A developmentally inspired combined mechanical and biochemical signaling approach on zonal lineage commitment of mesenchymal stem cells in articular cartilage regeneration

Abstract

Articular cartilage is organized into multiple zones including superficial, middle and calcified zones with distinct cellular and extracellular components to impart lubrication, compressive strength, and rigidity for load transmission to bone, respectively. During native cartilage tissue development, changes in biochemical, mechanical, and cellular factors direct the formation of stratified structure of articular cartilage. The objective of this work was to investigate the effect of combined gradients in cell density, matrix stiffness, and zone-specific growth factors on the zonal organization of articular cartilage. Human mesenchymal stem cells (hMSCs) were encapsulated in acrylate-functionalized lactide-chain-extended polyethylene glycol (SPELA) gels simulating cell density and stiffness of the superficial, middle and calcified zones. The cell-encapsulated gels were cultivated in medium supplemented with growth factors specific to each zone and the expression of zone-specific markers was measured with incubation time. Encapsulation of 60×106 cells/mL hMSCs in a soft gel (80 kPa modulus) and cultivation with a combination of TGF-β1 (3 ng/mL) and BMP-7 (100 ng/mL) led to the expression of markers for the superficial zone. Conversely, encapsulation of 15×106 cells/mL hMSCs in a stiff gel (320 MPa modulus) and cultivation with a combination of TGF-β1 (30 ng/mL) and hydroxyapatite (3%) led to the expression of markers for the calcified zone. Further, encapsulation of 20×106 cells/mL hMSCs in a gel with 2.1 MPa modulus and cultivation with a combination of TGF-β1 (30 ng/mL) and IGF-1 (100 ng/mL) led to up-regulation of the middle zone markers. Results demonstrate that a developmental approach with gradients in cell density, matrix stiffness, and zone-specific growth factors can potentially regenerate zonal structure of the articular cartilage.

Mechanical induction of dentin-like differentiation by adult mouse bone marrow stromal cells using compressive scaffolds

“…The compression-stimulated induction of differentiation appeared to be tooth-specific as it did not increase expression of the chondrogenic markers, Sox9 or Collagen II (Fig. 3), even though mesenchymal condensation is also a trigger for cartilage formation in the embryo [25]. Furthermore, the mRNA level of Pax9 and Msx1 in BMSCs injected in these collagen VI coated GRGDS-gels was found to be significantly higher than in cells in GRGDS-gels alone (p≤0.05) (Fig.…”

Comparative effect of physicomechanical and biomolecular cues on zone-specific chondrogenic differentiation of mesenchymal stem cells

“…The hydrogel precursor solution was prepared by mixing initiator solution (5 mg initiator in 1 mL PBS) with the solution of SPELA macromonomer and Ac-GRGD cell-adhesive peptide in PBS. The hydrogel precursor solution (with or without cells and nanofibers) was crosslinked by ultraviolet (UV) polymerization with an Omni Cure Series 1500 UV illumination system for 30 seconds as we previously described [18]. SPELA10, SPELA7.5 and SPELA5 gels with lactide to PEG molar feed ratio of 10, 7.5 and 5, respectively, with mass loss of 47%, 38%, and 28% after 21 days in PBS at 37°C for the gels simulating the superficial, middle, and calcified zones of articular cartilage, respectively [28] (Table 1).…”

“…For the middle zone, randomly-oriented fiber sheets were cut into small pieces, randomly dispersed in SPELA7.5 precursor solution and crosslinked. For the calcified zone, aligned fiber sheets were dip-coated in SPELA5 precursor solution, wrapped around a needle to simulate the vertical orientation of the fibers with respect to the chondral surface, and crosslinked [18]. Compressive modulus (E) of the hydrogels was measured with an AR2000 rheometer (TA Instruments, New Castle, DE) as we described previously [18].…”

“…Concurrent with increase in matrix stiffness, orientation of the collagen fibrils changes from parallel to the articulating surface in the superficial zone to random and perpendicular in the middle and calcified zones, respectively [13,17]. In addition to those gradients, the optimum TGF-β1 loading increases from 3 ng/mL in the superficial zone to 30 ng/mL in the middle and calcified zones [9,18]. Further, the expression of BMP-7 [9] and IGF-1 [19] is restricted to the superficial and middle zones, respectively, whereas the secretion of hydroxyapatite (HA) by hypertrophic chondrocytes further stimulates mineralization in the calcified zone [20].…”

“…We recently demonstrated using a developmentally inspired approach that the combination of TGF-β1 as a master regulator of chondrogenesis and zone-specific growth factor, matrix stiffness, and nanofiber orientation led to zone-specific chondrogenic differentiation of encapsulated human mesenchymal stem cells (hMSCs) and expression of corresponding zone-specific markers [18] for the superficial, middle, and calcified zones. The effects of biomolecular factors like TGF-β1, BMP-7, IGF-1, and HA, addition of nanofibers and their orientation, and mechanical stiffness on chondrogenic differentiation of MSCs and chondroprogenitor cells have been investigated extensively in the past [20–22].…”