Degenerative disease and damage to articular cartilage represents a growing concern in the aging population. New strategies for engineering cartilage have employed mesenchymal stem cells (MSCs) as a cell source. However, recent work has suggested that chondrocytes (CHs) produce extracellular matrix (ECM) with superior mechanical properties than MSCs do. Because MSC-biomaterial interactions are important for both initial cell viability and subsequent chondrogenesis, we compared the growth of MSC- and CH-based constructs in three distinct hydrogels-agarose (AG), photocrosslinkable hyaluronic acid (HA), and self-assembling peptide (Puramatrix, Pu). Bovine CHs and MSCs were isolated from the same group of donors and seeded in AG, Pu, and HA at 20 million cells/mL. Constructs were cultured for 8 weeks with biweekly analysis of construct physical properties, viability, ECM content, and mechanical properties. Correlation analysis was performed to determine quantitative relationships between formed matrix and mechanical properties for each cell type in each hydrogel. Results demonstrate that functional chondrogenesis, as evidenced by increasing mechanical properties, occurred in each MSC-seeded hydrogel. Interestingly, while CH-seeded constructs were strongly dependent on the 3D environment in which they were encapsulated, similar growth profiles were observed in each MSC-laden hydrogel. In every case, MSC-laden constructs possessed mechanical properties significantly lower than those of CH-seeded AG constructs. This finding suggests that methods for inducing MSC chondrogenesis have yet to be optimized to produce cells whose functional matrix-forming potential matches that of native CHs.

Objective Engineering cartilage requires that a clinically relevant cell type be situated within a 3D environment that supports cell viability, the production and retention of cartilage-specific ECM, and eventually, the establishment of mechanical properties that approach that of the native tissue. In this study, we investigated the ability of bone marrow derived mesenchymal stem cells (MSCs) to undergo chondrogenesis in crosslinked methacrylated hyaluronic acid (HA) hydrogels (MeHA) of different macromer concentrations (1, 2, and 5%). Design Over a six week culture period under pro-chondrogenic conditions, we evaluated cartilage-specific gene expression, ECM deposition within constructs and released to the culture media, and mechanical properties in both compression and tension. Further, we examined early matrix assembly and long term histological features of the forming tissues, as well as the ability of macromolecules to diffuse within hydrogels as a function of MeHA macromer concentration. Results Findings from this study show that variations in macromer density influence MSC chondrogenesis in distinct ways. Increasing HA macromer density promoted chondrogenesis and matrix formation and retention, but yielded functionally inferior constructs due to limited matrix distribution throughout the construct expanse. In 1% MeHA constructs, the equilibrium compressive modulus reached 0.12 MPa and s-GAG content reached nearly 3% of the wet weight, values that matched or exceeded those of control agarose constructs and that are 25% and 50% of native tissue levels, respectively. Conclusions These data provide new insight into how early matrix deposition regulates long term construct development, and defines new parameters for optimizing the formation of functional MSC-based engineered articular cartilage using HA hydrogels.

Study Design Develop construction algorithm in which electrospun nanofibrous scaffolds are coupled with a biocompatible hydrogel to engineer a mesenchymal stem cell (MSC)-based disc replacement. Objective Engineer a disc-like angle-ply structure (DAPS) that replicates the multi-scale architecture of the intervertebral disc. Summary of Background Data Successful engineering of a replacement for the intervertebral disc requires replication of its mechanical function and anatomic form. Despite many attempts to engineer a replacement for ailing and degenerated discs, no prior study has replicated the multi-scale hierarchical architecture of the native disc, and very few have assessed the mechanical function of formed neo-tissues. Methods A new algorithm for the construction of a disc analogue was developed, using agarose to form a central nucleus pulposus and electrospun nanofibrous scaffolds to form the annulus fibrosus region (AF, based on oriented nanofibrous scaffolds). Bovine MSCs were seeded into both regions and biochemical, histological, and mechanical maturation were observed with in vitro culture. Results We show that mechanical testing in compression and torsion, two loading modalities commonly used to assess disc mechanics, reveal equilibrium and time-dependent behaviors that are qualitatively similar to native tissue, although lesser in magnitude. Further, we demonstrate that cells seeded into the two regions adopt distinct morphologies that mirror those seen in native tissue, and that, in the AF region, this ordered community of cells deposited matrix that is organized in an angle-ply configuration. Finally, constructs demonstrated functional development with long-term in vitro culture. Conclusion These findings provide a new approach for disc tissue engineering that replicates multi-scale form and function of the intervertebral disc, providing a foundation from which to build a multi-scale, biologic, anatomically and hierarchically relevant composite disc analogue for eventual disc replacement.

These findings demonstrate that the tensile properties, an important and often overlooked metric of cartilage development, increase with time in culture in engineered hydrogel-based cartilage constructs. Under the free-swelling conditions employed in the present study, tensile moduli and toughness did not match that of the native tissue, though significant time-dependent increases were observed with the inclusion of TGF-beta3. Of note, MSC-seeded constructs achieved tensile properties that were comparable to chondrocyte-seeded constructs, confirming the utility of this alternative cell source in cartilage tissue engineering. Further work, including both modulation of the chemical and mechanical culture environment, is required to optimize the deposition of collagen and its remodeling to achieve tensile properties in engineered constructs matching the native tissue.

OverviewThe intent of this manuscript is to review recent advances in the use of mesenchymal stem cells (MSCs) for the engineering of functional cartilage replacement tissues. Mesenchymal stem cells are a multipotent cell type capable of differentiating toward a number of lineages of the musculoskeletal system, including bone, cartilage and fat (Baksh et al. 2004). This multipotential capacity was first described over three decades ago (Friedenstein et al. 1974), and since then, the potential use of MSCs for regenerative therapies has generated tremendous excitement and focus. The attractiveness of MSCs for tissue repair is self-evident: in addition to their ability to take on multiple phenotypes, MSCs are readily expandable in culture and retain their multipotential characteristics with expansion; further, MSCs and other similar progenitor cells can be isolated from a wide variety of tissue sources, thereby avoiding further damage to diseased/injured tissues.In this review, we outline seminal and recent work highlighting the potential of these unique cells in producing cartilage-like tissue equivalents. Specific focus is placed on the mechanical properties of engineered MSC-based cartilage and how these properties relate to that of engineered cartilage based on primary chondrocytes and to native tissue properties. We discuss current limitations and/or concerns that must be addressed for the clinical realization of MSCbased cartilage therapeutics, and provide some insight into potential underpinnings for the observed deviations from chondrocyte-based engineered constructs. We posit that these differences reveal specific deficits in terms of our description of chondrogenesis, and suggest that new benchmarks must be developed towards this end. Further, we describe the growing body of literature on the mechanobiology of MSC-based cartilage, highlighting positive findings with regards to the furtherance of the chondrogenic phenotype. We likewise discuss the failure of early successes to translate directly into engineered constructs with improved

Fibrocartilaginous tissues such as the meniscus serve critical load-bearing roles, relying on arrays of collagen fibers to resist tensile loads experienced with normal activity. As these structures are frequently injured and possess limited healing capacity, there exists great demand for tissue-engineered replacements. Toward recreating the structural features of these anisotropic tissues in vitro, we employ scaffolds composed of co-aligned nanofibers that direct mesenchymal stem cell (MSC) orientation and the formation of organized extracellular matrix (ECM). Concomitant with ECM synthesis, the mechanical properties of constructs increase with freeswelling culture, but ultimately failed to achieve equivalence with meniscal fibrocartilage. As mechanical forces are essential to the development and maintenance of musculoskeletal tissues, this work examined the effect of cyclic tensile loading on MSC-laden nanofibrous constructs. We hypothesized that loading would modulate the transcriptional behavior of MSCs, spur the deposition of ECM, and lead to enhancements in construct mechanical properties compared to free-swelling controls. Fiber-aligned scaffolds were seeded with MSCs and dynamically loaded daily in tension or maintained as nonloaded controls for 4 weeks. With mechanical stimulation, fibrous gene expression increased, collagen deposition increased, and the tensile modulus increased by 16% relative to controls. These results show that dynamic tensile loading enhances the maturation of MSC-laden aligned nanofibrous constructs, suggesting that recapitulation of the structural and mechanical environment of load-bearing tissues results in increases in functional properties that can be exploited for tissue engineering applications.

The long tendons of the limb extend from muscles that reside in the zeugopod (arm/leg) to their skeletal insertions in the autopod ( paw). How these connections are established along the length of the limb remains unknown. Here, we show that mouse limb tendons are formed in modular units that combine to form a functional contiguous structure; in muscle-less limbs, tendons develop in the autopod but do not extend into the zeugopod, and in the absence of limb cartilage the zeugopod segments of tendons develop despite the absence of tendons in the autopod. Analyses of cell lineage and proliferation indicate that distinct mechanisms govern the growth of autopod and zeugopod tendon segments. To elucidate the integration of these autopod and zeugopod developmental programs, we re-examined early tendon development. At E12.5, muscles extend across the full length of a very short zeugopod and connect through short anlagen of tendon progenitors at the presumptive wrist to their respective autopod tendon segment, thereby initiating musculoskeletal integration. Zeugopod tendon segments are subsequently generated by proximal elongation of the wrist tendon anlagen, in parallel with skeletal growth, underscoring the dependence of zeugopod tendon development on muscles for tendon anchoring. Moreover, a subset of extensor tendons initially form as fused structures due to initial attachment of their respective wrist tendon anlage to multiple muscles. Subsequent individuation of these tendons depends on muscle activity. These results establish an integrated model for limb tendon development that provides a framework for future analyses of tendon and musculoskeletal phenotypes.

Mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering and regenerative medicine. However, the use of these cells has been limited by their reduced ability to form functional tissue compared to chondrocytes when placed in three-dimensional culture systems. To optimize MSC functional chondrogenesis, we examined the effects of increasing seeding density and transient application of transforming growth factor beta 3 (TGF-b3), two factors previously shown to improve growth of chondrocyte-based constructs. Chondrocytes seeded in agarose at 20 million cells=mL and MSCs seeded at 20 or 60 million cells=mL agarose were cultured for 7 weeks under continuous or transient application of TGF-b3. In the transient group, cell-laden constructs were exposed to TGF-b3 for the initial 3 weeks, followed by 4 weeks of culture in medium without TGFb3. Compressive properties, biochemical content, and gene expression were assessed at 3, 5, and 7 weeks. Matrix distribution and collagen type was determined using histology and immunohistochemistry, and chondrogenic and osteogenic markers were assessed using real-time polymerase chain reaction. When maintained continuously with TGF-b3, chondrocyte-seeded constructs achieved a higher equilibrium compressive modulus than MSCs similarly maintained. Although properties of both groups increased with respect to starting values, there was no difference in bulk mechanical or biochemical properties with higher seeding density when MSCs were cultured with constant TGF-b3. Findings also showed that while transient application of TGF-b3 elicited robust growth from chondrocyte-laden gels, MSCs seeded at the same density failed to respond, although constructs maintained their previously accrued properties and continued to express cartilaginous genes after TGF-b3 removal. Conversely, MSCs seeded at 60 million cells=mL exhibited a strong anabolic response with transient TGF-b3 exposure, achieving an equilibrium modulus of approximately 200 kPa. Although this represents the highest modulus we have been able to achieve with MSC-seeded constructs using our culture system, further work remains to optimize MSC chondrogenesis for cartilage tissue engineering, particularly in terms of collagen content and dynamic mechanical properties.