Self-organized emergence of hyaline cartilage in hiPSC-derived multi-tissue organoids

Despite holding great therapeutic potential, existing protocols for in vitro chondrogenesis and hyaline cartilage production from human induced pluripotent stem cells (hiPSC) are laborious and complex with unclear long-term consequences. Here, we developed a simple xeno- and feeder-free protocol for human hyaline cartilage production in vitro using hydrogel-cultured multi-tissue organoids (MTOs). We investigate gene regulatory networks during spontaneous hiPSC-MTO differentiation using RNA sequencing and bioinformatic analyses. We find the interplays between BMPs and neural FGF pathways are associated with the phenotype transition of MTOs. We recognize TGF-beta/BMP and Wnt signaling likely contribute to the long-term maintenance of MTO cartilage growth and further adoption of articular cartilage development. By comparing the MTO transcriptome with human lower limb chondrocytes, we observe that the expression of chondrocyte-specific genes in MTO shows a strong correlation with fetal lower limb chondrocytes. Collectively, our findings describe the self-organized emergence of hyaline cartilage in MTO, its associated molecular pathways, and its spontaneous adoption of articular cartilage development trajectory.

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In Vitro Evaluation of the Anti-Inflammatory Effect of KMUP-1 and in Vivo Analysis of Its Therapeutic Potential in Osteoarthritis

Biomedicines ◽

10.3390/biomedicines9060615 ◽

2021 ◽

Vol 9 (6) ◽

pp. 615

Author(s):

Shang-En Huang ◽

Erna Sulistyowati ◽

Yu-Ying Chao ◽

Bin-Nan Wu ◽

Zen-Kong Dai ◽

...

Keyword(s):

Articular Cartilage ◽

Inflammatory Responses ◽

Therapeutic Potential ◽

Antioxidant Properties ◽

Serum Levels ◽

Gene Expressions ◽

Tnf Α ◽

Anti Inflammatory

Osteoarthritis is a degenerative arthropathy that is mainly characterized by dysregulation of inflammatory responses. KMUP-1, a derived chemical synthetic of xanthine, has been shown to have anti-inflammatory and antioxidant properties. Here, we aimed to investigate the in vitro anti-inflammatory and in vivo anti-osteoarthritis effects of KMUP-1. Protein and gene expressions of inflammation markers were determined by ELISA, Western blotting and microarray, respectively. RAW264.7 mouse macrophages were cultured and pretreated with KMUP-1 (1, 5, 10 μM). The productions of TNF-α, IL-6, MMP-2 and MMP- 9 were reduced by KMUP-1 pretreatment in LPS-induced inflammation of RAW264.7 cells. The expressions of iNOS, TNF-α, COX-2, MMP-2 and MMP-9 were also inhibited by KMUP-1 pretreatment. The gene expression levels of TNF and COX families were also downregulated. In addition, KMUP-1 suppressed the activations of ERK, JNK and p38 as well as phosphorylation of IκBα/NF-κB signaling pathways. Furthermore, SIRT1 inhibitor attenuated the inhibitory effect of KMUP-1 in LPS-induced NF-κB activation. In vivo study showed that KMUP-1 reduced mechanical hyperalgesia in monoiodoacetic acid (MIA)-induced rats OA. Additionally, KMUP-1 pretreatment reduced the serum levels of TNF-α and IL-6 in MIA-injected rats. Moreover, macroscopic and histological observation showed that KMUP-1 reduced articular cartilage erosion in rats. Our results demonstrated that KMUP-1 inhibited the inflammatory responses and restored SIRT1 in vitro, alleviated joint-related pain and cartilage destruction in vivo. Taken together, KMUP-1 has the potential to improve MIA-induced articular cartilage degradation by inhibiting the levels and expression of inflammatory mediators suggesting that KMUP-1 might be a potential therapeutic agent for OA.

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Differential Regulation of Articular Cartilage Biomechanical and Biochemical Properties by IGF-1 and TGF-β1 During In Vitro Growth

ASME 2009 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2009-205117 ◽

2009 ◽

Author(s):

Michael E. Stender ◽

Christian R. Flores ◽

Kristin J. Dills ◽

Gregory M. Williams ◽

Kevin M. Stewart ◽

...

Keyword(s):

Articular Cartilage ◽

Growth Models ◽

Biochemical Properties ◽

Low Friction ◽

Cartilage Growth ◽

Detailed Characterization ◽

Naturally Occurring ◽

Tgf Β1

Articular cartilage (AC) is a load bearing material that provides a low friction wear resistant interface in synovial joints. Naturally-occurring and stimulated intrinsic repair of damaged AC is ineffective. Thus, there is a desire to engineer effective replacement tissue that could be used for AC repair. Previous studies [1] have shown that culture of immature cartilage with medium including TGF-β1 will result in a more mature tissue than culture with IGF-1. Detailed characterization of tissue mechanical properties would be helpful for development of cartilage growth models [2].

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Long-term in vitro analysis of limb cartilage development: Involvement of Wnt signaling

Journal of Cellular Biochemistry ◽

10.1002/jcb.20190 ◽

2004 ◽

Vol 93 (3) ◽

pp. 526-541 ◽

Cited By ~ 41

Author(s):

Kathleen M. Daumer ◽

A. Cevik Tufan ◽

Rocky S. Tuan

Keyword(s):

Wnt Signaling ◽

Cartilage Development ◽

Vitro Analysis ◽

In Vitro Analysis

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Simulating the Growth of Articular Cartilage Explants in a Permeation Bioreactor to Aid in Experimental Protocol Design

Journal of Biomechanical Engineering ◽

10.1115/1.3049856 ◽

2009 ◽

Vol 131 (4) ◽

Cited By ~ 10

Author(s):

Timothy P. Ficklin ◽

Andrew Davol ◽

Stephen M. Klisch

Keyword(s):

Finite Element ◽

Steady State ◽

Articular Cartilage ◽

Element Model ◽

Cartilage Explants ◽

Solid Matrix ◽

Cartilage Growth ◽

Growth Laws ◽

Competing Hypotheses

Recently a cartilage growth finite element model (CGFEM) was developed to solve nonhomogeneous and time-dependent growth boundary-value problems (Davol et al., 2008, “A Nonlinear Finite Element Model of Cartilage Growth,” Biomech. Model. Mechanobiol., 7, pp. 295–307). The CGFEM allows distinct stress constitutive equations and growth laws for the major components of the solid matrix, collagens and proteoglycans. The objective of the current work was to simulate in vitro growth of articular cartilage explants in a steady-state permeation bioreactor in order to obtain results that aid experimental design. The steady-state permeation protocol induces different types of mechanical stimuli. When the specimen is initially homogeneous, it directly induces homogeneous permeation velocities and indirectly induces nonhomogeneous solid matrix shear stresses; consequently, the steady-state permeation protocol is a good candidate for exploring two competing hypotheses for the growth laws. The analysis protocols were implemented through the alternating interaction of the two CGFEM components: poroelastic finite element analysis (FEA) using ABAQUS and a finite element growth routine using MATLAB. The CGFEM simulated 12 days of growth for immature bovine articular cartilage explants subjected to two competing hypotheses for the growth laws: one that is triggered by permeation velocity and the other by maximum shear stress. The results provide predictions for geometric, biomechanical, and biochemical parameters of grown tissue specimens that may be experimentally measured and, consequently, suggest key biomechanical measures to analyze as pilot experiments are performed. The combined approach of CGFEM analysis and pilot experiments may lead to the refinement of actual experimental protocols and a better understanding of in vitro growth of articular cartilage.

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Altered swelling and ion fluxes in articular cartilage as a biomarker in osteoarthritis and joint immobilization: a computational analysis

Journal of The Royal Society Interface ◽

10.1098/rsif.2014.1090 ◽

2015 ◽

Vol 12 (102) ◽

pp. 20141090 ◽

Cited By ~ 12

Author(s):

Sara Manzano ◽

Raquel Manzano ◽

Manuel Doblaré ◽

Mohamed Hamdy Doweidar

Keyword(s):

Articular Cartilage ◽

Computational Model ◽

Three Dimensional ◽

Ion Distribution ◽

Tissue Degeneration ◽

Structural Deterioration ◽

Healthy Cartilage ◽

Tissue Degradation

In healthy cartilage, mechano-electrochemical phenomena act together to maintain tissue homeostasis. Osteoarthritis (OA) and degenerative diseases disrupt this biological equilibrium by causing structural deterioration and subsequent dysfunction of the tissue. Swelling and ion flux alteration as well as abnormal ion distribution are proposed as primary indicators of tissue degradation. In this paper, we present an extension of a previous three-dimensional computational model of the cartilage behaviour developed by the authors to simulate the contribution of the main tissue components in its behaviour. The model considers the mechano-electrochemical events as concurrent phenomena in a three-dimensional environment. This model has been extended here to include the effect of repulsion of negative charges attached to proteoglycans. Moreover, we have studied the fluctuation of these charges owning to proteoglycan variations in healthy and pathological articular cartilage. In this sense, standard patterns of healthy and degraded tissue behaviour can be obtained which could be a helpful diagnostic tool. By introducing measured properties of unhealthy cartilage into the computational model, the severity of tissue degeneration can be predicted avoiding complex tissue extraction and subsequent in vitro analysis. In this work, the model has been applied to monitor and analyse cartilage behaviour at different stages of OA and in both short (four, six and eight weeks) and long-term (11 weeks) fully immobilized joints. Simulation results showed marked differences in the corresponding swelling phenomena, in outgoing cation fluxes and in cation distributions. Furthermore, long-term immobilized patients display similar swelling as well as fluxes and distribution of cations to patients in the early stages of OA, thus, preventive treatments are highly recommended to avoid tissue deterioration.

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Modelling cartilage mechanobiology

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2003.1346 ◽

2003 ◽

Vol 358 (1437) ◽

pp. 1461-1471 ◽

Cited By ~ 100

Author(s):

Dennis R. Carter ◽

Marcy Wong

Keyword(s):

Articular Cartilage ◽

Single Phase ◽

Skeletal Development ◽

Fluid Pressure ◽

Superficial Layer ◽

Tissue Mechanics ◽

Joint Mechanics ◽

Cartilage Growth ◽

Skeletal Morphology

The growth, maintenance and ossification of cartilage are fundamental to skeletal development and are regulated throughout life by the mechanical cues that are imposed by physical activities. Finite element computer analyses have been used to study the role of local tissue mechanics on endochondral ossification patterns, skeletal morphology and articular cartilage thickness distributions. Using single–phase continuum material representations of cartilage, the results have indicated that local intermittent hydrostatic pressure promotes cartilage maintenance. Cyclic tensile strains (or shear), however, promote cartilage growth and ossification. Because single–phase material models cannot capture fluid exudation in articular cartilage, poroelastic (or biphasic) solid/fluid models are often implemented to study joint mechanics. In the middle and deep layers of articular cartilage where poroelastic analyses predict little fluid exudation, the cartilage phenotype is maintained by cyclic fluid pressure (consistent with the single–phase theory). In superficial articular layers the chondrocytes are exposed to tangential tensile strain in addition to the high fluid pressure. Furthermore, there is fluid exudation and matrix consolidation, leading to cell ‘flattening’. As a result, the superficial layer assumes an altered, more fibrous phenotype. These computer model predictions of cartilage mechanobiology are consistent with results of in vitro cell and tissue and molecular biology experiments.

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Incorporation of the factor IX Padua mutation into FIX-Triple improves clotting activity in vitro and in vivo

Thrombosis and Haemostasis ◽

10.1160/th13-02-0154 ◽

2013 ◽

Vol 110 (08) ◽

pp. 244-256 ◽

Cited By ~ 20

Author(s):

Chung-Yang Kao ◽

Shu-Jhu Yang ◽

Mi-Hua Tao ◽

Yung-Ming Jeng ◽

I-Shing Yu ◽

...

Keyword(s):

Gene Therapy ◽

Replacement Therapy ◽

Therapeutic Potential ◽

Factor Ix ◽

Adeno Associated Virus ◽

Viral Dose ◽

Better Than

SummaryUsing gain-of-function factor IX (FIX) for replacement therapy for haemophilia B (HB) is an attractive strategy. We previously reported a high-activity FIX, FIX-Triple (FIX-V86A/E277A/R338A) as a good substitute for FIX-WT (wild-type) in protein replacement therapy, gene therapy, and cell therapy. Here we generated a new recombinant FIXTripleL (FIX-V86A/E277A/R338L) by replacing the alanine at residue 338 of FIX-Triple with leucine as in FIX-Padua (FIX-R338L). Purified FIX-TripleL exhibited 22-fold higher specific clotting activity and 15-fold increased binding affinity to activated FVIII compared to FIXWT. FIX-TripleL increased the therapeutic potential of FIX-Triple by nearly 100% as demonstrated with calibrated automated thrombogram and thromboelastography. FIX-TripleL demonstrated a normal clearance rate in HB mice. The clotting activity of FIX-TripleL was consistently 2- to 3-fold higher in these mice than that of FIX-Triple or FIXR338L. Gene delivery of adeno-associated virus (AAV) in HB mice showed that FIX-TripleL had 15-fold higher specific clotting activity than FIX-WT, and this activity was significantly better than FIX-Triple (10-fold) or FIX-R338L (6-fold). At a lower viral dose, FIX-TripleL improved FIX activity from sub-therapeutic to therapeutic levels. Under physiological conditions, no signs of adverse thrombotic events were observed in long-term AAV-FIX-treated C57Bl/6 mice. Hepatocellular adenomas were observed in the high- but not the medium- or the lowdose AAV-treated mice expressing FIX-WT or FIX-Triple, indicating the advantages of using hyperfunctional FIX variants to reduce viral doses while maintaining therapeutic clotting activity. Thus, incorporation of the FIX Padua mutation significantly improves the clotting function of FIX-Triple so as to optimise protein replacement therapy and gene therapy.

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Roles and Mechanisms of Irisin in Attenuating Pathological Features of Osteoarthritis

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.703670 ◽

2021 ◽

Vol 9 ◽

Author(s):

Xiangfen Li ◽

Xiaofang Zhu ◽

Hongle Wu ◽

Thomas E. Van Dyke ◽

Xiaoyang Xu ◽

...

Keyword(s):

Developmental Stages ◽

Therapeutic Potential ◽

Gene Knockout ◽

Inflammatory Factors ◽

Proliferative Potential ◽

Opposite Pattern ◽

Tissue Samples ◽

Primary Chondrocytes ◽

Cartilage Development

To investigate the effects and mechanisms of irisin, a newly discovered myokine, in cartilage development, osteoarthritis (OA) pathophysiology and its therapeutic potential for treating OA we applied the following five strategical analyses using (1) murine joint tissues at different developmental stages; (2) human normal and OA pathological tissue samples; (3) experimental OA mouse model; (4) irisin gene knockout (KO) and knock in (KI) mouse lines and their cartilage cells; (5) in vitro mechanistic experiments. We found that Irisin was involved in all stages of cartilage development. Both human and mouse OA tissues showed a decreased expression of irisin. Intra-articular injection of irisin attenuated ACLT-induced OA progression. Irisin knockout mice developed severe OA while irisin overexpression in both irisin KI mice and intraarticular injection of irisin protein attenuated OA progression. Irisin inhibited inflammation and promoted anabolism in chondrogenic ADTC5 cells. Proliferative potential of primary chondrocytes from KI mice was found to be enhanced, while KO mice showed an inhibition under normal or inflammatory conditions. The primary chondrocytes from irisin KI mice showed reduced expression of inflammatory factors and the chondrocytes isolated from KO mice showed an opposite pattern. In conclusion, it is the first time to show that irisin is involved in cartilage development and OA pathogenesis. Irisin has the potential to ameliorate OA progression by decreasing cartilage degradation and inhibiting inflammation, which could lead to the development of a novel therapeutic target for treating bone and cartilage disorders including osteoarthritis.

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