Nanoparticle-Mediated TGF-ß Release from Microribbon-Based Hydrogels Accelerates Stem Cell-Based Cartilage Formation In Vivo.

Conventional nanoporous hydrogels often lead to slow cartilage deposition by MSCs in 3D due to physical constraints and requirement for degradation. Our group has recently reported macroporous gelatin microribbon (µRB) hydrogels, which substantially accelerate MSC-based cartilage formation in vitro compared to conventional gelatin hydrogels. To facilitate translating the use of µRB-based scaffolds for supporting stem cell-based cartilage regeneration in vivo, there remains a need to develop a customize-designed drug delivery system that can be incorporated into µRB-based scaffolds. Towards this goal, here we report polydopamine-coated mesoporous silica nanoparticles (MSNs) that can be stably incorporated within the macroporous µRB scaffolds, and allow tunable release of transforming growth factor (TGF)-ß3. We hypothesize that increasing concentration of polydopamine coating on MSNs will slow down TGF- ß3 release, and TGF-ß3 release from polydopamine-coated MSNs can enhance MSC-based cartilage formation in vitro and in vivo. We demonstrate that TGF-ß3 released from MSNs enhance MSC-based cartilage regeneration in vitro to levels comparable to freshly added TGF-ß3 in the medium, as shown by biochemical assays, mechanical testing, and histology. Furthermore, when implanted in vivo in a mouse subcutaneous model, only the group containing MSN-mediated TGF-ß3 release supported continuous cartilage formation, whereas control group without MSN showed loss of cartilage matrix and undesirable endochondral ossification. The modular design of MSN-mediated drug delivery can be customized for delivering multiple drugs with individually optimized release kinetics, and may be applicable to enhance regeneration of other tissue types.

Mixed Composition Microribbon Hydrogels Induce Rapid and Synergistic Cartilage Regeneration by Mesenchymal Stem Cells in 3D via Paracrine Signaling Exchange.

Hydrogels are widely used matrices for mesenchymal stem cell (MSC)-based cartilage regeneration but often result in slow cartilage deposition with inferior mechanical strength. We recently reported a gelatin-based microribbon (µRB) scaffold, which contains macroporosity and substantially enhances the speed of cartilage formation by MSCs in 3D. However, our previous method cannot be used to fabricate different polymers into µRBs, and the effects of varying µRB compositions on MSC cartilage regeneration in 3D remain unknown. Here, we report a method that allows fabricating different polymers [gelatin, chondroitin sulfate, hyaluronic acid, and polyethylene glycol (PEG)] into µRB structures, which can be mixed in any ratio and cross-linked into 3D scaffolds in a modular manner. Mixing glycosaminoglycan µRBs with gelatin or PEG µRBs induced great synergy, resulting in fast cartilage deposition. After only 3 weeks of culture, leading mixed µRB composition reached high compressive strength on par with native cartilage. Such synergy can be recapitulated via exchange of soluble factors secreted by MSCs seeded in different µRB compositions in a dose-dependent manner. Tuning the ratio of mixed µRB compositions allowed further optimization of the quantity and speed of cartilage regeneration by MSCs. Together, our results validate mixed µRB compositions as a novel biomaterial tool for inducing synergy and accelerating MSC-based cartilage regeneration with biomimetic mechanical properties through paracrine signal exchange.

The Effects of ROCK Inhibition on Mesenchymal Stem Cell Chondrogenesis Are Culture Model Dependent.

Rho-associated protein kinase (ROCK) signaling correlates with cell shape, with decreased cell spreading accompanied by decreased ROCK activity. However, how cell shape and ROCK activity impact the chondrogenesis of mesenchymal stem cells (MSCs) remains inconclusive. Here we examine the effects of ROCK inhibition on human MSC chondrogenesis in four different culture models, including three-dimensional (3D) microribbon (µRB) scaffolds, two-dimensional hydrogel (2D-HG) substrates, 3D hydrogels (3D-HGs), and pellet. For each culture model involving biomaterials, four polymers were compared, including gelatin, chondroitin sulfate, hyaluronic acid, and polyethylene glycol. ROCK inhibition decreased MSC chondrogenesis in µRB model, enhanced chondrogenesis in pellet, and had minimal effect in 2D-HG or 3D-HG models. Furthermore, we demonstrate that MSC chondrogenesis cannot be predicted using ROCK signaling alone. While varying biomaterial compositions can impact the amount or phenotype of resulting cartilage, varying biomaterials did not change the chondrogenic response to ROCK inhibition within each culture model. Regardless of culture model or ROCK expression, increased cartilage formation was always accompanied by enhanced N-cadherin expression and production, suggesting that N-cadherin is a robust marker to select culture conditions that promote chondrogenesis. Together, the results from this study may be used to enhance MSC-based cartilage regeneration in different culture models. Impact Statement Here we assessed the effects of Rho-associated protein kinase (ROCK) inhibition on mesenchymal stem cell (MSC) chondrogenesis in different culture models, including three-dimensional (3D) microribbon scaffolds, two-dimensional hydrogel substrates, 3D hydrogels, and pellet culture. Our results demonstrate that effects of ROCK inhibition on MSC chondrogenesis differ substantially depending on culture models. Furthermore, MSC chondrogenesis cannot be predicted using ROCK signaling alone. The results from this study fill in a gap of knowledge in the correlation between ROCK signaling and MSC chondrogenesis, which may be used to enhance MSC-based cartilage regeneration in different culture models.

Spatially patterned microribbon-based hydrogels induce zonally-organized cartilage regeneration by stem cells in 3D.

Regenerating cartilage with biomimetic zonal organization, which is critical for tissue function, remains a great challenge. The objective of this study was to evaluate the potential of spatially-patterned, multi-compositional, macroporous, extracellular matrix-based microribbon (µRB) µRB scaffolds to regenerate cartilage with biochemical, mechanical, and morphological zonal organization by mesenchymal stem cells (MSCs) compared to conventional multi-layer nanoporous hydrogels. MSCs were seeded in either trilayer microribbon (µRB) or hydrogel (HG) scaffolds that were composed of layered biomaterial compositions that had been chosen for their ability to differentiate MSCs into chondrocytes with zonal properties. To mimic the aligned collagen morphology in the superficial layer of native cartilage, an additional experimental group added MSC-laden aligned µRBs to the surface of the superficial layer of a µRB trilayer. Tuning µRB alignment and compositions in different zones led to zonal-specific responses of MSCs to create neocartilage with zonal biochemical, morphological, and mechanical properties, while trilayer HGs led to minimal cartilaginous deposition overall. Trilayer µRBs created neocartilage exhibiting significant increases in compressive modulus (up to 456 kPa) and > 4-fold increase in sGAG production from superficial to deep zones. Aligned gelatin µRBs in the superficial zone further enhanced biomimetic mimicry of the produced neocartilage by leading to robust collagen deposition and superficial zone protein production. STATEMENT OF SIGNIFICANCE: Regenerating cartilage with zonal organization using mesenchymal stem cells (MSCs) remains a great challenge. We developed a spatially-patterned, gradient, macroporous, trilayer microribbon (µRB) scaffold that we used to engineer MSC-based neocartilage with zonal trends that match native cartilage in many aspects, including collagen, sGAG, superficial zone protein, and compressive moduli. This is in direct contrast to conventional trilayer nanoporous hydrogels which led to minimal cartilage deposition and weak mechanical properties. It took only 21 days for MSC-seeded trilayer µRB scaffolds to reach cartilage-mimicking compressive moduli without requiring high cell seeding density, which has never been reported before. While this paper focuses on cartilage zonal organization, gradient µRB scaffolds can be used to repair other tissue interfaces such as osteochondral defects.

Tension stimulation drives tissue formation in scaffold-free systems.

Scaffold-free systems have emerged as viable approaches for engineering load-bearing tissues. However, the tensile properties of engineered tissues have remained far below the values for native tissue. Here, by using self-assembled articular cartilage as a model to examine the effects of intermittent and continuous tension stimulation on tissue formation, we show that the application of tension alone, or in combination with matrix remodelling and synthesis agents, leads to neocartilage with tensile properties approaching those of native tissue. Implantation of tension-stimulated tissues results in neotissues that are morphologically reminiscent of native cartilage. We also show that tension stimulation can be translated to a human cell source to generate anisotropic human neocartilage with enhanced tensile properties. Tension stimulation, which results in nearly sixfold improvements in tensile properties over unstimulated controls, may allow the engineering of mechanically robust biological replacements of native tissue.

Thyroid hormones enhance the biomechanical functionality of scaffold-free neocartilage.

INTRODUCTION: The aim of this study was to investigate the effects of thyroid hormones tri-iodothyronine (T3), thyroxine (T4), and parathyroid hormone (PTH) from the parathyroid glands, known to regulate the developing limb and growth plate, on articular cartilage tissue regeneration using a scaffold-free in vitro model. METHODS: In Phase 1, T3, T4, or PTH was applied during weeks 1 or 3 of a 4-week neocartilage culture. Phase 2 employed T3 during week 1, followed by PTH during week 2, 3, or weeks 2 to 4, to further enhance tissue properties. Resultant neotissues were evaluated biochemically, mechanically, and histologically. RESULTS: In Phase 1, T3 and T4 treatment during week 1 resulted in significantly enhanced collagen production; 1.4- and 1.3-times untreated neocartilage. Compressive and tensile properties were also significantly increased, as compared to untreated and PTH groups. PTH treatment did not result in notable tissue changes. As T3 induces hypertrophy, in Phase 2, PTH (known to suppress hypertrophy) was applied sequentially after T3. Excitingly, sequential treatment with T3 and PTH reduced expression of hypertrophic marker collagen X, while yielding neocartilage with significantly enhanced functional properties. Specifically, in comparison to no hormone application, these hormones increased compressive and tensile moduli 4.0-fold and 3.1-fold, respectively. CONCLUSIONS: This study demonstrated that T3, together with PTH, when applied in a scaffold-free model of cartilage formation, significantly enhanced functional properties. The novel use of these thyroid hormones generates mechanically robust neocartilage via the use of a scaffold-free tissue engineering model.

Promoting increased mechanical properties of tissue engineered neocartilage via the application of hyperosmolarity and 4α-phorbol 12,13-didecanoate (4αPDD).

Osteoarthritis, a degenerative disease of the load-bearing joints, greatly reduces quality of life for millions of Americans and places a tremendous cost on the American healthcare system. Due to limitations of current treatments, tissue engineering of articular cartilage may provide a promising therapeutic option to treat cartilage defects. However, cartilage tissue engineering has yet to recapitulate the functional properties of native tissue. During normal joint loading, cartilage tissue experiences variations in osmolarity and subsequent changes in ionic concentrations. Motivated by these known variations in the cellular microenvironment, this study sought to improve the mechanical properties of neocartilage constructs via the application of hyperosmolarity and transient receptor potential vanilloid 4 (TRPV4) channel activator 4α-phorbol 12,13-didecanoate (4αPDD). It was shown that 4αPDD elicited significant increases in compressive properties. Importantly, when combined, 4αPDD positively interacted with hyperosmolarity to modulate its effects on tensile stiffness and collagen content. Thus, this study supports 4αPDD-activated channel TRPV4 as a purported mechanosensor and osmosensor that can facilitate the cell and tissue level responses to improve the mechanical properties of engineered cartilage. To our knowledge, this study is the first to systematically evaluate the roles of hyperosmolarity and 4αPDD on the functional (i.e., mechanical and biochemical) properties of self-assembled neotissue. Future work may combine 4αPDD-induced channel activation with other chemical and mechanical stimuli to create robust neocartilages suitable for treatment of articular cartilage defects.