A bioprinted complex tissue model for myotendinous junction with biochemical and biophysical cues

Abstract

In the musculoskeletal system, the myotendinous junction (MTJ) is optimally designed from the aspect of force transmission generated from a muscle through a tendon onto the bone to induce movement. Although the MTJ is a key complex tissue in force transmission, the realistic fabrication, and formation of complex tissues can be limited. To obtain the MTJ construct, we prepared two bioinks, muscle- and tendon-derived decellularized extracellular matrix (dECM), which can induce myogenic and tenogenic differentiation of human adipose-derived stem cells (hASCs). By using a modified bioprinting process supplemented with a nozzle consisting of a single-core channel and double-sheath channels, we can achieve three different types of MTJ units, composed of muscle, tendon, and interface zones. Our results indicated that the bioprinted dECM-based constructs induced hASCs to myogenic and tenogenic differentiation. In addition, a significantly higher MTJ-associated gene expression was detected at the MTJ interface with a cell-mixing zone than in the other interface models. Based on the results, the bioprinted MTJ model can be a potential platform for understanding the interaction between muscle and tendon cells, and even the bioprinting method can be extensively applied to obtain complex tissues.

Keywords: 3D bioprinting; bioink; hASCs; myotendinous junction; tissue engineering.

FIGURE 1

(a) Schematic of fabrication of myotendinous junction (MTJ) unit using a 3D bioprinting system with a modified core/sheath nozzle with a single core (collagen) and double sheath (M‐ and T‐bioink). (b) MTJ unit (muscle, MTJ, and tendon). (c) Optical and DAPI (blue)/myosin heavy chain (MHC; green)/tenomodulin (TNMD; red) (at 28 days) images of the bioprinted MTJ unit

FIGURE 2

(a) Optical images showing native muscle/tendon tissues, decellularized extracellular matrices (dECMs), and their temperature‐dependent flowability. (b) 4′6‐diamidino‐2‐phenylindole (DAPI) (blue)/type I collagen (red) images of native and decellularized tissues. (c) DNA, collagen, elastin, and glycosaminoglycans (GAGs) contents of the native tissues, muscle‐derived dECM (mdECM), and tendon‐derived dECM (tdECM). (d) DAPI/TNMD (red) and DAPI/MHC (green) images of hASCs cultured on the tissue culture plates at 14 days, which were coated with collagen, mdECM, and tdECM, and laden in the bioinks (collagen, mdECM, and tdECM) at 28 days. Gene expression of myogenesis (Myod1 and Myh2) and tenogenesis (Scx and Tnmd) of hASCs, cultured (e) on 2D culture plates and (f) in the bioinks (n = 3; *p < 0.050, **p < 0.010, and ***p < 0.001)

FIGURE 3

(a) Storage modulus (G') for different collagen concentrations (3, 4, 5, and 7 wt%) and two bioinks [M‐bioink: Hascs (2 × 10⁷ cells/ml) + 3 wt% mdECM + 2.5 mg/ml fibronectin, T‐bioink: 3 Wt% tdECM] for temperature sweep. (b) 4′6‐diamidino‐2‐phenylindole (DAPI)/phalloidin images of cells in the sheath were fabricated using the two bioinks with different collagen concentrations (0, 4, and 7 wt%) in the core and their orientation factors. (c) Live/dead images and cell‐viability (at 7 days) and (d) DAPI/phalloidin images and orientation factor, calculated using the F‐Actin, (at 14 days) of the sheath constructs fabricated using different flow rates of the collagen solution in the core. (e) Cross‐sectional live/dead images (7 days) and DAPI/phalloidin images (14 days) of the sheath construct were fabricated using different flow rates of the M‐bioink in the sheath (n = 4; ***p < 0.001)

FIGURE 4

(a) Optical, scanning electron microscope, and in situ type‐I collagen immunofluorescence (Col‐I; red) images of the fabricated constructs using hASCs‐containing collagen, M‐bioink, and T‐bioink. (b) Gene expression result of Adipo, Opn, Acan, Neurod, Myh2, and Tnmd for the bioprinted constructs (at 28 days). The expression levels were normalized to the results for the cultured hASCs‐containing collagen bioink (without printing). (c) Live/dead (at 7 days), 4′6‐diamidino‐2‐phenylindole (DAPI)/F‐actin/integrin‐β1 (yellow) (at 14 days), and DAPI/MHC (green) (at 28 days) images of the cell‐containing collagen construct (control) and M‐bioink. (d) Myogenesis‐related gene expression results, including Pax7, Myf5, Myod1, and Myh2, of the collagen control and M‐bioink at 28 days. (e) Live /dead (at 7 days), DAPI/F‐actin/scleraxis (SCX; red) (at 14 days), and DAPI/TNMD (red) (at 28 days) images of the collagen control and T‐bioink. (f) Expression levels of Scx and Tnmd for the collagen control and T‐bioink at 28 days. (g) Stress–strain curves and Young's modulus for the structures fabricated using M‐bioink and T‐bioink (n = 4; *p < 0.050, **p < 0.010, and ***p < 0.001)

FIGURE 5

(a) Schematics showing the fabrication procedure for three types of myotendinous junction (MTJ) unit (type 1: Direct contact between muscle and tendon, type 2: Contact in mixing zone, and type 3: Interdigitated contact). (b) Optical, 4′6‐diamidino‐2‐phenylindole (DAPI)/F‐Actin (at 14 days), DAPI/integrin‐β1 (red) (at 14 days), DAPI/F‐Actin/integrin‐β1 (at 14 days), and DAPI/MHC/TNMD (at 28 days) images for the three types of MTJ interfaces. (c) Schematic showing the MTJ ECM composition and crosstalk between the muscle and tendon cells. (d) Level of integrin‐β1 expression using fluorescence images (n = 4). (e) Expression level of the MTJ‐associated genes (Scx, Pxn, Tln1, Thbs1, Col1a1, Lama1, and Myh2) for the three fabricated types of MTJ structure (n = 3) (*p < 0.050, **p < 0.010, and ***p < 0.001)

FIGURE 6

(a) Load–displacement curves for junction types 1, 2, and 3 at 1, 28, and 42 days of cell culture. (b) Maximum load and stiffness of myotendinous junction (MTJ) structures estimated using the load–displacement curves. (c) Captured images of the samples at initial and break point. (d) A load–displacement curve and stiffness of MTJ unit of a rabbit medial biceps brachii (n < 4, *p < 0.050, **p < 0.010, and ***p < 0.001)