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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 ...

University of Pittsburgh School of Medicine

Human mesenchymal stem cells (hMSCs) are considered a highly promising candidate cell type for cell-based tissue engineering and regeneration because of their self-renewal and multi-lineage differentiation characteristics. Increased levels of reactive oxygen/nitrogen species (ROS/RNS) are associated with tissue injury and inflammation, impact a number of cellular processes, including cell adhesion, migration, and proliferation, and have been linked to cellular senescence in MSCs, potentially compromising their activities. Naturally occurring polyphenolic compounds (polyphenols), epigallocatechin-3-gallate (EGCG), and curcumin, block ROS/RNS and are potent inflammation-modulating agents. However, their potential protective effects against oxidative stress in hMSCs have not been examined. In this study, we carried out a systematic analysis of the effects of polyphenols on hMSCs in their response to oxidative stress in the form of treatment with H(2)O(2) and S-nitroso-N-acetylpenicillamine (SNAP), respectively. Parameters measured included colony forming activity, apoptosis, and the levels of antioxidant enzymes and free reactive species. We found that polyphenols reversed H(2)O(2) -induced loss of colony forming activity in hMSCs. In a dose-dependent manner, polyphenols inhibited increased levels of ROS and NO, produced by H(2)O(2) or SNAP, respectively, in MSCs. Notably, polyphenols rapidly and almost completely blocked H(2)O(2) -induced ROS in the absence of significant direct effect on H(2)O(2) itself. Polyphenols also protected the antioxidant enzymes and reduced apoptotic cell death caused by H(2)O(2) exposure. Taken together, these findings demonstrate that EGCG and curcumin are capable of suppressing inducible oxidative stress in hMSCs, and suggest a possible new approach to maintain MSC viability and potency for clinical application.

Polyphenols suppress hydrogen peroxide-induced oxidative stress in human bone-marrow derived mesenchymal stem cells.

Osteomyelitis is a devastating condition whose treatment relies on the detection of bacteria. The current standard of microbiology culture may not be adequate. Molecular biology based diagnostic procedures for detecting bacteria in orthopaedic infections was previously established, but has not been applied to the setting of chronic osteomyelitis. We aim to determine the applicability of molecular diagnostic procedures for monitoring chronic osteomyelitis, and to evaluate if these procedures are superior to standard culture methods of osteomyelitis detection.

Polymerase Chain Reaction molecular diagnostic technology for monitoring chronic osteomyelitis.

There have been limited advances in the treatment of bone and joint infections, which currently involves a combination of surgery and antibiotic administration. There is a timely need in orthopedics to develop more effective and less invasive forms of antimicrobial prophylaxis and treatment. The antibacterial effect of adult tissue-derived mesenchymal stem cells (MSCs) has recently been investigated against Escherichia coli and Staphylococcus aureus. The main mechanism of action is postulated to be via MSC production of the cationic antimicrobial peptide, LL-37.

Antimicrobial activity of mesenchymal stem cells against Staphylococcus aureus.

Periprosthetic joint infections (PJIs) and osteomyelitis are clinical challenges that are difficult to eradicate. Well-characterized large animal models necessary for testing and validating new treatment strategies for these conditions are lacking. The purpose of this study was to develop a rabbit model of chronic PJI in the distal femur.

Development of a large animal rabbit model for chronic periprosthetic joint infection.

Biomaterials that can harness the intrinsic osteogenic potential of stem cells offer a promising strategy to accelerate bone regeneration and repair. Previously, we had used methacrylated gelatin (GelMA)-based scaffolds to achieve bone formation from human mesenchymal stem cells (hMSCs). In this study, we aimed to further enhance hMSC osteogenesis by incorporating graphene oxide (GO)-based nanosheets into GelMA. In vitro results showed high viability and metabolic activities in hMSCs encapsulated in the newly developed nanocomposites. Incorporation of GO markedly increased mineralization within hMSC-laden constructs, which was further increased by replacing GO with silica-coated graphene oxide (SiGO). Mechanistic analysis revealed that the nanosheet enhanced the production, retention, and biological activity of endogenous bone morphogenetic proteins (BMPs), resulting in robust osteogenesis in the absence of exogenous osteoinductive growth factors. Specifically, the osteoinductive effect of the nanosheets was abolished by inhibiting the BMP signaling pathway with LDN-193189 treatment. The bone formation potential of the technology was further tested in vivo using a mouse subcutaneous implantation model, where hMSCs-laden GO/GelMA and SiGO/GelMA samples resulted in bone volumes 108 and 385 times larger, respectively, than the GelMA control group. Taken together, these results demonstrate the biological activity and mechanism of action of GO-based nanosheets in augmenting the osteogenic capability of hMSCs, and highlights the potential of leveraging nanomaterials such as GO and SiGO for bone tissue engineering applications.

Graphene oxide-functionalized nanocomposites promote osteogenesis of human mesenchymal stem cells via enhancement of BMP-SMAD1/5 signaling pathway.

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-1β 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.

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

Reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in chondrocyte senescence and cartilage aging, pathogenesis of osteoarthritis (OA), and rheumatoid arthritis. Naturally occurring polyphenolic compounds (PPCs), such as curcumin (turmeric), resveratrol (grape), and epigallocatechin-3-gallate (EGCG) (green tea), have been known for their anti-inflammatory and chondroprotective effects. However, the potential protective effects of these PPCs against oxidative stress in chondrocytes are unclear. To investigate this, bovine articular chondrocytes and human osteoarthritic chondrocytes were pre-treated with PPCs at varying concentrations, and then exposed to hydrogen peroxide (HO) as an ROS inducer or S-nitroso-N-acetylpenicillamine (SNAP) as a NO donor. Alternatively, chondrocytes were co-treated with polyphenols and HO. Intracellular ROS/NO were measured using a fluorescent dye technique (HDCF-DA for ROS; DAF-FM for NO). Our findings showed that PPC pre-/co-treatment inhibited both HO-induced ROS and SNAP-induced NO at different concentrations in both bovine chondrocytes and human osteoarthritic chondrocytes. Curcumin also increased glutathione peroxidase activity in the presence of HO in bovine chondrocytes. Taken together, these findings indicate that PPCs are capable of suppressing oxidative stress- induced responses in chondrocytes, which may have potential therapeutic value for OA clinical application.

Haruyo Yagi

Department of Orthopaedic Surgery

University of Pittsburgh School of Medicine

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

Haruyo Yagi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Orthopaedic Surgery

University of Pittsburgh School of Medicine

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

Department of Orthopaedic Surgery