Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs.

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either "systemic administration" through all the medium streams or "intraarticular administration" by adding the drugs to only the shared "synovial fluid"-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

In vitro study to identify ligand-independent function of estrogen receptor-α in suppressing DNA damage-induced chondrocyte senescence.

In osteoarthritis (OA), chondrocytes undergo many pathological alternations that are linked with cellular senescence. However, the exact pathways that lead to the generation of a senescence-like phenotype in OA chondrocytes are not clear. Previously, we found that loss of estrogen receptor-α (ERα) was associated with an increased senescence level in human chondrocytes. Since DNA damage is a common cause of cellular senescence, we aimed to study the relationship among ERα levels, DNA damage, and senescence in chondrocytes. We first examined the levels of ERα, representative markers of DNA damage and senescence in normal and OA cartilage harvested from male and female human donors, as well as from male mice. The influence of DNA damage on ERα levels was studied by treating human chondrocytes with doxorubicin (DOX), which is an often-used DNA-damaging agent. Next, we tested the potential of overexpressing ERα in reducing DNA damage and senescence levels. Lastly, we explored the interaction between ERα and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Results indicated that the OA chondrocytes contained DNA damage and displayed senescence features, which were accompanied by significantly reduced ERα levels. Overexpression of ERα reduced the levels of DNA damage and senescence in DOX-treated normal chondrocytes and OA chondrocytes. Moreover, DOX-induced the activation of NF-κB pathway, which was partially reversed by overexpressing ERα. Taken together, our results demonstrated the critical role of ERα in maintaining the health of chondrocytes by inhibiting DNA damage and senescence. This study also suggests that maintaining the ERα level may represent a new avenue to prevent and treat OA.

Efficacy and Safety of Tramadol for Knee or Hip Osteoarthritis: A Systematic Review and Network Meta-Analysis of Randomized Controlled Trials.

OBJECTIVE: To examine efficacy and safety of tramadol for knee or hip osteoarthritis (OA). METHODS: PubMed, Embase, Cochrane Library, and Web of Science were searched up to May 2020 for randomized controlled trials (RCTs) comparing any of the following interventions: tramadol 100 mg/day, 200 mg/day, and 300 mg/day, and placebo for knee or hip OA. Pain and function were measured at or near 12 weeks for efficacy. Gastrointestinal, cardiovascular, and central nervous system (CNS) adverse events (AEs), and withdrawals were measured for safety. Bayesian network meta-analysis was conducted. RESULTS: Six RCTs (3,611 participants) were included. Tramadol 100 mg/day (standardized mean difference [SMD] -0.16 [95% confidence interval (95% CI) -0.34, 0.00]), 200 mg/day (SMD -0.21 [95% CI -0.37, -0.06]), and 300 mg/day (SMD -0.30 [95% CI -0.48, -0.14]) were statistically more effective than placebo in pain relief, but only tramadol 300 mg/day was better than placebo in functional improvement (SMD -0.24 [95% CI -0.47, -0.03]). Tramadol 100 mg/day (relative risk [RR] 2.29 [95% credible interval (CrI) 1.22, 4.25]), 200 mg/day (RR 4.35 [95% CrI 2.31, 8.01]), and 300 mg/day (RR 6.02 [95% CrI 3.22, 11.1]) involved a higher risk of gastrointestinal AEs. Similarly, tramadol 100-300 mg/day showed a higher risk of CNS AEs and withdrawals. However, the risk of cardiovascular AEs remained unclear. CONCLUSION: Only tramadol 300 mg/day showed minimal improvement in pain and function but with increasing AEs compared with placebo. Tramadol may not be sufficiently recommended for knee or hip OA based on the presented evidence, especially in patients with the risk of gastrointestinal and CNS AEs.

Differences in the intrinsic chondrogenic potential of human mesenchymal stromal cells and iPSC-derived multipotent cells.

BACKGROUND: Human multipotent progenitor cells (hiMPCs) created from induced pluripotent stem cells (iPSCs) represent a new cell source for cartilage regeneration. In most studies, bone morphogenetic proteins (BMPs) are needed to enhance transforming growth factor-ß (TGFß)-induced hiMPC chondrogenesis. In contrast, TGFß alone is sufficient to result in robust chondrogenesis of human primary mesenchymal stromal cells (hMSCs). Currently, the mechanism underlying this difference between hiMPCs and hMSCs has not been fully understood. METHODS: In this study, we first tested different growth factors alone or in combination in stimulating hiMPC chondrogenesis, with a special focus on chondrocytic hypertrophy. The reparative capacity of hiMPCs-derived cartilage was assessed in an osteochondral defect model created in rats. hMSCs isolated from bone marrow were included in all studies as the control. Lastly, a mechanistic study was conducted to understand why hiMPCs and hMSCs behave differently in responding to TGFß. RESULTS: Chondrogenic medium supplemented with TGFß3 and BMP6 led to robust in vitro cartilage formation from hiMPCs with minimal hypertrophy. Cartilage tissue generated from this new method was resistant to osteogenic transition upon subcutaneous implantation and resulted in a hyaline cartilage-like regeneration in osteochondral defects in rats. Interestingly, TGFß3 induced phosphorylation of both Smad2/3 and Smad1/5 in hMSCs, but only activated Smad2/3 in hiMPCs. Supplementing BMP6 activated Smad1/5 and significantly enhanced TGFß's compacity in inducing hiMPC chondrogenesis. The chondro-promoting function of BMP6 was abolished by the treatment of a BMP pathway inhibitor. CONCLUSIONS: This study describes a robust method to generate chondrocytes from hiMPCs with low hypertrophy for hyaline cartilage repair, as well as elucidates the difference between hMSCs and hiMPCs in response to TGFß. Our results also indicated the importance of activating both Smad2/3 and Smad1/5 in the initiation of chondrogenesis.

Sugar transporter TaSTP3 activation by TaWRKY19/61/82 enhances stripe rust susceptibility in wheat.

Sugar efflux from host plants is essential for pathogen survival and proliferation. Sugar transporter-mediated redistribution of host sugar contributes to the outcomes of plant-pathogen interactions. However, few studies have focused on how sugar translocation is strategically manipulated during host colonization. To elucidate this question, the wheat sugar transport protein (STP) TaSTP3 responding to Puccinia striiformis f. sp. tritici (Pst) infection was characterized for sugar transport properties in Saccharomyces cerevisiae and its potential role during Pst infection by RNA interference and overexpression in wheat. In addition, the transcription factors regulating TaSTP3 expression were further determined. The results showed that TaSTP3 is localized to the plasma membrane and functions as a sugar transporter of hexose and sucrose. TaSTP3 confers enhanced wheat susceptibility to Pst, and overexpression of TaSTP3 resulted in increased sucrose accumulation and transcriptional suppression of defense-related genes. Furthermore, TaWRKY19, TaWRKY61 and TaWRKY82 were identified as positive transcriptional regulators of TaSTP3 expression. Our findings reveal that the Pst-induced sugar transporter TaSTP3 is transcriptionally activated by TaWRKY19/61/82 and facilitates wheat susceptibility to stripe rust possibly through elevated sucrose concentration, and suggest TaSTP3 as a strong target for engineering wheat resistance to stripe rust.

Human Mesenchymal Stem Cell-Derived Miniature Joint System for Disease Modeling and Drug Testing.

Diseases of the knee joint such as osteoarthritis (OA) affect all joint elements. An in vitro human cell-derived microphysiological system capable of simulating intraarticular tissue crosstalk is desirable for studying etiologies/pathogenesis of joint diseases and testing potential therapeutics. Herein, a human mesenchymal stem cell-derived miniature joint system (miniJoint) is generated, in which engineered osteochondral complex, synovial-like fibrous tissue, and adipose tissue are integrated into a microfluidics-enabled bioreactor. This novel design facilitates different tissues communicating while still maintaining their respective phenotypes. The miniJoint exhibits physiologically relevant changes when exposed to interleukin-1ß mediated inflammation, which are similar to observations in joint diseases in humans. The potential of the miniJoint in predicting in vivo efficacy of drug treatment is confirmed by testing the "therapeutic effect" of the nonsteroidal anti-inflammatory drug, naproxen, as well as four other potential disease-modifying OA drugs. The data demonstrate that the miniJoint recapitulates complex tissue interactions, thus providing a robust organ chip model for the study of joint pathology and the development of novel therapeutic interventions.

Engineering pre-vascularized bone-like tissue from human mesenchymal stem cells through simulating endochondral ossification.

Currently, most in vitro engineered bone tissues do not contain viable blood vessel systems, so the vascularization depends on post-implantation angiogenesis from the host, which is often insufficient for repairing large bone defects. In this study, we aimed to create pre-vascularized bone-like tissue from human bone marrow-derived mesenchymal stem cells (HBMSCs) within the self-generated extracellular matrix by simulating the developmental endochondral ossification. Afterward, a three-dimensional (3D) culture of human umbilical vein endothelial cells (HUVECs)/HBMSCs was introduced to cover bone-like constructs surface for vascularization. Lastly, the pre-vascularized bone-like tissues were subcutaneously implanted into mice and the quality of newly formed blood vessels and bones were later assessed. We particularly examined whether the pre-existing HUVECs/HBMSCs vascular networks within the implants were able to integrate with the host's blood vessels and facilitate bone formation. Our results showed that this developmentally informed procedure resulted in a robust osteogenic differentiation of HBMSCs. Moreover, the bone-like constructs markedly promoted HUVEC/HBMSCs network formation in vitro. After 28 days of implantation in mice, the experimental group, in which bone-like constructs were pre-vascularized with HUVEC/HBMSCs networks, exhibited significantly more functional blood vessels than the control group that contained HUVEC and HBMSC single cells. Interestingly, increased levels of bone formation and absorption markers were also observed in the pre-vascularized bone-like constructs. Taken together, these findings demonstrated the potential of pre-vascularized bone-like constructs in repairing bone defects.

Mesenchymal stem cell-derived extracellular matrix (mECM): a bioactive and versatile scaffold for musculoskeletal tissue engineering.

Mesenchymal stem cell-derived extracellular matrix (mECM) has received increased attention in the fields of tissue engineering and scaffold-assisted regeneration. mECM exhibits many unique characteristics, such as robust bioactivity, biocompatibility, ease of use, and the potential for autologous tissue engineering. As the use of mECM has increased in musculoskeletal tissue engineering, it should be noted that mECM generated from current methods has inherited insufficiencies, such as low mechanical properties and lack of internal architecture. In this review, we first summarize the development and use of mECM as a scaffold for musculoskeletal tissue regeneration and highlight our current progress on moving this technology toward clinical application. Then we review recent methods to improve the properties of mECM that will overcome current weaknesses. Lastly, we propose future studies that will pave the road for mECM application in regenerating tissues in humans.

Does symptomatic knee osteoarthritis increase the risk of all-cause mortality? Data from four international population-based longitudinal surveys of aging.

OBJECTIVE: This study aimed at examining the association between symptomatic knee osteoarthritis and all-cause mortality based on four population-based longitudinal surveys. METHOD: Data were retrieved from the English Longitudinal Study of Aging (ELSA), the Survey of Health, Aging and Retirement in Europe (SHARE), the Korean Longitudinal Study of Aging (KLoSA), and the Indonesian Family Life Survey (IFLS). The association between symptomatic knee osteoarthritis and all-cause mortality over the 8- to 12-year follow-up period was assessed using Cox-proportional hazard models. RESULTS: In the entire sample of 59,522 participants (4823 with symptomatic knee osteoarthritis; 54,699 without symptomatic knee osteoarthritis [control group]; mean age: 61.8 years; female percentage: 55.3%), 8375 died (937 in the symptomatic knee osteoarthritis group, 7438 in the control group) during the follow-up period. Patients with symptomatic knee osteoarthritis had a higher risk of all-cause mortality than control group without adjusting for potential confounders in each survey, and the unadjusted hazard ratios (HRs) of all-cause mortality were 1.32 (95% confidence interval [CI] 1.18 to 1.47) in ELSA, 1.40 (95%CI 1.24 to 1.56) in SHARE, 1.25 (95%CI 1.06 to 1.47) in KLoSA, and 1.65 (95%CI 1.31 to 2.07) in IFLS. However, with adjustment of potential confounders, the corresponding HRs dropped to 1.07 (95%CI 0.94 to 1.20) in ELSA, 1.08 (95%CI 0.97 to 1.22) in SHARE, 0.91 (95%CI 0.77 to 1.08) in KLoSA, and 0.89 (95%CI 0.66 to 1.21) in IFLS, respectively. CONCLUSIONS: In these four population-based longitudinal studies, no association between symptomatic knee osteoarthritis and increased risk of all-cause mortality was observed after adjusting for potential confounders. Key Points • This study evaluated the association between symptomatic knee OA and the risk of all-cause mortality among the participants retrieved from four large population-based longitudinal studies across the world. • No association between symptomatic knee osteoarthritis and increased risk of all-cause mortality was observed after considering potential confounders, and our findings were consistent with the results derived from four independent longitudinal studies. • The present study included four international population-based longitudinal studies, comprising both developed and developing areas, which allowed the findings to be interpreted under larger circumstance.

DTWscore: differential expression and cell clustering analysis for time-series single-cell RNA-seq data.

BACKGROUND: The development of single-cell RNA sequencing has enabled profound discoveries in biology, ranging from the dissection of the composition of complex tissues to the identification of novel cell types and dynamics in some specialized cellular environments. However, the large-scale generation of single-cell RNA-seq (scRNA-seq) data collected at multiple time points remains a challenge to effective measurement gene expression patterns in transcriptome analysis. RESULTS: We present an algorithm based on the Dynamic Time Warping score (DTWscore) combined with time-series data, that enables the detection of gene expression changes across scRNA-seq samples and recovery of potential cell types from complex mixtures of multiple cell types. CONCLUSIONS: The DTWscore successfully classify cells of different types with the most highly variable genes from time-series scRNA-seq data. The study was confined to methods that are implemented and available within the R framework. Sample datasets and R packages are available at https://github.com/xiaoxiaoxier/DTWscore .

Effects of Ca/P molar ratios on regulating biological functions of hybridized carbon nanofibers containing bioactive glass nanoparticles.

Biological functions of hybridized carbon nanofibers (CNFs) depend closely on the incorporated bioactive components. For hybridized CNFs containing bioactive glass (BG) nanoparticles (CNF/BG), chemical compositions of BG nanoparticles might have decisive effects on their cell affinity and osteocompatibility. Herein, three hybridized CNF/BGs were produced by incorporating 68S-type BG nanoparticles with different Ca/P molar ratios (1.0, 1.67 or 2.5) into CNFs via a sol-gel/electrospinning and carbonization method. Structural evolution of these hybridized CNF/BGs was studied in relation to their Ca/P molar ratios. Crystalline wollastonite was found to be the dominant phase at a high feeding Ca/P molar ratio (i.e. 2.5), but weak crystallized hydroxyapatite was the main phase at the low feeding Ca/P molar ratio (i.e. 1.0). These findings were correlated to the biological functions of the resulted CNF/BG hybrids including apatite formation ability in simulated body fluid and osteoblast behaviors in in vitro culture. All the CNF/BG hybrids displayed a strong affinity for inducing apatite deposition, showing insignificant difference after the initial nucleation stage, while they behaved differently in promoting the proliferation and osteogenic differentiation of osteoblasts. The fastest proliferation rate and the highest expression of alkaline phosphatase activity was found on the CNF/BG (Ca/P = 1.0). The results suggested a feasible way to upregulate osteoblast behaviors is by changing the feeding Ca/P molar ratios in the preparation of CNF/BG hybrids for potential bone repairing applications.

Cell studies of hybridized carbon nanofibers containing bioactive glass nanoparticles using bone mesenchymal stromal cells.

Bone regeneration required suitable scaffolding materials to support the proliferation and osteogenic differentiation of bone-related cells. In this study, a kind of hybridized nanofibrous scaffold material (CNF/BG) was prepared by incorporating bioactive glass (BG) nanoparticles into carbon nanofibers (CNF) via the combination of BG sol-gel and polyacrylonitrile (PAN) electrospinning, followed by carbonization. Three types (49 s, 68 s and 86 s) of BG nanoparticles were incorporated. To understand the mechanism of CNF/BG hybrids exerting osteogenic effects, bone marrow mesenchymal stromal cells (BMSCs) were cultured directly on these hybrids (contact culture) or cultured in transwell chambers in the presence of these materials (non-contact culture). The contributions of ion release and contact effect on cell proliferation and osteogenic differentiation were able to be correlated. It was found that the ionic dissolution products had limited effect on cell proliferation, while they were able to enhance osteogenic differentiation of BMSCs in comparison with pure CNF. Differently, the proliferation and osteogenic differentiation were both significantly promoted in the contact culture. In both cases, CNF/BG(68 s) showed the strongest ability in influencing cell behaviors due to its fastest release rate of soluble silicium-relating ions. The synergistic effect of CNF and BG would make CNF/BG hybrids promising substrates for bone repairing.