Mechanical activation drives tenogenic differentiation of human mesenchymal stem cells in aligned dense collagen hydrogels

Abstract

Tendons are force transmitting mechanosensitive tissues predominantly comprised of highly aligned collagen type I fibres. In this study, the recently introduced gel aspiration-ejection method was used to rapidly fabricate aligned dense collagen (ADC) hydrogel scaffolds. ADCs provide a biomimetic environment compared to traditional collagen hydrogels that are mechanically unstable and comprised of randomly oriented fibrils. The ADC scaffolds were shown to be anisotropic with comparable stiffness to immature tendons. Furthermore, the application of static and cyclic uniaxial loading, short-term (48 h) and high-strain (20%), resulted in a 3-fold increase in both the ultimate tensile strength and modulus of ADCs. Similar mechanical activation of human mesenchymal stem cell (MSC) seeded ADCs in serum- and growth factor-free medium induced their tenogenic differentiation. Both static and cyclic loading profiles resulted in a greater than 12-fold increase in scleraxis gene expression and either suppressed or maintained osteogenic and chondrogenic expressions. Following the 48 h mechanoactivation period, the MSC-seeded scaffolds were matured by tethering in basal medium without further external mechanical stimulation for 19 days, altogether making up 21 days of culture. Extensive cell-induced matrix remodeling and deposition of collagens type I and III, tenascin-C and tenomodulin were observed, where initial cyclic loading induced significantly higher tenomodulin protein content. Moreover, the initial short-term mechanical stimulation elongated and polarized seeded MSCs and overall cell alignment was significantly increased in those under static loading. These findings indicate the regenerative potential of the ADC scaffolds for short-term mechanoactivated tenogenic differentiation, which were achieved even in the absence of serum and growth factors that may potentially increase clinical translatability.Graphical abstract

Continuous two-phase in vitro co-culture model of the enthesis

“…The tensile properties of the scaffolds were also investigated to confirm the continuous nature of the hydrogel (Figure 2C-D). The tensile strength was found to be 0.13 ± 0.05 MPa and apparent modulus was 0.54 ± 0.2 MPa, which were not significantly different from previous single-phase findings (p = 0.75 and 0.78, respectively) [26]. To confirm the two-phased nature of seeded cells within this modified GAE method, the distribution of fluorescent protein expressing cells were investigated in the two-phased ADC hydrogels.…”

“…ACL fibroblast-osteoblast seeded ADC scaffolds were fabricated and cultured by tethering in custom moulds and cultured for 14 days (Figure 1C III) [26]. The percentage of live cells within these co-culture scaffolds was 85 ± 7.5%, which was not significantly different compared to their single-culture controls (p > 0.05) (Figure 3A and B).…”

“…The precursor gels were subjected to the GAE method to simultaneously compact and align the collagen fibrils [23,24], while maintaining the two-phases that were then cultured for 14 days. The anisotropic nature of ADCs resembled the structure of native ligament tissues and showed comparable mechanical properties to immature ligament, whose tensile modulus ranges between 1-3 MPa [34] and seeded mesenchymal stem cells was observed to undergo tenogenesis [26]. These ADCs have also been identified as osteoid-mimicking scaffolds [21], and have been shown to form ectopic bone in vivo [35].…”

“…Neutralized collagen hydrogel concentrations are often between 2-6 mg/mL, and form highly hydrated collagen gels of randomly oriented fibrils [21], which are mechanically unstable [12]. By manipulating these highly hydrated collagen hydrogels, the collagen fibrils can be densified and aligned to form scaffolds that mimic the properties of immature musculoskeletal tissues [22][23][24][25][26]. By applying pressure differentials through a needle connected to an angioplasty inflation device to the highly hydrated collagen gels, simultaneous densification and alignment of the collagen fibers has been achieved.…”

“…This gel aspiration-ejection (GAE) method aspirates the gel into the needle and removes the excess casting fluid, during which the shear forces that are applied to the gel aligns the collagen fibrils. Aligned dense collagen (ADC) scaffolds have previously been investigated in vitro seeded with single cell types, including fibroblasts, mesenchymal stem cells, vascular smooth muscle cells, and Schwann cells [21,[24][25][26], and as a co-culture model using Schwann cells and neuronal cells [25]. The latter, however, did not form zones for the co-culture but seeded secondary cells on the scaffold surface.…”