creation-knee-joint-on-chip-for-modeling-joint-diseases-testing

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

Three-dimensional hydrogel constructs incorporated with live stem cells that support chondrogenic differentiation and maintenance offer a promising regenerative route towards addressing the limited self-repair capabilities of articular cartilage. In particular, hydrogel scaffolds that augment chondrogenesis and recapitulate the native physical properties of cartilage, such as compressive strength, can potentially be applied in point-of-care procedures. We report here the synthesis of two new materials, [poly-l-lactic acid/polyethylene glycol/poly-l-lactic acid] (PLLA-PEG 1000) and [poly-d,l-lactic acid/polyethylene glycol/poly-d,l-lactic acid] (PDLLA-PEG 1000), that are biodegradable, biocompatible (>80% viability post fabrication), and possess high, physiologically relevant mechanical strength (∼1500 to 1800kPa). This study examined the effects of physiologically relevant cell densities (4, 8, 20, and 50×10/mL) and hydrogel stiffnesses (∼150kPa to∼1500kPa Young's moduli) on chondrogenesis of human bone marrow stem cells incorporated in hydrogel constructs fabricated with these materials and a previously characterized PDLLA-PEG 4000. Results showed that 20×10cells/mL, under a static culture condition, was the most efficient cell seeding density for extracellular matrix (ECM) production on the basis of hydroxyproline and glycosaminoglycan content. Interestingly, material stiffness did not significantly affect chondrogenesis, but rather material concentration was correlated to chondrogenesis with increasing levels at lower concentrations based on ECM production, chondrogenic gene expression, and histological analysis. These findings establish optimal cell densities for chondrogenesis within three-dimensional cell-incorporated hydrogels, inform hydrogel material development for cartilage tissue engineering, and demonstrate the efficacy and potential utility of PDLLA-PEG 1000 for point-of-care treatment of cartilage defects.

Chondrogenesis of human bone marrow mesenchymal stem cells in 3-dimensional, photocrosslinked hydrogel constructs: Effect of cell seeding density and material stiffness.

Adult mesenchymal stem cells (MSCs) are an important resource for tissue growth, repair, and regeneration. To utilize MSCs more effectively, a clear understanding of how they react to environmental cues is essential. Currently, relatively little is known about how the composition of the plasma membranes affects stem cell phenotype and properties. The presence of lipid molecules, including cholesterol in particular, in the plasma membrane plays a crucial role in regulating a variety of physiological processes in cells. In this study, we examined the effects of perturbations in cholesterol/caveolin-1 (CAV-1)/caveolae homeostasis on the membrane properties and adhesive characteristics of MSCs. Findings from this study will contribute to the understanding of how cholesterol/CAV-1/caveolae regulates aspects of the cell membrane important to cell adhesion, substrate sensing, and microenvironment interaction.

Influence of cholesterol/caveolin-1/caveolae homeostasis on membrane properties and substrate adhesion characteristics of adult human mesenchymal stem cells.

Cell-loaded hydrogels are frequently applied in cartilage tissue engineering for their biocompatibility, ease of application, and ability to conform to various defect sites. As a bioactive adjunct to the biomaterial, transforming growth factor beta (TGF-β) has been shown to be essential for cell differentiation into a chondrocyte phenotype and maintenance thereof, but the low amounts of endogenous TGF-β in the in vivo joint microenvironment necessitate a mechanism for controlled delivery and release of this growth factor. In this study, TGF-β3 was directly loaded with human bone marrow-derived mesenchymal stem cells (MSCs) into poly-d,l-lactic acid/polyethylene glycol/poly-d,l-lactic acid (PDLLA-PEG) hydrogel, or PDLLA-PEG with the addition of hyaluronic acid (PDLLA/HA), and cultured in vitro. We hypothesize that the inclusion of HA within PDLLA-PEG would result in a controlled release of the loaded TGF-β3 and lead to a robust cartilage formation without the use of TGF-β3 in the culture medium. ELISA analysis showed that TGF-β3 release was effectively slowed by HA incorporation, and retention of TGF-β3 in the PDLLA/HA scaffold was detected by immunohistochemistry for up to 3 weeks. By means of both in vitro culture and in vivo implantation, we found that sulfated glycosaminoglycan production was higher in PDLLA/HA groups with homogenous distribution throughout the scaffold than PDLLA groups. Finally, with an optimal loading of TGF-β3 at 10 μg/mL, as determined by RT-PCR and glycosaminoglycan production, an almost twofold increase in Young's modulus of the construct was seen over a 4-week period compared to TGF-β3 delivery in the culture medium. Taken together, our results indicate that the direct loading of TGF-β3 and stem cells in PDLLA/HA has the potential to be a one-step point-of-care treatment for cartilage injury. STATEMENT OF SIGNIFICANCE: Stem cell-seeded hydrogels are commonly used in cell-based cartilage tissue engineering, but they generally fail to possess physiologically relevant mechanical properties suitable for loading. Moreover, degradation of the hydrogel in vivo with time further decreases mechanical suitability of the hydrogel due in part to the lack of TGF-β3 signaling. In this study, we demonstrated that incorporation of hyaluronic acid (HA) into a physiologically stiff PDLLA-PEG hydrogel allowed for slow release of one-time preloaded TGF-β3, and when loaded with adult mesenchymal stem cells and cultured in vitro, it resulted in higher chondrogenic gene expression and constructs of significantly higher mechanical strength than constructs cultured in conventional TGF-β3-supplemented medium. Similar effects were also observed in constructs implanted in vivo. Our results indicate that direct loading of TGF-β3 combined with HA in the physiologically stiff PDLLA-PEG hydrogel has the potential to be used for one-step point-of-care treatment of cartilage injury.

Enhancing chondrogenesis and mechanical strength retention in physiologically relevant hydrogels with incorporation of hyaluronic acid and direct loading of TGF-β.

There is no therapy currently available for fully repairing articular cartilage lesions. Our laboratory has recently developed a visible light-activatable methacrylated gelatin (mGL) hydrogel, with the potential for cartilage regeneration. In this study, we further optimized mGL scaffolds by supplementing methacrylated hyaluronic acid (mHA), which has been shown to stimulate chondrogenesis via activation of critical cellular signalling pathways. We hypothesized that the introduction of an optimal ratio of mHA would enhance the biological properties of mGL scaffolds and augment chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs). To test this hypothesis, hybrid scaffolds consisting of mGL and mHA at different weight ratios were fabricated with hBMSCs encapsulated at 20 × 10  cells/ml and maintained in a chondrogenesis-promoting medium. The chondrogenenic differentiation of hBMSCs, within different scaffolds, was estimated after 8 weeks of culture. Our results showed that mGL/mHA at a 9:1 (%, w/v) ratio resulted in the lowest hBMSC hypertrophy and highest glycosaminoglycan production, with a slightly increased volume of the entire construct. The applicability of this optimally designed mGL/mHA hybrid scaffold for cartilage repair was then examined in vivo. A full-thickness cylindrical osteochondral defect was surgically created in the rabbit femoral condyle, and a three-dimensional cell-biomaterial construct was fabricated by in situ photocrosslinking to fully fill the lesion site. The results showed that implantation of the mGL/mHA (9:1) construct resulted in both cartilage and subchondral bone regeneration after 12 weeks, supporting its use as a promising scaffold for repair and resurfacing of articular cartilage defects, in the clinical setting.

Optimization of photocrosslinked gelatin/hyaluronic acid hybrid scaffold for the repair of cartilage defect.

Musculoskeletal injuries and associated conditions are the leading cause of physical disability worldwide. The concept of tissue engineering has opened up novel approaches to repair musculoskeletal defects in a fast and/or efficient manner. Biomaterials, cells, and signaling molecules constitute the tissue engineering triad. In the past 40 years, significant progress has been made in developing and optimizing all three components, but only a very limited number of technologies have been successfully translated into clinical applications. A major limiting factor of this barrier to translation is the insufficiency of two-dimensional cell cultures and traditional animal models in informing the safety and efficacy of in-human applications. In recent years, microphysiological systems, often referred to as organ or tissue chips, generated according to tissue engineering principles, have been proposed as the next-generation drug testing models. This chapter aims to first review the current tissue engineering-based approaches that are being applied to fabricate and develop the individual critical elements involved in musculoskeletal organ/tissue chips. We next highlight the general strategy of generating musculoskeletal tissue chips and their potential in future regenerative medicine research. Exemplary microphysiological systems mimicking musculoskeletal tissues are described. With sufficient physiological accuracy and relevance, the human cell-derived, three-dimensional, multi-tissue systems have been used to model a number of orthopedic disorders and to test new treatments. We anticipate that the novel emerging tissue chip technology will continually reshape and improve our understanding of human musculoskeletal pathophysiology, ultimately accelerating the development of advanced pharmaceutics and regenerative therapies.

Tissue Engineering for Musculoskeletal Regeneration and Disease Modeling.

Autologous chondrocyte implantation (ACI) is a regenerative procedure used to treat focal articular cartilage defects in knee joints. However, age has been considered as a limiting factor and ACI is not recommended for patients older than 40-50 years of age. One reason for this may be due to the reduced capacity of aged chondrocytes in generating new cartilage. Currently, the underlying mechanism contributing to aging-associated functional decline in chondrocytes is not clear and no proven approach exists to reverse chondrocyte aging. Given that chondrocytes in healthy hyaline cartilage typically display a spherical shape, believed to be essential for chondrocyte phenotype stability, we hypothesize that maintaining aged chondrocytes in a suspension culture that forces the cells to adopt a round morphology may help to "rejuvenate" them to a younger state, thus, leading to enhanced cartilage regeneration. Chondrocytes isolated from aged donors displayed reduced proliferation potential and impaired capacity in generating hyaline cartilage, compared to cells isolated from young donors, indicated by increased hypertrophy and cellular senescence. To test our hypothesis, the "old" chondrocytes were seeded as a suspension onto an agarose-based substratum, where they maintained a round morphology. After the 3-day suspension culture, aged chondrocytes displayed enhanced replicative capacity, compared to those grown adherent to tissue culture plastic. Moreover, chondrocytes subjected to suspension culture formed new cartilage in vitro with higher quality and quantity, with enhanced cartilage matrix deposition, concomitant with lower levels of hypertrophy and cellular senescence markers. Mechanistic analysis suggested the involvement of the RhoA and ERK1/2 signaling pathways in the "rejuvenation" process. In summary, our study presents a robust and straightforward method to enhance the function of aged human chondrocytes, which can be conveniently used to generate a large number of high-quality chondrocytes for ACI application in the elderly.

Enhancing the potential of aged human articular chondrocytes for high-quality cartilage regeneration.

Human bone marrow-derived mesenchymal stem cells (hBMSCs) can differentiate into adipocytes upon stimulation and are considered an appropriate cell source for adipose tissue engineering. In addition to biochemical cues, the stiffness of a substrate that cells attach to has also been shown to affect hBMSC differentiation potential. Of note, most current studies are conducted on monolayer cultures which do not directly inform adipose tissue engineering, where 3-dimensional (3D) scaffolds are often used to create proper tissue architecture. In this study, we aim to examine the adipogenic differentiation of hBMSCs within soft or stiff scaffolds and investigate the molecular mechanism mediating the response of hBMSCs to substrate stiffness in 3D culture, specifically the involvement of the integral membrane protein, caveolin-1 (CAV1), known to regulate signaling in MSCs via compartmentalizing and concentrating signaling molecules.

Caveolin-1 mediates soft scaffold-enhanced adipogenesis of human mesenchymal stem cells.

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.

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.

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

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.

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-β (TGFβ)-induced hiMPC chondrogenesis. In contrast, TGFβ 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.

Article	Year
Chondrogenesis of human bone marrow mesenchymal stem cells in 3-dimensional, photocrosslinked hydrogel constructs: Effect of cell seeding density and material stiffness. Acta biomaterialia\| PubMed ID: 28611002	2017
Influence of cholesterol/caveolin-1/caveolae homeostasis on membrane properties and substrate adhesion characteristics of adult human mesenchymal stem cells. Stem cell research & therapy\| PubMed ID: 29615119	2018
Enhancing chondrogenesis and mechanical strength retention in physiologically relevant hydrogels with incorporation of hyaluronic acid and direct loading of TGF-β. Acta biomaterialia\| PubMed ID: 30458242	2019
Optimization of photocrosslinked gelatin/hyaluronic acid hybrid scaffold for the repair of cartilage defect. Journal of tissue engineering and regenerative medicine\| PubMed ID: 31066519	2019
Tissue Engineering for Musculoskeletal Regeneration and Disease Modeling. Handbook of experimental pharmacology\| PubMed ID: 33471201	2021
Enhancing the potential of aged human articular chondrocytes for high-quality cartilage regeneration. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 33617078	2021
Caveolin-1 mediates soft scaffold-enhanced adipogenesis of human mesenchymal stem cells. Stem cell research & therapy\| PubMed ID: 34127047	2021
Graphene oxide-functionalized nanocomposites promote osteogenesis of human mesenchymal stem cells via enhancement of BMP-SMAD1/5 signaling pathway. Biomaterials\| PubMed ID: 34464823	2021
Engineering pre-vascularized bone-like tissue from human mesenchymal stem cells through simulating endochondral ossification. Biomaterials\| PubMed ID: 35259584	2022
Human Mesenchymal Stem Cell-Derived Miniature Joint System for Disease Modeling and Drug Testing. Advanced science (Weinheim, Baden-Wurttemberg, Germany)\| PubMed ID: 35436042	2022
Differences in the intrinsic chondrogenic potential of human mesenchymal stromal cells and iPSC-derived multipotent cells. Clinical and translational medicine\| PubMed ID: 36536500	2022

Madalyn R. Fritch

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