Megan J. Farrell

Mechanics and Mechanobiology of Mesenchymal Stem Cell-Based Engineered Cartilage

In this review, we outline seminal and recent work highlighting the potential of mesenchymal stem cells (MSCs) 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 MSC-based 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 molecular changes to translate directly into engineered constructs with improved mechanical properties. Finally, we highlight recent work from our group and others that may point to new strategies for enhancing the formation of engineered cartilage based on MSCs.

High Mesenchymal Stem Cell Seeding Densities in Hyaluronic Acid Hydrogels Produce Engineered Cartilage with Native Tissue Properties

Engineered cartilage based on adult mesenchymal stem cells (MSCs) is an alluring goal for the repair of articular defects. However, efforts to date have failed to generate constructs with sufficient mechanical properties to function in the demanding environment of the joint. Our findings with a novel photocrosslinked hyaluronic acid (HA) hydrogel suggest that stiff gels (high HA concentration, 5% w/v) foster chondrogenic differentiation and matrix production, but limit overall functional maturation due to the inability of the formed matrix to diffuse away from the point of production and form a contiguous network. In the current study, we hypothesized that increasing the MSC seeding density would decrease the required diffusional distance, and so expedite the development of functional properties. To test this hypothesis bovine MSCs were encapsulated at seeding densities of either 20,000,000 or 60,000,000 cells ml(-1) in 1%, 3%, and 5% (w/v) HA hydrogels. Counter to our hypothesis the higher concentration HA gels (3% and 5%) did not develop more rapidly with increased MSC seeding density. However, the biomechanical properties of the low concentration (1%) HA constructs increased markedly (nearly 3-fold with a 3-fold increase in seeding density). To ensure that optimal nutrient access was delivered, we next cultured these constructs under dynamic culture conditions (with orbital shaking) for 9 weeks. Under these conditions 1% HA seeded at 60,000,000 MSCs ml(-1) reached a compressive modulus in excess of 1 MPa (compared with 0.3-0.4 MPa for free swelling constructs). This is the highest level we have reported to date in this HA hydrogel system, and represents a significant advance towards functional stem cell-based tissue engineered cartilage.

Functional properties of bone marrow-derived MSC-based engineered cartilage are unstable with very long-term in vitro culture

The success of stem cell-based cartilage repair requires that the regenerate tissue reach a stable state. To investigate the long-term stability of tissue engineered cartilage constructs, we assessed the development of compressive mechanical properties of chondrocyte and mesenchymal stem cell (MSC)-laden three dimensional agarose constructs cultured in a well defined chondrogenic in vitro environment through 112 days. Consistent with previous reports, in the presence of TGF-β, chondrocytes outperformed MSCs through day 56, under both free swelling and dynamic culture conditions, with MSC-laden constructs reaching a plateau in mechanical properties between days 28 and 56. Extending cultures through day 112 revealed that MSCs did not simply experience a lag in chondrogenesis, but rather that construct mechanical properties never matched those of chondrocyte-laden constructs. After 56 days, MSC-laden constructs underwent a marked reversal in their growth trajectory, with significant declines in glycosaminoglycan content and mechanical properties. Quantification of viability showed marked differences in cell health between chondrocytes and MSCs throughout the culture period, with MSC-laden construct cell viability falling to very low levels at these extended time points. These results were not dependent on the material environment, as similar findings were observed in a photocrosslinkable hyaluronic acid (HA) hydrogel system that is highly supportive of MSC chondrogenesis. These data suggest that, even within a controlled in vitro environment that is conducive to chondrogenesis, there may be an innate instability in the MSC phenotype that is independent of scaffold composition, and may ultimately limit their application in functional cartilage repair.

Hypoxic regulation of functional extracellular matrix elaboration by nucleus pulposus cells in long‐term agarose culture

Degeneration of the intervertebral discs is strongly implicated as a cause of low back pain. Since current treatments for discogenic low back pain show poor long‐term efficacy, a number of new biological strategies are being pursued. For such therapies to succeed, it is critical that they be validated in conditions that mimic the unique biochemical microenvironment of the nucleus pulposus (NP), which include low oxygen tension. Therefore, the objective of this study was to investigate the effects of oxygen tension on NP cell functional extracellular matrix elaboration in 3D culture. Bovine NP cells were encapsulated in agarose constructs and cultured for 14 or 42 days in either 20% or 2% oxygen in defined media containing transforming growth factor beta‐3. At each time point, extracellular matrix composition, biomechanics, and mRNA expression of key phenotypic markers were evaluated. Results showed that while bulk mechanics and composition were largely independent of oxygen level, low oxygen promoted improved restoration of the NP phenotype, higher mRNA expression of extracellular matrix and NP specific markers, and more uniform matrix elaboration. These findings indicate that culture under physiological oxygen levels is an important consideration for successful development of cell and growth factor‐based regenerative strategies for the disc.

A microengineered model of RBC transfusion-induced pulmonary vascular injury

Red blood cell (RBC) transfusion poses significant risks to critically ill patients by increasing their susceptibility to acute respiratory distress syndrome. While the underlying mechanisms of this life-threatening syndrome remain elusive, studies suggest that RBC-induced microvascular injury in the distal lung plays a central role in the development of lung injury following blood transfusion. Here we present a novel microengineering strategy to model and investigate this key disease process. Specifically, we created a microdevice for culturing primary human lung endothelial cells under physiological flow conditions to recapitulate the morphology and hemodynamic environment of the pulmonary microvascular endothelium in vivo. Perfusion of the microengineered vessel with human RBCs resulted in abnormal cytoskeletal rearrangement and release of intracellular molecules associated with regulated necrotic cell death, replicating the characteristics of acute endothelial injury in transfused lungs in vivo. Our data also revealed the significant effect of hemodynamic shear stress on RBC-induced microvascular injury. Furthermore, we integrated the microfluidic endothelium with a computer-controlled mechanical stretching system to show that breathing-induced physiological deformation of the pulmonary microvasculature may exacerbate vascular injury during RBC transfusion. Our biomimetic microsystem provides an enabling platform to mechanistically study transfusion-associated pulmonary vascular complications in susceptible patient populations.

Enhanced nutrient transport improves the depth-dependent properties of tri-layered engineered cartilage constructs with zonal co-culture of chondrocytes and MSCs.

Biomimetic design in cartilage tissue engineering is a challenge given the complexity of the native tissue. While numerous studies have generated constructs with near-native bulk properties, recapitulating the depth-dependent features of native tissue remains a challenge. Furthermore, limitations in nutrient transport and matrix accumulation in engineered constructs hinders maturation within the central core of large constructs. To overcome these limitations, we fabricated tri-layered constructs that recapitulate the depth-dependent cellular organization and functional properties of native tissue using zonally derived chondrocytes co-cultured with MSCs. We also introduced porous hollow fibers (HFs) and HFs/cotton threads to enhance nutrient transport. Our results showed that tri-layered constructs with depth-dependent organization and properties could be fabricated. The addition of HFs or HFs/threads improved matrix accumulation in the central core region. With HF/threads, the local modulus in the deep region of tri-layered constructs nearly matched that of native tissue, though the properties in the central regions remained lower. These constructs reproduced the zonal organization and depth-dependent properties of native tissue, and demonstrate that a layer-by-layer fabrication scheme holds promise for the biomimetic repair of focal cartilage defects. STATEMENT OF SIGNIFICANCE Articular cartilage is a highly organized tissue driven by zonal heterogeneity of cells, extracellular matrix proteins and fibril orientations, resulting in depth-dependent mechanical properties. Therefore, the recapitulation of the functional properties of native cartilage in a tissue engineered construct requires such a biomimetic design of the morphological organization, and this has remained a challenge in cartilage tissue engineering. This study demonstrates that a layer-by-layer fabrication scheme, including co-cultures of zone-specific articular CHs and MSCs, can reproduce the depth-dependent characteristics and mechanical properties of native cartilage while minimizing the need for large numbers of chondrocytes. In addition, introduction of a porous hollow fiber (combined with a cotton thread) enhanced nutrient transport and depth-dependent properties of the tri-layered construct. Such a tri-layered construct may provide critical advantages for focal cartilage repair. These constructs hold promise for restoring native tissue structure and function, and may be beneficial in terms of zone-to-zone integration with adjacent host tissue and providing more appropriate strain transfer after implantation.

Dynamic Culture Improves Mechanical Functionality of MSC-Laden Tissue Engineered Constructs in a Depth-Dependent Manner

Limitations associated with the use of autologous chondrocytes (CH) for cartilage tissue engineering beget the need for alternative cell sources. Mesenchymal stem cells (MSC) are clinically attractive due to their ability to undergo chondrogenesis in three-dimensional culture [1,2]; however, when compared to CH, MSC fail to develop functional equivalence [2,3]. We have previously shown a marked depth-dependence in local equilibrium modulus of MSC-laden gels, with the superficial zones (where maximal media exchange occurs) considerably stiffer than regions removed from nutrient supply (center and bottom of construct); less dramatic depth-dependence was observed in CH-laden gels [4]. Similarly, other studies have shown depth-dependent properties in CH-laden gels with the construct edge generally stiffer than the center [5]. Given this apparent influence of nutrient supply, the objective of the current study was to assess the impact of dynamic culture (via orbital shaking) on the development of depth-dependent mechanical properties in both MSC and CH-laden hydrogels. Furthermore, we assessed cell viability and matrix content throughout the construct depth to determine the mechanism by which this depth-dependency arises. We hypothesized that improved nutrient transport would reduce construct inhomogeneity (particularly for MSC-laden constructs) and improve bulk mechanical properties.Copyright

Functional Consequences of Glucose and Oxygen Deprivation in Engineered MSC-Based Cartilage Constructs

Clinical implementation of stem cell-based cartilage repair techniques has been limited by the inability of these cells to produce cartilaginous tissue equivalent to that produced by native chondrocytes. We have recently shown that while bulk mechanical properties of mesenchymal stem cell (MSC)-laden constructs are lower than chondrocyte-laden constructs, MSCs can in fact produce tissue that matches or exceeds the biochemical and mechanical properties produced by chondrocytes in regions where there is maximal nutrient supply [1]. We also noted that in the central regions of constructs, where nutrient and oxygen availability is lowest (due to consumption through the construct depth), MSC viability was markedly lower than in the outer regions and drastically lower than the center of chondrocyte-laden constructs maintained similarly. These data suggest that MSCs can achieve a high anabolic functionality when they undergo chondrogenesis (via the provision of TGF-β3) and in doing so can produce tissue of equivalent or greater properties than chondrocytes. However, unlike chondrocytes, MSCs appear thrive only when they are provided with a sufficient nutrient supply. To further delineate the role of microenvironmental stressors [2, 3, 4] on MSC viability and functional capacity, we evaluated the impact of glucose and oxygen deprivation, in the presence and absence of TGF-β, during long term culture. Furthermore, since MSC isolation procedures result in a heterogeneous cell population [5,6], we investigated whether different clonal populations respond to these microenvironmental stressors in a distinct fashion.Copyright

Mesenchymal stem cell death in three-dimensional agarose culture for cartilage tissue engineering applications: Progression, factors, and prevention

Engineered cartilage based on adult derived mesenchymal stem cells (MSCs) would be an ideal treatment for cartilage damage and osteoarthritis. However, insufficient mechanical properties in constructs formed using this cell source limits in vivo application. Here we identify a rapid and then progressive decline in MSC viability in 3D agarose tissue engineered constructs. We show that this decline in viability is linked to metabolic activity and waste production by these cells, and that they are poorly equipped to handle this demanding environment compared to cells derived from the native tissue. Blockade of early cell death may be controlled through small molecule inhibitors of apoptosis.

Micromechanical Deformation of Chondrogenic Mesenchymal Stem Cells in 3D Hydrogels is Modulated by Time in Culture and Matrix Connectivity

Mesenchymal stem cells (MSCs) are a clinically attractive alternative to chondrocytes for the development of engineered cartilage tissue owing to their ease of isolation and chondrogenic potential [1]. However, the mechanical properties of MSC-based constructs have yet to match those of native cartilage or of chondrocyte-based constructs cultured similarly [1]. One route for improving these properties may be the application of mechanical stimulation, as normal cartilage development and homeostatic maintenance is dependent on force transduction. In a tissue engineering context, dynamic compression applied to chondrocyte-seeded hydrogels modulates both matrix production and mechanical properties [2, 3]. Similarly, when MSCs are embedded in 3D hydrogels, expression of chondrogenic markers and cartilaginous ECM synthesis are differentially regulated by dynamic compressive loading [4, 5]. Indeed, we have recently shown that long-term dynamic loading initiated after a pre-culture period of 21 days in pro-chondrogenic medium improves matrix distribution and the compressive properties of MSC-seeded constructs [5]. Interestingly, when loading was initiated after a single day of culture, mechanical properties failed to develop [6, 7], suggesting that elaboration of matrix was required prior to dynamic loading in order to positively direct construct maturation. When chondrocytes are embedded in agarose, the initial growth phase is characterized by the establishment of a dense pericellular matrix (PCM). At early times in culture, before these islands of PCM become connected into an interterritorial matrix, cells are protected from bulk deformation applied to the gel [8]. In a recent study, we showed that clonal heterogeneity in stem cell populations determines the rate at which this PCM forms, with some MSC clones rapidly establishing a dense PCM, while others fail to develop a robust PCM (and so continue to deform with gel deformation) through several weeks in culture [9]. To further this investigation, this study charted the culture time-dependent changes in ECM connectivity and MSC deformation under basal and chondrogenic conditions.Copyright