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Dysregulated balance between bone resorption and formation mediates the onset and progression of osteoporosis. The administration of prednisolone is known to induce osteoporosis, whereas alendronate is commonly used to reverse the process. However, the assessment of the effects of such medicines on the nanostructure of bone remodeling and mechanical properties remains a major technical challenge. The aim of this study was to apply various analytical approaches to evaluate the compositional, morphological, and mechanical properties of regenerative zebrafish caudal fin bony rays affected by prednisolone and alendronate. Adult wild-type AB strain zebrafish were first exposed to 125μM of prednisolone for 14 days to develop glucocorticoid-induced osteoporosis. Fish fins were then amputated and let to regenerate for 21 days in tank water containing 30μM of alendronate or no alendronate. The lepidotrichia in the proximal and distal regions were evaluated separately using confocal microscope, scanning electron microscope, electron-dispersive spectroscopy, Raman spectroscopy, atomic force microscopy, and a triboindenter. As expected, prednisolone led to significant osteoporotic phenotypes. A decrease of Ca/P ratio was observed in the osteoporotic subjects (1.46 ± 0.04) as compared to the controls (1.74 ± 0.10). Subsequent treatment of alendronate overmineralized the bony rays during regeneration. Enhanced phosphate deposition was detected in the proximal part with alendronate treatment. Moreover, prednisolone attenuated the reduced elastic modulus and hardness levels (5.60 ± 5.04 GPa and 0.12 ± 0.17 GPa, respectively), whereas alendronate recovered them to the pre-amputation condition (8.68 ± 8.74 GPa and 0.34 ± 0.47 GPa, respectively). As an emerging model of osteoporosis, regrowth of zebrafish caudal fin was shown to be a reliable assay system to investigate the various effects of medicines in the de novo mineralization process. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research., (© 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.)

Drug lag-delayed approval or reimbursement-is a major barrier to accessing cutting-edge drugs. Unlike approval lag, reimbursement lag is under-researched. We investigated the key determinants of reimbursement lag under Taiwan National Health Insurance (NHI), and compared this lag with those in the United Kingdom, Canada, Australia, Japan, and South Korea. Using retrospective data on 190 new NHI-reimbursed drugs from 2007 to 2014, we studied reimbursement lag in Taiwan vs. other countries, and investigated associated factors using generalized linear models (GLMs). The median reimbursement lags during before ("first-generation") and after ("second-generation") NHI drug reimbursement scheme in Taiwan were 378 and 458 days, respectively. The "first-generation" lag was shorter only than that in South Korea, whereas the "second-generation" lag only exceeded those of the United Kingdom and Japan. In GLM models, higher drug expenditure and the introduction of the "second-generation" NHI were two statistically significant parameters associated with reimbursement lag among antineoplastic and immunomodulating agents. For other drug classes, the reimbursement price proposed by pharmaceutical companies and use of price-volume agreements were two statistically significant parameters associated with longer reimbursement lags. The current reimbursement lag in Taiwan is longer than 1 year, but only longer than those of the United Kingdom and Japan. The determinants differ between drug categories. A specific review process for antineoplastic and immunomodulating drugs may expedite reimbursement. There is a clear need for systematic data collection and analysis to ascertain factors associated with reimbursement lag and thereby inform future policy making., (© 2020 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of the American Society of Clinical Pharmacology and Therapeutics.)

We present in vivo sequence-specific RNA base editing via adenosine deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). To achieve this, we systematically engineered adRNAs to harness ADARs, and comprehensively evaluated the specificity and activity of the toolsets in vitro and in vivo via two mouse models of human disease. We anticipate that this platform will enable tunable and reversible engineering of cellular RNAs for diverse applications.

Background: Given the shortage of available organs for whole or partial liver transplantation, hepatocyte cell transplantation has long been considered a potential strategy to treat patients suffering from various liver diseases. Some of the earliest approaches that attempted to deliver hepatocytes via portal vein or spleen achieved little success due to poor engraftment. More recent efforts include transplantation of cell sheets or thin hepatocyte-laden synthetic hydrogels. However, these implants must remain sufficiently thin to ensure that nutrients can diffuse into the implant., Methods: To circumvent these limitations, we investigated the use of a vascularizable dual-compartment hydrogel system for minimally invasive transplantation of primary hepatocytes. The dual-compartment system features a macroporous outer polyethylene glycol diacrylate/hyaluronic acid methacrylate hydrogel compartment for seeding supportive cells and facilitating host cell infiltration and vascularization and a hollow inner core to house the primary human hepatocytes., Results: We show that the subcutaneous implantation of these cell-loaded devices in NOD/SCID mice facilitated vascular formation while supporting viability of the transplanted cells. Furthermore, the presence of human serum albumin in peripheral blood and the immunostaining of excised implants indicated that the hepatocytes maintained function in vivo for at least 1 month, the longest assayed time point., Conclusions: Cell transplantation devices that assist the anastomosis of grafts with the host can be potentially used as a minimally invasive ectopic liver accessory to augment liver-specific functions as well as potentially treat various pathologies associated with compromised functions of liver, such as hemophilia B or alpha-1 antitrypsin deficiency.

Synthetic biomimetic matrices with osteoconductivity and osteoinductivity have been developed to regenerate bone tissues. However, whether such systems harbor donor marrow in vivo and support mixed chimerism remains unknown. We devised a strategy to engineer bone tissues with a functional bone marrow (BM) compartment in vivo by using a synthetic biomaterial with spatially differing cues. Specifically, we have developed a synthetic matrix recapitulating the dual-compartment structures by modular assembly of mineralized and nonmineralized macroporous structures. Our results show that these matrices incorporated with BM cells or BM flush transplanted into recipient mice matured into functional bone displaying the cardinal features of both skeletal and hematopoietic compartments similar to native bone tissue. The hematopoietic function of bone tissues was demonstrated by its support for a higher percentage of mixed chimerism compared with i.v. injection and donor hematopoietic cell mobilization in the circulation of nonirradiated recipients. Furthermore, hematopoietic cells sorted from the engineered bone tissues reconstituted the hematopoietic system when transplanted into lethally irradiated secondary recipients. Such engineered bone tissues could potentially be used as ectopic BM surrogates for treatment of nonmalignant BM diseases and as a tool to study hematopoiesis, donor-host cell dynamics, tumor tropism, and hematopoietic cell transplantation., Competing Interests: The authors declare no conflict of interest.

The abilities of human pluripotent stem cells (hPSCs) to proliferate without phenotypic alteration and to differentiate into tissue-specific progeny make them a promising cell source for regenerative medicine and development of physiologically relevant in vitro platforms. Despite this potential, efficient conversion of hPSCs into tissue-specific cells still remains a challenge. Herein, we report direct conversion of hPSCs into functional osteoblasts through the use of adenosine, a naturally occurring nucleoside in the human body. The hPSCs treated with adenosine not only expressed the molecular signatures of osteoblasts but also produced calcified bone matrix. Our findings show that the adenosine-mediated osteogenesis of hPSCs involved the adenosine A2bR. When implanted in vivo, using macroporous synthetic matrices, the human induced pluripotent stem cell (hiPSC)-derived donor cells participated in the repair of critical-sized bone defects through the formation of neobone tissue without teratoma formation. The newly formed bone tissues exhibited various attributes of the native tissue, including vascularization and bone resorption. To our knowledge, this is the first demonstration of adenosine-induced differentiation of hPSCs into functional osteoblasts and their subsequent use to regenerate bone tissues in vivo. This approach that uses a physiologically relevant single small molecule to generate hPSC-derived progenitor cells is highly appealing because of its simplicity, cost-effectiveness, scalability, and impact in cell manufacturing, all of which are decisive factors for successful translational applications of hPSCs.

To understand the inhomogeneity of cells in biological systems, there is a growing demand on the capability of characterizing the properties of individual single cells. Since single-cell studies require continuous monitoring of the cell behaviors, an effective single-cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single-cell technologies cannot provide proper environments to sustain cell growth and, proliferation of single cells and convenient, noninvasive tests of single-cell behaviors from molecular markers. Here, a highly versatile single-cell assay is presented that can accommodate different cellular types, enable easy and efficient single-cell loading and culturing, and be suitable for the study of effects of in vitro environmental factors in combination with drug screening. One salient feature of the assay is the noninvasive collection and surveying of single-cell secretions at different time points, producing unprecedented insight of single-cell behaviors based on the biomarker signals from individual cells under given perturbations. Above all, the acquired information is quantitative, for example, measured by the number of exosomes each single-cell secretes for a given time period. Therefore, our single-cell assay provides a convenient, low-cost, and enabling tool for quantitative, time lapsed studies of single-cell properties., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Human pluripotent stem cells such as embryonic stem cells (hESCs) and multipotent stem cells like mesenchymal stem cells (hMSCs) hold great promise as potential cell sources for bone tissue regeneration. Comparing the in vivo osteogenesis of hESCs and hMSCs by biomaterial-based cues provides insight into the differentiation kinetics of these cells as well as their potential to contribute to bone tissue repair in vivo. Here, we compared in vivo osteogenic differentiation of hESCs and hMSCs within osteoinductive calcium phosphate (CaP)-bearing biomineralized scaffolds that recapitulate a bone-specific mineral microenvironment. Both hESCs and hMSCs underwent osteogenic differentiation responding to the biomaterial-based instructive cues. Furthermore, hMSCs underwent earlier in vivo osteogenesis compared to hESCs, but both stem cell types acquired a similar osteogenic maturation by 8 weeks of implantation.

Cell-responsive hydrogels hold tremendous potential as cell delivery devices in regenerative medicine. In this study, we developed a hydrogel-based cell delivery vehicle, in which the encapsulated cell cargo control its own release from the vehicle in a protease-independent manner. Specifically, we have synthesized a modified poly(ethylene glycol) (PEG) hydrogel that undergoes degradation responding to cell-secreted molecules by incorporating disulfide moieties onto the backbone of the hydrogel precursor. Our results show the disulfide-modified PEG hydrogels disintegrate seamlessly into solution in presence of cells without any external stimuli. The rate of hydrogel degradation, which ranges from hours to months, is found to be dependent upon the type of encapsulated cells, cell number, and fraction of disulfide moieties present in the hydrogel backbone. The differentiation potential of human mesenchymal stem cells released from the hydrogels is maintained in vitro. The in vivo analysis of these cell-laden hydrogels, through a dorsal window chamber and intramuscular implantation, demonstrated autonomous release of cells to the host environment. The hydrogel-mediated implantation of cells resulted in higher cell retention within the host tissue when compared to that without a biomaterial support. Biomaterials that function as a shield to protect cell cargos and assist their delivery in response to signals from the encapsulated cells could have a wide utility in cell transplantation and could improve the therapeutic outcomes of cell-based therapies., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Human embryonic stem cells (hESCs) are attractive cell sources for tissue engineering and regenerative medicine due to their self-renewal and differentiation ability. Design of biomaterials with an intrinsic ability that promotes hESC differentiation to the targeted cell type boasts significant advantages for tissue regeneration. We have previously developed biomineralized calcium phosphate (CaP) matrices that inherently direct osteogenic differentiation of hESCs without the need of osteogenic-inducing chemicals or growth factors. Here, we show that CaP matrix-driven osteogenic differentiation of hESCs occurs through A2b adenosine receptor (A2bR). The inhibition of the receptor with an A2bR-specific antagonist attenuated mineralized matrix-mediated osteogenic differentiation of hESCs. In addition, when cultured on matrices in an environment deficient of CaP minerals, exogenous adenosine promoted osteogenic differentiation of hESCs, but was attenuated by the inhibition of A2bR. Such synthetic matrices that intrinsically support osteogenic commitment of hESCs are not only beneficial for bone tissue engineering but can also be used as a platform to study the effect of the physical and chemical cues to the extracellular milieu on stem cell commitment. Insights into the cell signaling during matrix-induced differentiation of stem cells will also help define the key processes and enable discovery of new targets that promote differentiation of pluripotent stem cells for bone tissue engineering.

Advances in tissue engineering have offered new opportunities to restore anatomically and functionally compromised tissues. Although traditional tissue engineering approaches that utilize biomaterials and cells to create tissue constructs for implantation or biomaterials as a scaffold to deliver cells are promising, strategies that can activate endogenous cells to promote tissue repair are more clinically attractive. Here, we demonstrate that an engineered injectable matrix mimicking a calcium phosphate (CaP)-rich bone-specific microenvironment can recruit endogenous cells to form bone tissues in vivo. Comparison of matrix alone with that of bone marrow-soaked or bFGF-soaked matrix demonstrates similar extent of neo-bone formation and bridging of decorticated transverse processes in a posterolateral lumbar fusion rat model. Synthetic biomaterials that stimulate endogenous cells without the need for biologics to assist tissue repair could circumvent limitations associated with conventional tissue engineering approaches, including ex vivo cell processing and laborious efforts, thereby accelerating the translational aspects of regenerative medicine., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Stem cell differentiation is determined by a repertoire of signals from its microenvironment, which includes the extracellular matrix (ECM) and soluble cues. The ability of mesenchymal stem cells (MSCs), a common precursor for the skeletal system, to differentiate into osteoblasts and adipocytes in response to their local cues plays an important role in skeletal tissue regeneration and homeostasis. In this study, we investigated whether a bone-specific calcium phosphate (CaP) mineral environment could induce osteogenic differentiation of human MSCs, while inhibiting their adipogenic differentiation, in the presence of adipogenic-inducing medium. We also examined the mechanism through which the mineralized matrix suppresses adipogenesis of hMSCs to promote their osteogenic differentiation. Our results show that hMSCs cultured on mineralized matrices underwent osteogenic differentiation despite being cultured in the presence of adipogenic medium, which indicates the dominance of matrix-based cues of the mineralized matrix in directing osteogenic commitment of stem cells. Furthermore, the mineralized matrix-driven attenuation of adipogenesis was reversed with the inhibition of A2b adenosine receptor (A2bR), implicating a role of adenosine signaling in mineralized environment-mediated inhibition of adipogenesis. Such synthetic matrices with an intrinsic ability to direct differentiation of multipotent adult stem cells toward a targeted phenotype while inhibiting their differentiation into other lineages not only will be a powerful tool in delineating the role of complex microenvironmental cues on stem cell commitment but also will contribute to functional tissue engineering and their translational applications.

The physical and chemical properties of a matrix play an important role in determining various cellular behaviors, including lineage specificity. We demonstrate that the differentiation commitment of human embryonic stem cells (hESCs), both in vitro and in vivo, can be solely achieved through synthetic biomaterials. hESCs were cultured using mineralized synthetic matrices mimicking a calcium phosphate (CaP)-rich bone environment differentiated into osteoblasts in the absence of any osteogenic inducing supplements. When implanted in vivo , these hESC-laden mineralized matrices contributed to ectopic bone tissue formation. In contrast, cells within the corresponding non-mineralized matrices underwent either osteogenic or adipogenic fate depending upon the local cues present in the microenvironment. To our knowledge, this is the first demonstration where synthetic matrices are shown to induce terminal cell fate specification of hESCs exclusively by biomaterial-based cues both in vitro and in vivo . Technologies that utilize tissue specific cell-matrix interactions to control stem cell fate could be a powerful tool in regenerative medicine. Such approaches can be used as a tool to advance our basic understanding and assess the translational potential of stem cells.

The ability of human embryonic stem cells (hESCs) to differentiate into skeletal muscle cells is an important criterion in using them as a cell source to ameliorate skeletal muscle impairments. However, differentiation of hESCs into skeletal muscle cells still remains a challenge, often requiring introduction of transgenes. Here, we describe the use of WNT3A protein to promote in vitro myogenic commitment of hESC-derived cells and their subsequent in vivo function. Our findings show that the presence of WNT3A in culture medium significantly promotes myogenic commitment of hESC-derived progenitors expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), as evident from the expression of myogenic markers, including DES, MYOG, MYH1, and MF20. In vivo transplantation of these committed cells into cardiotoxin-injured skeletal muscles of NOD/SCID mice reveals survival and engraftment of the donor cells. The cells contributed to the regeneration of damaged muscle fibers and the satellite cell compartment. In lieu of the limited cell source for treating skeletal muscle defects, the hESC-derived PDGFRA(+) cells exhibit significant in vitro expansion while maintaining their myogenic potential. The results described in this study provide a proof-of-principle that myogenic progenitor cells with in vivo engraftment potential can be derived from hESCs without genetic manipulation.

Background: Traditional Chinese medicine (TCM) is one of the most common complementary and alternative medicines used in the treatment of patients with breast cancer. However, the clinical effect of TCM on survival, which is a major concern in these individuals, lacks evidence from large-scale clinical studies., Methods: The authors used the Taiwan National Health Insurance Research Database to conduct a retrospective population-based cohort study of patients with advanced breast cancer between 2001 and 2010. The patients were separated into TCM users and nonusers, and Cox regression models were applied to determine the association between the use of TCM and patient survival., Results: A total of 729 patients with advanced breast cancer receiving taxanes were included in the current study. Of this cohort, the mean age was 52.0 years; 115 patients were TCM users (15.8%) and 614 patients were TCM nonusers. The mean follow-up was 2.8 years, with 277 deaths reported to occur during the 10-year period. Multivariate analysis demonstrated that, compared with nonusers, the use of TCM was associated with a significantly decreased risk of all-cause mortality (adjusted hazards ratio [HR], 0.55 [95% confidence interval, 0.33-0.90] for TCM use of 30-180 days; adjusted HR, 0.46 [95% confidence interval, 0.27-0.78] for TCM use of >180 days). Among the frequently used TCMs, those found to be most effective (lowest HRs) in reducing mortality were Bai Hua She She Cao, Ban Zhi Lian, and Huang Qi., Conclusions: The results of the current observational study suggest that adjunctive TCM therapy may lower the risk of death in patients with advanced breast cancer. Future randomized controlled trials are required to validate these findings., (© 2014 American Cancer Society.)

Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases.

Current research in pulmonary pathology has focused on inflammatory reactions initiated by immunological responses to allergens and irritants. In addition to these biochemical stimuli, physical forces also play an important role in regulating the structure, function, and metabolism of the lung. Hyperstretch of lung tissues can contribute to the inflammatory responses in asthma, but the mechanisms of mechanically induced inflammation in the lung remain unclear. Our results demonstrate that excessive stretch increased the secretion of inflammatory cytokines by human bronchial epithelial cells (hBECs), including IL-8. This increase of IL-8 secretion was due to an elevated microRNA-155 (miR-155) expression, which caused the suppression of Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) production and the subsequent activation of JNK signaling. In vivo studies in our asthmatic mouse model also showed such changes in miR-155, IL-8, and SHIP1 expressions that reflect inflammatory responses. Co-culture with human mesenchymal stem cells (hMSCs) reversed the stretch-induced hBEC inflammatory responses as a result of IL-10 secretion by hMSCs to down-regulate miR-155 expression in hBECs. In summary, we have demonstrated that mechanical stretch modulates the homeostasis of the hBEC secretome involving miR-155 and that hMSCs can be used as a potential therapeutic approach to reverse bronchial epithelial inflammation in asthma.

The effect of substrate-mediated signals on osteogenic differentiation of hMSCs is studied using a synthetic bone-like material comprising both organic and inorganic components that supports adhesion, spreading, and proliferation of hMSCs. hMSCs undergo osteogenic differentiation even in the absence of osteogenesis-inducing supplements. They exhibit higher expressions of Runx2, BSP, and OCN compared to their matrix-rigidity-matched, non-mineralized hydrogel counterparts. The mineralized-hydrogel-assisted osteogenic differentiation of hMSCs could be attributed to their exposure to high local concentrations of calcium and phosphate ions in conjunction with chemical and topological cues arising from the hydrogel-bound calcium phosphate mineral layer., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Extracellular stimuli imposed on stem cells enable efficient initiation of mechanotransductive signaling to regulate stem cell fates; however, how such physical cues conferred by the stereo-topographical matrix govern the fate of stem cells still remains unknown. The purpose of this study is to delineate the effects of stereotopography and its various relevant physical properties on the fate regulation of human mesenchymal stem cells (hMSCs). Stereo-topographical silicon nanowires (SiNWs) that were precisely controlled with respect to their various dimensions and their growth orientation were used in this study. hMSCs cultured on stereo SiNWs of different lengths in the absence of biochemical osteogenic induction cues displayed a spherical and less-elongated morphology and showed an approximately 10% loss of cell viability compared to those grown on two-dimensional (2-D) flat Si. Moreover, osteogenic gene expression of COL1A1 and Runx2 in hMSCs cultured on the shortest SiNWs was significantly higher than those grown on the longer SiNWs and 2-D flat Si. hMSCs grown on shorter SiNWs also demonstrated higher expression levels for F-actin, phosphorylated focal adhesion kinase (pFAK), vinculin and alpha 2 integrin. Stereo-topographical cues provided by SiNWs are able to regulate osteogenic differentiation of hMSCs via cytoskeleton remodeling and this is correlated with the differential expression of alpha 2/beta 1 integrin heterodimers and the focal adhesion molecules pFAK and vinculin. The findings in this study provide insights in terms of the design of stereo-topographical structures for use in tissue engineering, bone regeneration and relevant medical applications., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Human mesenchymal stromal cells (hMSCs) are promising candidates for cell therapy and tissue regeneration. Knowledge of the molecular mechanisms governing hMSC commitment into osteoblasts is critical to the development of therapeutic applications for human bone diseases. Because protein phosphorylation plays a critical role in signaling transduction network, the purpose of this study is to elucidate the phosphoproteomic changes in hMSCs during early osteogenic lineage commitment. hMSCs cultured in osteogenic induction medium for 0, 1, 3, and 7 days were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Surprisingly, we observed a dramatic loss of protein phosphorylation level after 1 day of osteogenic induction. Pathways analysis of these reduced phosphoproteins exhibited a high correlation with cell proliferation and protein synthesis pathways. During osteogenic differentiation, differentially expressed phosphoproteins demonstrated the dynamic alterations in cytoskeleton at the early stages of differentiation. The fidelity of our quantitative phosphoproteomic analyses were further confirmed by Western blot analyses, and the changes from protein expression or its phosphorylation level were distinguished. In addition, several ion channels and transcription factors with differentially expressed phosphorylation sites during osteogenic differentiation were identified and may serve as potentially unexplored transcriptional regulators of the osteogenic phenotype of hMSCs. Taken together, our results have demonstrated the dynamic changes in phosphoproteomic profiles of hMSCs during osteogenic differentiation and unraveled potential candidates mediating the osteogenic commitment of hMSCs. The findings in this study may also shed light on the development of new therapeutic targets for metabolic bone diseases such as osteoporosis and osteomalacia.

Mesenchymal stem cells (MSCs), which can be isolated from bone marrow and other somatic tissues, are residing in an environment with relative low oxygen tension. The purpose of this study is to investigate the effects of hypoxia on MSCs, and we hypothesize that oxygen concentration regulates the intricate balance between cellular proliferation and commitment towards differentiation. In this study, human bone marrow-derived MSCs were cultured under hypoxia with 1% O(2). The proliferation ability of MSCs was increased after a 7-day hypoxic culture period. Migration assay showed that hypoxia enhanced the migration capabilities of MSCs. Moreover, expression of stemness genes Oct4, Nanog, Sall4 and Klf4 was increased under hypoxia. Furthermore, the differentiation ability of MSCs under hypoxia favored osteogenesis while adipogenesis was inhibited during a 4-week induction period. Cytokine antibody array analysis showed that a number of growth factors were up-regulated after a 7-day hypoxic incubation and the differential expression of growth factors may account for the increased proliferation and osteogenic potentials of MSCs under hypoxic condition. Taken together, hypoxia provides a favorable culture condition to promote proliferation as well as osteogenesis of MSCs through differential growth factor production., (Copyright © 2011 Orthopaedic Research Society.)

Mesenchymal stem cells (MSCs) cultured on extracellular matrices with different stiffness have been shown to possess diverse lineage commitment owing to the extracellular mechanical stimuli sensed by the cells. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through the mechanotransducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MSCs. MSCs were cultured in osteogenic medium on tunable polyacrylamide hydrogels coated with type I collagen with elasticities corresponding to Young's modulus of 7.0 ± 1.2 and 42.1 ± 3.2 kPa. Osteogenic differentiation was increased on stiffer matrices, as evident by type I collagen, osteocalcin, and Runx2 gene expressions and alizarin red S staining for mineralization. Western blot analysis demonstrated an increase in kinase activities of ROCK, FAK, and ERK1/2 on stiffer matrices. Inhibition of FAK, an important mediator of osteogenic differentiation, and inhibition of ROCK, a known mechanotransducer of matrix stiffness during osteogenesis, resulted in decreased expression of osteogenic markers during osteogenic induction. In addition, FAK affects osteogenic differentiation through ERK1/2, whereas ROCK regulates both FAK and ERK1/2. Furthermore, α(2)-integrin was upregulated on stiffer matrices during osteogenic induction, and its knockdown by siRNA downregulated the osteogenic phenotype through ROCK, FAK, and ERK1/2. Taken together, our results provide evidence that the matrix rigidity affects the osteogenic outcome of MSCs through mechanotransduction events that are mediated by α(2)-integrin., (Copyright © 2011 American Society for Bone and Mineral Research.)

Objectives: Somatic stem cells can be obtained from a variety of adult human tissues. However, it was not clear whether human parathyroid glands, which secrete parathyroid hormones and are essential in maintaining homeostasis levels of calcium ions in the circulation, contained stem cells. We aimed to investigate the possibility of isolating such parathyroid-derived stem cells (PDSC)., Materials and Methods: Surgically removed parathyroid glands were obtained with informed consent. Cell cytogenetics was used to observe chromosomal abnormalities. Surface phenotypes were characterized by flow cytometry. Telomerase repeat amplification protocol (TRAP) assay was performed to observe the telomerase activity. RT-PCR and real-time PCR was was used to detect gene expressions. Real-time calcium uptake imaging was performed for extent of calcium uptake and transmission electron microscopy and immunofluorecent staining for smooth muscle actin., Results: After enzymatic digestion and primary culture, plastic-adherent, fibroblast-like cells appeared in culture and a morphologically homogeneous population was derived from subsequent limiting dilution and clonal expansion. Karyotyping was normal and doubling time of clonal cell growth was estimated to be 70.7 +/- 14.5 h (mean +/- standard deviation). The surface phenotype of the cells was positive for CD73, CD166, CD29, CD49a, CD49b, CD49d, CD44, CD105, and MHC class I, and negative for CD34, CD133, CD117, CD114, CD31, CD62P, EGF-R, ICAM-3, CD26, CXCR4, CD106, CD90 and MHC class II, similar to mesenchymal stem cells (MSC). Detectable levels of telomerase activity along with pluripotency Sall4 gene expression were observed from the isolated PDSCs. Expression of calcium-sensing receptor gene along with alpha-smooth muscle actin was induced and cellular uptake of extracellular calcium ions was observed. Furthermore, PDSCs possessed osteogenic, chondrogenic and adipogenic differentiation potentials., Conclusions: Our results reveal that PDSCs were similar phenotypically to MSCs and further studies are needed to formulate induction conditions to differentiate PDSCs into parathyroid hormone-secreting chief cells.

Background & Aims: Liver transplantation is the primary treatment for various end-stage hepatic diseases but is hindered by the lack of donor organs and by complications associated with rejection and immunosuppression. There is increasing evidence to suggest the bone marrow is a transplantable source of hepatic progenitors. We previously reported that multipotent bone marrow-derived mesenchymal stem cells differentiate into functional hepatocyte-like cells with almost 100% induction frequency under defined conditions, suggesting the potential for clinical applications. The aim of this study was to critically analyze the various parameters governing the success of bone marrow-derived mesenchymal stem cell-based therapy for treatment of liver diseases., Methods: Lethal fulminant hepatic failure in nonobese diabetic severe combined immunodeficient mice was induced by carbon tetrachloride gavage. Mesenchymal stem cell-derived hepatocytes and mesenchymal stem cells were then intrasplenically or intravenously transplanted at different doses., Results: Both mesenchymal stem cell-derived hepatocytes and mesenchymal stem cells, transplanted by either intrasplenic or intravenous route, engrafted recipient liver, differentiated into functional hepatocytes, and rescued liver failure. Intravenous transplantation was more effective in rescuing liver failure than intrasplenic transplantation. Moreover, mesenchymal stem cells were more resistant to reactive oxygen species in vitro, reduced oxidative stress in recipient mice, and accelerated repopulation of hepatocytes after liver damage, suggesting a possible role for paracrine effects., Conclusions: Bone marrow-derived mesenchymal stem cells can effectively rescue experimental liver failure and contribute to liver regeneration and offer a potentially alternative therapy to organ transplantation for treatment of liver diseases.

The multidifferentiation ability of mesenchymal stem cells holds great promise for cell therapy. Numerous studies have focused on the establishment of differentiation protocols, whereas little attention has been paid to the metabolic changes during the differentiation process. Mitochondria, the powerhouse of mammalian cells, vary in their number and function in different cell types with different energy demands, but how these variations are associated with cell differentiation remains elusive. In this study, we investigated the changes of mitochondrial biogenesis and bioenergetic function using human mesenchymal stem cells (hMSCs) because of their well-defined differentiation potentials. Upon osteogenic induction, the copy number of mitochondrial DNA, protein subunits of the respiratory enzymes, oxygen consumption rate, and intracellular ATP content were increased, indicating the upregulation of aerobic mitochondrial metabolism. On the other hand, undifferentiated hMSCs showed higher levels of glycolytic enzymes and lactate production rate, suggesting that hMSCs rely more on glycolysis for energy supply in comparison with hMSC-differentiated osteoblasts. In addition, we observed a dramatic decrease of intracellular reactive oxygen species (ROS) as a consequence of upregulation of two antioxidant enzymes, manganese-dependent superoxide dismutase and catalase. Finally, we found that exogenous H(2)O(2) and mitochondrial inhibitors could retard the osteogenic differentiation. These findings suggested an energy production transition from glycolysis to oxidative phosphorylation in hMSCs upon osteogenic induction. Meanwhile, antioxidant enzymes were concurrently upregulated to prevent the accumulation of intracellular ROS. Together, our findings suggest that coordinated regulation of mitochondrial biogenesis and antioxidant enzymes occurs synergistically during osteogenic differentiation of hMSCs.

Background: The elderly segment of the population in Taiwan is increasing rapidly. According to the latest information from the Directorate-General of Budget, Accounting and Statistics, Executive Yuan, Taiwan, 1,553,367 civilians of the total population of 22,879,510 (6.8%) are seventy years old and older in 2007. The proportion of high-risk patients has increased dramatically owing to a greater number of elderly patients and increased number of average patients with heart diseases presenting for cardiac surgery. We analyzed the preoperative risk factors for in-hospital mortality, morbidity and the likelihood of prolonged intensive care unit (ICU) stay in elderly patients after cardiac surgery., Methods: We retrospectively studied 952 adult patients who received cardiac surgery during a three-year period (from August 1, 2004 to December 31, 2006) at Taichung Veterans General Hospital. Patients were divided into a control group and a study group. The study group (n=395) exclusively consisted of patients aged seventy or over while the rest of the patients under study served as reference group (n=557). Continuous variables were compared using Student's t test, and categorical variables were compared using Pearson chi-square test. Variables associated with in-hospital mortality, major morbidity and prolonged ICU stay in univariate analysis with P < 0.05 were entered into multivariate analysis using logistic regression with a stepwise forward selection procedure to determine independent variables and identify variables associated with major adverse outcomes., Results: Fifty-six (14.2%) patients died during their hospitalization in the study group and 46 (8.3%) in the reference group. Major mortality in the study group was 58.0% versus 39.7% in the reference group. The patients of the study group spent more days in the ICU than did patients of the reference group (8.7 +/- 12.0 versus 6.1 +/- 10.0 days, P < 0.05). In addition, 114 (28.9%) patients of the study group and 85 (15.3%) of the reference group spent more than 7 days in the ICU. Using multiple logistic regression analysis, risk factors affecting in-hospital mortality in the study group include impairment of renal function, reoperation, congestive heart failure (CHF), catastrophic event. Impaired renal function, complexity of surgical procedure, CHF, chronic obstructive pulmonary disease (COPD) and catastrophic state were significant factors affecting morbidity in the study group. CHF, COPD and catastrophic event contributed to prolonged ICU stay in the study group., Conclusions: The perioperative risk of cardiac surgery increases in older patient groups, and some risk factors have different influences on mortality, morbidity and ICU stay. Risk assessment in older cardiosurgical patients with convenient risk factors helps the clinicians to apply rational and cost-effective treatment strategies into practice.

Background/purpose: Recent advances in medical treatment have altered the profile of patients referred for cardiac surgery. The proportion of high risk patients has increased dramatically. Numerous multifactorial risk scores have been developed to predict outcomes after cardiac surgery. However, these additive risk models were all developed outside of Asia and have never been validated in Taiwan. We applied the Parsonnet score, Tu score and logistic regression to a population in Taiwan who received cardiac surgery to predict the mortality, morbidity and likelihood of prolonged stay in the intensive care unit (ICU)., Methods: This retrospective study included 622 adult patients who received cardiac surgery during a 2-year period at Taichung Veterans General Hospital. The patients were randomly divided into a reference set (n = 423) and a validation set (n = 199). The Parsonnet score and Tu score were calibrated separately with the reference set to determine mortality, morbidity and likelihood of prolonged ICU stay. We developed a separate logistic regression model for each of the three outcomes by using the reference set. The validation set was used to test these models., Results: The area under the receiver operating characteristic (ROC) curve (AUC) of the Parsonnet score, Tu score and logistic regression for predicting in-hospital mortality were 0.843, 0.714 and 0.867, respectively. The AUC of the Parsonnet score, Tu score and logistic regression for predicting major morbidity were 0.784, 0.736 and 0.808, respectively. The AUC of the Parsonnet score, Tu score and logistic regression for predicting likelihood of prolonged ICU stay were 0.701, 0.689 and 0.764, respectively., Conclusion: The Parsonnet score performed as well as the logistic regression models in predicting major adverse outcomes. The Parsonnet score appears to be a very suitable model for clinicians to use in risk stratification of cardiac surgery.

Purpose: Exogenous thymosin beta-4 (Tbeta(4)) has been shown to inhibit the apoptosis in nontransformed human corneal epithelial cells that is triggered by ethanol. The purpose of this study is to examine whether exogenous Tbeta(4) protects SV40-immortalized human corneal epithelial T (HCE-T) cells against the toxic effects of Fas ligand (FasL) and hydrogen peroxide (H(2)O(2)) and to elucidate its mechanism of action., Methods: HCE-T cells were incubated without or with the recombinant histidine-tagged Tbeta(4) produced by Escherichia coli before the addition of FasL or H(2)O(2). Cell viability was determined by MTT or MTS assay, and activation of caspase-8, -9, and -3 was examined by colorimetric and fluorescent substrate cleavage assays. The internalization of exogenous Tbeta(4) in HCE-T cells was analyzed by immunofluorescence staining. Cytochalasin D, an actin depolymerization agent, was added to examine whether the actin cytoskeleton is involved in Tbeta(4) entry and whether the internalization of this peptide is crucial for its cytoprotection., Results: The death of HCE-T cells induced by both FasL and H(2)O(2) was dramatically reduced by the recombinant Tbeta(4) pretreatment. Moreover, FasL-mediated activation of caspases-8 and -3 as well as H(2)O(2)-triggered stimulation of caspases-9 and -3 in these cells was abolished by preincubating them with the exogenous Tbeta(4). Of note, internalization of this G-actin-sequestering peptide into HCE-T cells was found to be essential in cell death prevention, in that disruption of the cellular entry of Tbeta(4) by cytochalasin D abrogated its cytoprotective effects., Conclusions: This is the first report to demonstrate that the internalization of exogenous Tbeta(4) is essential for its antiapoptotic activity in human corneal epithelial cells.

We reconstituted type I collagen nanofibers prepared by electrospin technology and examined the morphology, growth, adhesion, cell motility, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) on three nano-sized diameters (50-200, 200-500, and 500-1,000 nm). Results from scanning electron microscopy showed that cells on the nanofibers had a more polygonal and flattened cell morphology. MTS (3-[4,5-dimethythiazol-2-yl]-5-[3-carboxy-methoxyphenyl]-2-[4-sul-fophenyl]-2H-tetrazolium compound) assay demonstrated that the MSCs grown on 500-1,000-nm nanofibers had significantly higher cell viability than the tissue culture polystyrene control. A decreased amount of focal adhesion formation was apparent in which quantifiable staining area of the cytoplasmic protein vinculin for the 200-500-nm nanofibers was 39% less compared with control, whereas the area of quantifiable vinculin staining was 45% less for both the 200-500-nm and 500-1,000-nm nanofibers. The distances of cell migration were quantified on green fluorescent protein-nucleofected cells and was 56.7%, 37.3%, and 46.3% for 50-200, 200-500, and 500-1,000 nm, respectively, compared with those on the control. Alkaline phosphatase activity demonstrated no differences after 12 days of osteogenic differentiation, and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed comparable osteogenic gene expression of osteocalcin, osteonectin, and ostepontin between cells differentiated on polystyrene and nanofiber surfaces. Moreover, single-cell RT-PCR of type I collagen gene expression demonstrated higher expression on cells seeded on the nanofibers. Therefore, type I collagen nanofibers support the growth of MSCs without compromising their osteogenic differentiation capability and can be used as a scaffold for bone tissue engineering to facilitate intramembranous bone formation. Further efforts are necessary to enhance their biomimetic properties.

Acute intermittent porphyria (AIP) is an inherited metabolic disorder caused by deficiency of porphobilinogen deaminase, an enzyme found in the synthetic pathway of heme. Acute attack of AIP may be precipitated by many factors during operation and anesthesia, including fasting, dehydration, stress, infection, and drugs. Acute attack of AIP is likely fatal. Therefore, the drugs recommended as being safe in anesthesia for porphyria patients are up-to-dately refreshed and renovated and the identification of whether a drug is safe or not is based on cumulative anecdotal experiences. Here, we report the safe use of rocuronium and sevoflurane for long exposure in a patient affected with acute intermittent porphyria.

Pulmonary embolism is not uncommonly encountered in orthopedic patients with high risks, such as prolonged immobility, obesity, past or family history of thromboembolism, pelvic and long bone fractures. Here we report a young male patient with a residual cerebral arteriovenous malformation post-craniotomy suffered from acute severe hypoxemia after sustaining a simple fracture of the left femur shaft from a motorcycle accident. The emergent surgery was deferred in view of suspectable pulmonary embolism. Under supportive treatment, the condition spontaneously resolved, and the surgery was later performed uneventfully. To the best of our knowledge, this was the first instance that a pulmonary embolism (suspected subacute fat embolism syndrome) which ran in a natural course to spontaneous resolution was observed. We would like to report our clinical observation, and discuss the principle of anesthetic management in the text.

In this paper we describe the application and characterization of zinc oxide (ZnO) nanowires in an infrared (IR) chemical sensing system for the detection of volatile organic compounds (VOCs). Under suitable conditions, we grew ZnO nanowires on the surfaces of IR internal reflection elements (IREs) and obtained successful results for the detection of VOCs. ZnO nanowires offer a large surface area to effectively adsorb the examined species; the sensitivity of these IR sensing systems was increased by 3- to 15-fold after surface treatment with the ZnO nanowires. To explore the performance of this type of sensor, we correlated the morphologies of the ZnO nanowires grown on the surfaces of the IREs with the adsorption behavior observed during the sensing of the VOCs. To characterize the properties of the ZnO nanowires during the detection of VOCs having a range of functionalities, we classified the VOCs and examined their enrichment factors by comparing the IR signals detected in the presence and absence of the ZnO nanowires. Our results indicate that the ZnO nanowires exhibited better performance for the detection of aromatic-type VOCs than they did for non-aromatic compounds. For quantitative analyses, we examined several compounds for their responses toward varying quantities of injected VOCs. Our results indicate that the IREs treated with ZnO nanowires display acceptable linearity in their standard curves; the linear regression coefficients were higher than 0.995 for a range of volatile compounds.

Forehead lift plastic surgery is regarded as a rather safe operation without serious cardiovascular complications. Although cardiac arrhythmias (e.g., sinus bradycardia) during such an esthetically plastic surgery have been sporadically reported in literature, repetitive junctional rhythm has not ever been recounted. In this case report, we present a 45-year-old woman who developed repetitive junctional rhythm during forehead lift plastic surgery. The possible offenders causative of the arrhythmia (e.g., oculocardiac reflex induced by repeated surgical manipulation) and the principle of perioperative management were discussed in the text.