Spatially Regulated Multiphenotypic Differentiation of Stem Cells in 3D via Engineered Mechanical Gradient.

Within the osteochondral interface, cellular and extracellular matrix gradients provide a biomechanical and biochemical niche for homeostatic tissue functions. Postnatal joint loading is critical for the development of such tissue gradients, leading to the formation of functional osteochondral tissues composed of superficial, middle, and deep zones of cartilage, and underlying subchondral bone, in a depth-dependent manner. In this regard, a novel, variable core-shell electrospinning strategy was employed to generate spatially controlled strain gradients within three-dimensional scaffolds under dynamic compressive loading, enabling the local strain-magnitude dependent, multiphenotypic stem cell differentiation. Human mesenchymal stem cells (hMSCs) were cultured in electrospun scaffolds with a linear or biphasic mechanical gradient, which was computationally engineered and experimentally validated. The cell/scaffold constructs were subjected to various magnitudes of dynamic compressive strains in a scaffold depth-dependent manner at a frequency of 1 Hz for 2 h daily for up to 42 days in osteogenic media. Spatially upregulated gene expression of chondrogenic markers (ACAN, COL2A1, PRG4) and glycosaminoglycan deposition was observed in the areas of greater compressive strains. In contrast, osteogenic markers (COL1A1, SPARC, RUNX2) and calcium deposition were downregulated in response to high local compressive strains. Dynamic mechanical analysis showed the maintenance of the engineered mechanical gradients only under dynamic culture conditions, confirming the potent role of biomechanical gradients in developing and maintaining a tissue gradient. These results demonstrate that multiphenotypic differentiation of hMSCs can be controlled by regulating local mechanical microenvironments, providing a novel strategy to recapitulate the gradient structure in osteochondral tissues for successful regeneration of damaged joints in vivo and facile development of interfacial tissue models in vitro.

Magnitude-dependent and inversely-related osteogenic/chondrogenic differentiation of human mesenchymal stem cells under dynamic compressive strain.

Biomechanical forces have been shown to significantly affect tissue development, morphogenesis, pathogenesis and healing, especially in orthopaedic tissues. Such biological processes are critically related to the differentiation of human mesenchymal stem cells (hMSCs). However, the mechanistic details regarding how mechanical forces direct MSC differentiation and subsequent tissue formation are still elusive. Electrospun three-dimensional scaffolds were used to culture and subject hMSCs to various magnitudes of dynamic compressive strains at 5, 10, 15 or 20% (ε = 0.05, 0.10, 0.15, 0.20) at a frequency of 1 Hz for 2 h daily for up to 28 days in osteogenic media. Gene expression of chondrogenic markers (ACAN, COL2A1, SOX9) and glycosaminoglycan (GAG) synthesis were upregulated in response to the increased magnitudes of compressive strain, whereas osteogenic markers (COL1A1, SPARC, RUNX2) and calcium deposition had noticeable decreases by compressive loading in a magnitude-dependent manner. Dynamic mechanical analysis showed enhanced viscoelastic modulus with respect to the increased dynamic strain peaking at 15%, which coincides with the maximal GAG synthesis. Furthermore, polarization-sensitive optical coherence tomography revealed that mechanical loading enhanced the alignment of extracellular matrix to the greatest level by 15% strain as well. Overall, we show that the degree of differentiation of hMSCs towards osteogenic or chondrogenic lineage is inversely related, and it depends on the magnitude of dynamic compressive strain. These results demonstrate that multiphenotypic differentiation of hMSCs can be controlled by varying the strain regimens, providing a novel strategy to modulate differentiation specification and tissue morphogenesis. Copyright © 2016 John Wiley & Sons, Ltd.

Electrospun thermosensitive hydrogel scaffold for enhanced chondrogenesis of human mesenchymal stem cells.

Hydrogels have shown great potential for cartilage tissue engineering applications due to their capability to encapsulate cells within biomimetic, 3-dimensional (3D) microenvironments. However, the multi-step fabrication process that is necessary to produce cell/scaffold constructs with defined dimensions, limits their off-the-shelf translational usage. In this study, we have developed a hybrid scaffolding system which combines a thermosensitive hydrogel, poly(ethylene glycol)-poly(N-isopropylacrylamide) (PEG-PNIPAAm), with a biodegradable polymer, poly(ε-caprolactone) (PCL), into a composite, electrospun microfibrous structure. A judicious optimization of material composition and electrospinning process produced a structurally self-supporting hybrid scaffold. The reverse thermosensitivity of PEG-PNIPAAm allowed its dissolution/hydration upon cell seeding within a network of PCL microfibers while maintaining the overall scaffold shape at room temperature. A subsequent temperature elevation to 37 °C induced the hydrogel's phase transition to a gel state, effectively encapsulating cells in a 3D hydrogel without the use of a mold. We demonstrated that the hybrid scaffold enhanced chondrogenic differentiation of human mesenchymal stem cells (hMSCs) based on chondrocytic gene and protein expression, which resulted in superior viscoelastic properties of the cell/scaffold constructs. The hybrid scaffold enables a facile, single-step cell seeding process to inoculate cells within a 3D hydrogel with the potential for cartilage tissue engineering. STATEMENT OF SIGNIFICANCE: Hydrogels have demonstrated the excellent ability to enhance chondrogenesis of stem cells due to their hydrated fibrous nanostructure providing a cellular environment similar to native cartilage. However, the necessity for multi-step processes, including mixing of hydrogel precursor with cells and subsequent gelation in a mold to form a defined shape, limits their off-the-shelf usage. In this study, we developed a hybrid scaffold by combining a thermosensitive hydrogel with a mechanically stable polymer, which provides a facile means to inoculate cells in a 3D hydrogel with a mold-less, single step cell seeding process. We further showed that the hybrid scaffold enhanced chondrogenesis of mesenchymal stem cells, demonstrating its potential for cartilage tissue engineering.

Physico-electrochemical Characterization of Pluripotent Stem Cells during Self-Renewal or Differentiation by a Multi-modal Monitoring System.

Monitoring pluripotent stem cell behaviors (self-renewal and differentiation to specific lineages/phenotypes) is critical for a fundamental understanding of stem cell biology and their translational applications. In this study, a multi-modal stem cell monitoring system was developed to quantitatively characterize physico-electrochemical changes of the cells in real time, in relation to cellular activities during self-renewal or lineage-specific differentiation, in a non-destructive, label-free manner. The system was validated by measuring physical (mass) and electrochemical (impedance) changes in human induced pluripotent stem cells undergoing self-renewal, or subjected to mesendodermal or ectodermal differentiation, and correlating them to morphological (size, shape) and biochemical changes (gene/protein expression). An equivalent circuit model was used to further dissect the electrochemical (resistive and capacitive) contributions of distinctive cellular features. Overall, the combination of the physico-electrochemical measurements and electrical circuit modeling collectively offers a means to longitudinally quantify the states of stem cell self-renewal and differentiation.

Microstructure-dependent mechanical properties of electrospun core-shell scaffolds at multi-scale levels.

Mechanical factors among many physiochemical properties of scaffolds for stem cell-based tissue engineering significantly affect tissue morphogenesis by controlling stem cell behaviors including proliferation and phenotype-specific differentiation. Core-shell electrospinning provides a unique opportunity to control mechanical properties of scaffolds independent of surface chemistry, rendering a greater freedom to tailor design for specific applications. In this study, we synthesized electrospun core-shell scaffolds having different core composition and/or core-to-shell dimensional ratios. Two independent biocompatible polymer systems, polyetherketoneketone (PEKK) and gelatin as the core materials while maintaining the shell polymer with polycaprolactone (PCL), were utilized. The mechanics of such scaffolds was analyzed at the microscale and macroscales to determine the potential implications it may hold for cell-material and tissue-material interactions. The mechanical properties of individual core-shell fibers were controlled by core-shell composition and structure. The individual fiber modulus correlated with the increase in percent core size ranging from 0.55±0.10GPa to 1.74±0.22GPa and 0.48±0.12GPa to 1.53±0.12GPa for the PEKK-PCL and gelatin-PCL fibers, respectively. More importantly, it was demonstrated that mechanical properties of the scaffolds at the macroscale were dominantly determined by porosity under compression. The increase of scaffold porosity from 70.2%±1.0% to 93.2%±0.5% by increasing the core size in the PEKK-PCL scaffold resulted in the decrease of the compressive elastic modulus from 227.67±20.39kPa to 14.55±1.43kPa while a greater changes in the porosity of gelatin-PCL scaffold from 54.5%±4.2% to 89.6%±0.4% resulted in the compressive elastic modulus change from 484.01±30.18kPa to 17.57±1.40kPa. On the other hand, the biphasic behaviors under tensile mechanical loading result in a range from a minimum of 5.42±1.05MPa to a maximum of 12.00±1.96MPa for the PEKK-PCL scaffolds, and 10.19±4.49MPa to 22.60±2.44MPa for the gelatin-PCL scaffolds. These results suggest a feasible approach for precisely controlling the local and global mechanical characteristics, in addition to independent control over surface chemistry, to achieve a desired tissue morphogenesis using the core-shell electrospinning.