Isaac E. Erickson

Differential Maturation and Structure–Function Relationships in Mesenchymal Stem Cell- and Chondrocyte-Seeded Hydrogels

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.

Macromer density influences mesenchymal stem cell chondrogenesis and maturation in photocrosslinked hyaluronic acid hydrogels

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 extracellular matrix (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 hydrogels (MeHA) of different macromer concentrations (1, 2, and 5%). DESIGN Over a 6 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 0.12MPa 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.

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.

Transient exposure to TGF-β3 improves the functional chondrogenesis of MSC-laden hyaluronic acid hydrogels

Tissue engineering with adult stem cells is a promising approach for the restoration of focal defects in articular cartilage. For this, progenitor cells would ideally be delivered to (and maintained within) the defect site via a biocompatible material and in combination with soluble factors to promote initial cell differentiation and subsequent tissue maturation in vivo. While growth factor delivery methods are continually being optimized, most offer only a short (days to weeks) delivery profile at high doses. To address this issue, we investigated mesenchymal stem cell (MSC) differentiation and maturation in photocrosslinkable hyaluronic acid (HA) hydrogels with transient exposure to the pro-chondrogenic molecule transforming growth factor-beta3 (TGF-β3), at varying doses (10, 50 and 100 ng/mL) and durations (3, 7, 21 and 63 days). Mechanical, biochemical, and histological outcomes were evaluated through 9 weeks of culture. Results showed that a brief exposure (7 days) to a very high level (100 ng/mL) of TGF-β3 was sufficient to both induce and maintain cartilage formation in these 3D constructs. Indeed, this short delivery resulted in constructs with mechanical and biochemical properties that exceeded that of continuous exposure to a lower level (10 ng/mL) of TGF-β3 over the entire 9-week time course. Of important note, the total TGF delivery in these two scenarios was roughly equivalent (200 vs. 180 ng), but the timing of delivery differed markedly. These data support the idea that acute exposure to a high dose of TGF will induce functional and long-term differentiation of stem cell populations, and further our efforts to improve cartilage repair in vivo.

Improved cartilage repair via in vitro pre-maturation of MSC-seeded hyaluronic acid hydrogels

Functional repair of focal cartilage defects requires filling the space with neotissue that has compressive properties comparable to native tissue and integration with adjacent host cartilage. While poor integration is a common complication with current clinical treatments, reports of tissue engineering advances in the development of functional compressive properties rarely include analyses of their potential for integration. Our objective was thus to assess both the maturation and integration of mesenchymal stem cell (MSC)-laden hyaluronic acid (HA) hydrogels in an in vitro cartilage defect model. Furthermore, we considered the effects of an initial period of pre-maturation as well as various material formulations to maximize both construct compressive properties and integration strength. MSCs were encapsulated in 1%, 3% and 5% methacrylated HA (MeHA) or 2% agarose (Ag) and gelled directly (in situ) within an in vitro cartilage defect or were formed and then pre-cultured for 4 weeks before implantation. Results showed that the integration strength of pre-cultured repair constructs was equal to (1% MeHA) or greater than (2% Ag) the integration of in situ repaired cartilage. Moreover, MSC chondrogenesis and maturation was restricted by the in situ repair environment with constructs maturing to a much lesser extent than pre-matured constructs. These results indicate that construct pre-maturation may be an essential element of functional cartilage repair.

Optimization of macromer density in human MSC-laden hyaluronic acid (HA) hydrogels

Mesenchymal stem cells are attractive cell type and can undergo chondrogenesis in various 3D platforms. Hyaluronic acid (HA) hydrogel, a natural constituent of the cartilage extracellular matrix, provides a biologically relevant interface for encapsulated cells. While MSC-laden HA constructs can produce native mechanical properties using cells from animal sources, clinical repair will depend on successful translation of these findings to human MSCs (hMSCs). To optimize chondrogenesis, we assessed the ability of hMSCs to undergo chondrogenesis in varying macromer concentration HA gels. Variation in this parameter influenced construct mechanical and biochemical properties. In 1% methacrylated HA (MeHA), equilibrium modulus and GAG content were higher (86kPa (EY) and 2.16%) than in 2% MeHA constructs (50 kPa, 1.62% GAG). However, greater contractility occurred in 1% MeHA (-36.25%/-24.25%; Thickness/diameter) compared to 2% MeHA (-20.57%/1.02%) constructs. This study provides new insight into optimized macromer densities for hMSC-based cartilage tissue engineering using HA hydrogels.

Dynamic Compression Promotes Cartilage-Like Functional Properties in MSC-Seeded Hyaluronic Acid Hydrogels

The specialized function of articular cartilage in distributing stresses during normal joint movement must be recapitulated in a successful engineered cartilage repair. Chondrocytes can generate in vitro cartilage constructs with mechanical properties at or near native levels [1–3] when cultured in specialized media formulations. While these advances in chondrocyte-based tissue engineering are highly instructive, the difficulty of obtaining sufficient numbers of healthy autologous chondrocytes represents a considerable challenge. To circumvent this limitation, many have evaluated mesenchymal stem cells (MSCs), an autologous cell type that can be expanded in vitro and with a demonstrated capacity for chondrogenic differentiation. Despite their potential, MSC-based engineered cartilage has yet to achieve functional properties comparable to those produced by chondrocytes in 3D culture [4–6].Copyright

Transient Exposure to TGF-β3 Improves the Functional Properties of MSC-Seeded Photocrosslinked Hyaluronic Acid Hydrogels

Articular cartilage is the primary compressive load bearing soft tissue in diarthrodial joints. While the tissue can function remarkably well in a demanding environment over a lifetime of use, focal defects and other trauma can initiate progressive degeneration. Cartilage tissue engineering approaches have been developed with the goal of forming biologic replacement materials with functional mechanical properties [1]. While chondrocytes are a popular cell source for such approaches, and can produce constructs with near-native functional properties [2], mesenchymal stem cells (MSCs) derived from bone marrow have emerged as an attractive alternative cell type. MSCs are multi-potent and easy to expand, and so are available in a nearly unlimited supply, and in an autologous fashion. While MSCs can undergo functional chondrogenesis in a variety of 3D contexts [3], we are particularly interested in the translational capacity of hyaluronic acid (HA). Hydrogels formed from this natural constituent of the cartilage extracellular matrix provide a biologically relevant interface for encapsulated cells and gel properties are readily tunable [4, 5]. Indeed, using a methacrylated (and so photo-crosslinkable) HA macromer, we have optimized gel formation and functional matrix production by MSCs with variations in both macromer (1%, [5]) and MSC (∼60 million cells/mL, [6]) concentration, consistently producing cartilage-like constructs with near native compressive properties. Additionally, we have reported that transient exposure of TGF-β3 (for three weeks) to MSCs in agarose constructs at a high-density induced a stable chondrogenic phenotype, with functional properties at six weeks greater than continual exposure to this pro-chondrogenic factor [7]. Transient exposure presents an interesting paradigm with clinical relevance, in vivo defect filling will require robust maturation of the engineered tissue driven by TGF-β3 delivered from the material itself in a controlled and sustained fashion. The purpose of this study was to determine the minimal TGF-β3 dosage and duration of exposure required to promote the most robust chondrogenesis and functional maturation of MSCs in this HA hydrogel system.Copyright

Effects of Aging and TGF-Beta 3 on Chondrocyte and Mesenchymal Stem Cell Matrix Formation

Articular cartilage pathology is common in the aged population. Numerous studies have shown that aged chondrocytes (CHs) are inferior to juvenile CHs in their ability to proliferate and produce cartilage-specific extracellular matrix proteins, potentially limiting their use in tissue engineering applications for cartilage restoration [1,2]. Mesenchymal stem cells (MSCs) are an alternative cell type that can be expanded in vitro while maintaining their ability to differentiate into cell types comparable to articular chondrocytes. However, organismal aging also influences human MSC proliferation [3,4] and multi-potential differentiation [5], though for chondrogenesis these findings are mixed, with some suggesting that aged progenitor cells retain their chondrogenic capacity [6]. The objective of this study was to assess age related differences in donor-matched CH and MSC potential for chondrogenic repair. In addition, the effects of the chondrogenic growth factor TGF-β3 on CHs and MSCs were evaluated.Copyright

Differential Chondrogenic Potential of Human and Bovine Mesenchymal Stem Cells in Agarose and Photocrosslinked Hyaluronic Acid Hydrogels

Articular cartilage has a limited regenerative capacity, and there exist no methodologies to restore structure and function after damage or degeneration. This has focused intense work on cell-based therapies for cartilage repair, with considerable literature demonstrating that chondrocytes in vitro and in vivo can generate cartilage-like tissue replacements. However, use of primary cells is limited by the amount and quality of autologous donor cells and tissue. Multipotential mesenchymal stem cells (MSCs) derived from bone marrow offer an alternative cell source for cartilage tissue engineering. MSCs are easily accessible and expandable in culture, and differentiate towards a chondrocyte-like phenotype with exposure to TGF-β [1]. For example, we have shown that bovine MSCs undergo chondrogenic differentiation and mechanical maturation in agarose, self-assembling peptide, and photocrosslinkable hyaluronic acid (HA) hydrogels [2]. HA hydrogels are particularly advantageous as they are biologically relevant and easily modified to generate a range of hydrogel properties [3]. Indeed, bovine MSCs show a strong dependence of functional outcomes on the macromer density of the HA gel [4]. To further the clinical application of this material, the purpose of this study was to investigate functional chondrogenesis of human MSCs in HA compared to agarose hydrogels. To carry out this study, juvenile bovine and human MSCs were encapsulated and cultured in vitro in HA and agarose hydrogels, and cell viability, biochemical, biomechanical, and histological properties were evaluated over 4 weeks of culture.© 2010 ASME