. Parameters for osteoblasts, osteoclasts, and matrix resorption/formation were also correlated. E-64 disrupted the correlation between resorption and formation by potentially inhibiting the emergence of spherical osteoblasts, which are speculated to be reversal cells in the resorption sites. CONCLUSION: These new findings help clarify coupling mechanisms and will contribute to the development of new drugs for osteoporosis.

Quantitative analyses of matrices, osteoblasts, and osteoclasts during bone remodeling using an in vitro system.

INTRODUCTION: Bone remodeling plays a central role in the maintenance of bone homeostasis. Our group has established an in vitro system by which the cellular events during bone remodeling can be observed longitudinally. This study used this system to quantitatively analyze osteoblasts, osteoclasts, and matrices to elucidate their temporal changes and correlations. MATERIALS AND METHODS: Osteoblasts from EGFP mice were cultured to form calcified nodules, followed by co-culture with bone marrow macrophages from Tnfrsf11aCre/+ x Ai14 mice for 3 weeks (resorption phase). Then cells were cultured with osteoblast differentiation medium for 3 weeks (formation phase). The same sites were observed weekly using 2-photon microscopy. Matrices were detected using second harmonic generation. Parameters related to matrices, osteoblasts, and osteoclasts were quantified and statistically analyzed. RESULTS: Resorption and replenishment of the matrix were observed at the same sites by 2 photon microscopy. Gross quantification revealed that matrix and osteoblast parameters decreased in the resorption phase and increased in the formation phase, while osteoclast parameters showed the opposite pattern. When one field of view was divided into 16 regions of interest (ROIs) and correlations between parameters were analyzed in each ROI, decreased and increased matrix volumes were moderately correlated. Parameters of matrices and osteoblasts, and those of matrices and osteoclasts exhibited moderate correlations, while those of osteoblasts and osteoclasts were only weakly correlated. CONCLUSION: Several correlations between cells and matrix during remodeling were demonstrated quantitatively. This system may be a powerful tool for the research of bone remodeling.

Hematopoietic-Mesenchymal Signals Regulate the Properties of Mesenchymal Stem Cells.

It is well known that the properties of hematopoietic stem/progenitor cells (HSCs), such as their self-renewal ability and multipotency, are maintained through interactions with mesenchymal stem/stromal cells (MSCs). MSCs are rare cells that are present in the bone marrow and are useful for clinical applications due to their functional ability. To obtain the necessary number of cells, MSCs must be cultured to expand, but this causes a remarkable decrease in stem cell properties, such as multipotency and proliferation ability. In this study, we show that the c-Mpl signal, which is related to the maintenance of hematopoietic stem cells, has an important effect on the proliferation and differentiation ability of MSCs. Utilizing a co-culture system comprising MSCs and HSCs, it is suggested that signaling from hematopoietic cells to MSCs supports cell proliferation. Interestingly, the enhanced proliferation ability of the HSCs was decreased in c-Mpl knock-out HSCs (c-Mpl-KO). In addition, the MSCs co-cultured with c-Mpl-KO HSCs had reduced MSC marker expression (PDGFRa and Sca-1) compared to the MSCs co-cultured with c-Mpl-wild-type HSCs. These results suggest that a hematopoietic-mesenchymal signal exists, and that the state of the HSCs is important for the stability of MSC properties.

Roles of macrophage migration inhibitory factor in cartilage tissue engineering.

To obtain stable outcomes in regenerative medicine, understanding and controlling immunological responses in transplanted tissues are of great importance. In our previous study, auricular chondrocytes in tissue-engineered cartilage transplanted in mice were shown to express immunological factors, including macrophage migration inhibitory factor (MIF). Since MIF exerts pleiotropic functions, in this study, we examined the roles of MIF in cartilage regenerative medicine. We made tissue-engineered cartilage consisting of auricular chondrocytes of C57BL/6J mouse, atellocollagen gel and a PLLA scaffold, and transplanted the construct subcutaneously in a syngeneic manner. Localization of MIF was prominent in cartilage areas of tissue-engineered cartilage at 2 weeks after transplantation, though it became less apparent by 8 weeks. Co-culture with RAW264 significantly increased the expression of MIF in chondrocytes, suggesting that the transplanted chondrocytes in tissue-engineered cartilage could enhance the expression of MIF by stimulation of surrounding macrophages. When MIF was added in the culture of chondrocytes, the expression of type II collagen was increased, indicating that MIF could promote the maturation of chondrocytes. Meanwhile, toluidine blue staining of constructs containing wild type (Mif+/+) chondrocytes showed increased metachromasia compared to MIF-knockout (Mif-/-) constructs at 2 weeks. However, this tendency was reversed by 8 weeks, suggesting that the initial increase in cartilage maturation in Mif+/+ constructs deteriorated by 8 weeks. Since the Mif+/+ constructs included more iNOS-positive inflammatory macrophages at 2 weeks, MIF might induce an M1 macrophage-polarized environment, which may eventually worsen the maturation of tissue-engineered cartilage in the long term.

Biological aspects of tissue-engineered cartilage.

Cartilage regenerative medicine has been progressed well, and it reaches the stage of clinical application. Among various techniques, tissue engineering, which incorporates elements of materials science, is investigated earnestly, driven by high clinical needs. The cartilage tissue engineering using a poly lactide scaffold has been exploratorily used in the treatment of cleft lip-nose patients, disclosing good clinical results during 3-year observation. However, to increase the reliability of this treatment, not only accumulation of clinical evidence on safety and usefulness of the tissue-engineered products, but also establishment of scientific background on biological mechanisms, are regarded essential. In this paper, we reviewed recent trends of cartilage tissue engineering in clinical practice, summarized experimental findings on cellular and matrix changes during the cartilage regeneration, and discussed the importance of further studies on biological aspects of tissue-engineered cartilage, especially by the histological and the morphological methods.

Clinical Findings of a Cantilever Iliac Bone Graft for Secondary Correction of Cleft Lip-Nose Deformities.

The authors performed a cantilever iliac bone graft for the secondary correction of severe cleft lip-nose deformities after the completion of growth. For the purpose of clarifying effects of the cantilever iliac bone grafts and the adverse events with regard to their time course changes after this procedure, the authors retrospectively surveyed long-term morphologic changes in 65 cleft lip, alveolus, and palate patients in whom cleft lip-nose deformities were treated with a cantilever iliac bone graft (age at surgery: 14-45 years old). All postsurgical documents of facial photographs and radiologic images were reviewed to evaluate the effects and adverse events. The main adverse events were deviations of the apex of the nose, excess resorption of the grafted iliac bone, protruding deformations of the grafted iliac bone at the root of the nose, and fracture of the grafted iliac bone. Additional surgery was necessary in 10.7% of patients. Postsurgical changes in facial profiles became favorable, measured on lateral view of cephalometric radiography, achieving morphologic improvements. A cantilever iliac bone graft was effective for improving nasal deformities in cleft lip, alveolus, and palate patients, although the counter measures should be taken to these adverse events.

[Cartilage/chondrocyte research and osteoarthritis. Intra-articular injection of mesenchymal stem cells for the treatment of osteoarthritis.]

Mesenchymal stem cells(MSCs)are reported to have not only multipotency, but also anti-inflammatory and tissue repairing effects. Assays using animal models for osteoarthritis show that MSCs suppress the degeneration of cartilage. Based on these data, clinical researches and clinical trials have been performed for the treatment of osteoarthritis using MSCs. Results of these trials show the safety of the treatment, improvement of the symptoms, and findings indicating the cartilage repair. For the industrialization of the treatment using MSCs, application of allogenic cells has many advantages, while issues such as the immune reaction must be overcome.

In vivo subcellular imaging of tumors in mouse models using a fluorophore-conjugated anti-carcinoembryonic antigen antibody in two-photon excitation microscopy.

Recently, there has been growing interest in applying fluorescence imaging techniques to the study of various disease processes and complex biological phenomena in vivo. To apply these methods to clinical settings, several groups have developed protocols for fluorescence imaging using antibodies against tumor markers conjugated to fluorescent substances. Although these probes have been useful in macroscopic imaging, the specificity and sensitivity of these methods must be improved to enable them to detect micro-lesions in the early phases of cancer, resulting in better treatment outcomes. To establish a sensitive and highly specific imaging method, we used a fluorophore-conjugated anti-carcinoembryonic antigen (CEA) antibody to perform macroscopic and microscopic in vivo imaging of inoculated cancer cells expressing GFP with or without CEA. Macroscopic imaging by fluorescence zoom microscopy revealed that bio-conjugation of Alexa Fluor 594 to the anti-CEA antibody allowed visualization of tumor mass consisting of CEA-expressing human cancer cells, but the background levels were unacceptably high. In contrast, microscopic imaging using a two-photon excitation microscope and the same fluorescent antibody resulted in subcellular-resolution imaging that was more specific and sensitive than conventional imaging using a fluorescence zoom microscope. These results suggest that two-photon excitation microscopy in conjunction with fluorophore-conjugated antibodies could be widely adapted to detection of cancer-specific cell-surface molecules, both in cancer research and in clinical applications.

Intravital multiphoton fluorescence imaging and optical manipulation of spinal cord in mice, using a compact fiber laser system.

BACKGROUND AND OBJECTIVE: Near-infrared ultrafast lasers are widely used for multiphoton excited fluorescence microscopy in living animals. Ti:Sapphire lasers are typically used for multiphoton excitation, but their emission wavelength is restricted below 1,000 nm. The aim of this study is to evaluate the performance of a compact Ytterbium-(Yb-) fiber laser at 1,045 nm for multiphoton excited fluorescence microscopy in spinal cord injury. MATERIALS AND METHODS: In this study, we employed a custom-designed microscopy system with a compact Yb-fiber laser and evaluated the performance of this system in in vivo imaging of brain cortex and spinal cord in YFP-H transgenic mice. RESULTS: For in vivo imaging of brain cortex, sharp images of basal dendrites, and pyramidal cells expressing EYFP were successfully captured using the Yb-fiber laser in our microscopy system. We also performed in vivo imaging of axon fibers of spinal cord in the transgenic mice. The obtained images were almost as sharp as those obtained using a conventional ultrafast laser system. In addition, laser ablation and multi-color imaging could be performed simultaneously using the Yb-fiber laser. CONCLUSION: The high-peak pulse Yb-fiber laser is potentially useful for multimodal bioimaging methods based on a multiphoton excited fluorescence microscopy system that incorporates laser ablation techniques. Our results suggest that microscopy systems of this type could be utilized in studies of neuroscience and clinical use in diagnostics and therapeutic tool for spinal cord injury in the future.