Journal: Scientific Reports

Article Title: 3D Bioprinted Human Skeletal Muscle Constructs for Muscle Function Restoration

doi: 10.1038/s41598-018-29968-5

Figure Lengend Snippet: Bioprinting of skeletal muscle. ( A ) Design concept using 3D CAD modeling and ( B ) motion program generation of the bioprinted muscle construct. The code includes XYX stage movement and actuating pneumatic pressure. ( C,D ) Bioprinting process using ITOP system. ( C ) The motion program was transferred to the operating computer of ITOP. The cell-laden bioink containing hMPCs, the acellular sacrificing hydrogel, and the supporting PCL pillar were loaded in the multi-dispensing modules. ( D ) All three components were printed in a layer-by-layer fashion. ( E ) The bioprinted skeletal muscle constructs composed of multi-layered myofiber bundles were fabricated up to 15 × 15 × 15 mm 3 in dimension. The thickness of the printed muscle construct was determined by controlling the number of stacking myofiber bundles. ( F ) Microchannels in the constructs created after the removal of the sacrificial patterns to maintain the viability of printed cells.

Article Snippet: The outcomes indicate the structural and functional skeletal muscle regeneration using the 3D biomimetic bioprinted muscle constructs, while the non-printed construct and cell-free construct (gel only) did not provide any therapeutic effects.

Techniques: Construct

Journal: Scientific Reports

Article Title: 3D Bioprinted Human Skeletal Muscle Constructs for Muscle Function Restoration

doi: 10.1038/s41598-018-29968-5

Figure Lengend Snippet: In vitro evaluations of bioprinted muscle constructs compared with non-printed constructs. ( A ) Representative Live/Dead staining images and ( B ) cell viability (%) at 1 and 5 days in culture (n = 4, 4 random fields/sample, * P < 0.05, **not measurable because of too confluence - % viability was over 90%). ( C ) Immunofluorescent staining for MHC after 7 day-differentiation and ( D ) quantification of area of MHC + myofibers ( n = 3, 4–7 random images/sample, * P < 0.05). Human MPCs in the construct showed enhanced myofiber formation with unidirectional cell alignment. ( E ) Double-immunostaining for α-SA (red)/laminin (green) indicates the presence of cross-striated myofibers surrounded by laminin matrix in the printed construct. Quantification of ( F ) α-SA + area (%) and ( G ) laminin + area (%) (n = 3, * P < 0.05).

Article Snippet: The outcomes indicate the structural and functional skeletal muscle regeneration using the 3D biomimetic bioprinted muscle constructs, while the non-printed construct and cell-free construct (gel only) did not provide any therapeutic effects.

Techniques: In Vitro, Construct, Staining, Double Immunostaining

Journal: Scientific Reports

Article Title: 3D Bioprinted Human Skeletal Muscle Constructs for Muscle Function Restoration

doi: 10.1038/s41598-018-29968-5

Figure Lengend Snippet: In vitro cell density optimization. ( A ) Live/dead staining images and ( B ) quantification of bioprinted muscle constructs with cell densities of 10, 20, 30, and 50 × 10 6 cells/ml at 1 day in culture ( n = 6, 5 random fields/sample, no significant difference). ( C ) TUNEL assay of bioprinted muscle constructs after 6 days in culture. Apoptotic cells were calculated with different cell densities ( n = 3, 5 random fields/sample, no significant difference). ( D ) MHC immunofluorescent images of bioprinted muscle constructs at 6 days in culture (after 5-day differentiation). Representative immunofluorescent images for MHC (red) showed that bioprinted hMPCs in the constructs with different cell densities were formed into longitudinally aligned myofibers.

Article Snippet: The outcomes indicate the structural and functional skeletal muscle regeneration using the 3D biomimetic bioprinted muscle constructs, while the non-printed construct and cell-free construct (gel only) did not provide any therapeutic effects.

Techniques: In Vitro, Staining, Construct, TUNEL Assay

Journal: Scientific Reports

Article Title: 3D Bioprinted Human Skeletal Muscle Constructs for Muscle Function Restoration

doi: 10.1038/s41598-018-29968-5

Figure Lengend Snippet: In vivo cell density optimization on dimensional maintenance. ( A ) H&E-stained images of longitudinal cross-sections of bioprinted muscle constructs with different cell densities at 1, 2 and 4 weeks after implantation. ( B ) The thickness of bioprinted muscle constructs as measured by H&E-stained sections ( n = 4, 3 random regions/sample). Thickness of the constructs increased with cell density, but there was no significant difference between 30 and 50 × 10 6 cells/ml at 2 weeks and 4 weeks (* P < 0.05 compared with 10 × 10 6 cells/ml, ** P < 0.05 with 10 × 10 6 cells/ml and 20 × 10 6 cells/ml).

Article Snippet: The outcomes indicate the structural and functional skeletal muscle regeneration using the 3D biomimetic bioprinted muscle constructs, while the non-printed construct and cell-free construct (gel only) did not provide any therapeutic effects.

Techniques: In Vivo, Staining, Construct

Journal: Scientific Reports

Article Title: 3D Bioprinted Human Skeletal Muscle Constructs for Muscle Function Restoration

doi: 10.1038/s41598-018-29968-5

Figure Lengend Snippet: Ectopic skeletal muscle regeneration. ( A ) Representative immunofluorescent images for MHC (red)/HLA (green) at 2 weeks after implantation. Double-immunostained MHC + /HLA + myofibers in bioprinted constructs indicate newly formed skeletal muscle. ( B ) Numbers of MHC + myofibers and ( C ) areas of MHC + myofibers ( n = 4, 3 random regions/sample). There is an increasing trend of skeletal muscle tissue formation with increasing cell density, but no significant difference between 30 and 50 × 10 6 cells/ml at 1 week and 2 weeks after implantation (* P < 0.05 compared with 10 × 10 6 cells/ml at 1 week, ** P < 0.05 compared with 10 and 20 × 10 6 cells/ml at 1 week, † P < 0.05 compared with 10 × 10 6 cells/ml at 2 weeks, and †† P < 0.05 compared with 10 and 20 × 10 6 cells/ml at 2 weeks). ( D ) Numbers of HLA + myofibers and ( E ) areas of HLA + myofibers ( n = 4, 3 random regions/sample). There is no significant difference between 30 × 10 6 cells/ml and 50 × 10 6 cells/ml at 2 weeks after implantation.

Article Snippet: The outcomes indicate the structural and functional skeletal muscle regeneration using the 3D biomimetic bioprinted muscle constructs, while the non-printed construct and cell-free construct (gel only) did not provide any therapeutic effects.

Techniques: Construct

Journal: Scientific Reports

Article Title: 3D Bioprinted Human Skeletal Muscle Constructs for Muscle Function Restoration

doi: 10.1038/s41598-018-29968-5

Figure Lengend Snippet: Rat TA muscle defect model. ( A ) Tetanic force (N · mm/kg) and ( B ) TA muscle weight (% of contralateral normal TA muscle) were measured at 4 and 8 weeks after implantation (n = 3 per group, triple measures per sample). Tetanic force and muscle weight of bioprinted muscle constructs-implanted group had significantly increased when compared with other groups (* P < 0.05 compared with non-treated group at 4 weeks, ** P < 0.05 compared with non-treated, gel only, and non-printed groups at 4 weeks, and † P < 0.05 compared with non-treated, gel only, and non-printed groups at 8 weeks).

Article Snippet: The outcomes indicate the structural and functional skeletal muscle regeneration using the 3D biomimetic bioprinted muscle constructs, while the non-printed construct and cell-free construct (gel only) did not provide any therapeutic effects.

Techniques: Construct

Journal: Scientific Reports

Article Title: 3D Bioprinted Human Skeletal Muscle Constructs for Muscle Function Restoration

doi: 10.1038/s41598-018-29968-5

Figure Lengend Snippet: Histological examinations. Histological images showed highly aligned newly formed myofibers in bioprinted constructs with superior muscle volume maintenance at 4 and 8 weeks post-implantation, while severe muscle atrophy and limited muscle regeneration were determined in other groups. Squares in left column indicate areas shown in detail with high magnifications. MTS, Masson’s trichrome staining.

Article Snippet: The outcomes indicate the structural and functional skeletal muscle regeneration using the 3D biomimetic bioprinted muscle constructs, while the non-printed construct and cell-free construct (gel only) did not provide any therapeutic effects.

Techniques: Construct, Staining

Journal: Scientific Reports

Article Title: 3D Bioprinted Human Skeletal Muscle Constructs for Muscle Function Restoration

doi: 10.1038/s41598-018-29968-5

Figure Lengend Snippet: Immunofluorescent analysis of bioprinted muscle constructs on newly formed muscle. ( A ) Double-immunostaining of MHC/HLA of the retrieved TA muscles (MHC: red, HLA: green). ( B ) Double-immunostaining of MHC/HLA of the implanted regions. ( C ) Quantification of co-localized HLA + /MHC + cells (% of HLA + cells per MHC + cells ( n = 3, * P < 0.05 compared with non-printed group and ** P < 0.05 between 4 and 8 weeks). Higher skeletal muscle regeneration in bioprinted construct was observed at 4 and 8 weeks of implantation. MHC + /HLA + newly formed myofibers in bioprinted constructs indicate that the implanted hMPCs contributed to skeletal muscle regeneration in the defect region (MHC: red, HLA: green, MHC − /HLA + cells: white arrow, MHC + /HLA + cells: yellow arrow). ( D ) A cross-sectional view of double-immunostaining of MHC/HLA of the bioprinted muscle constructs and ( E ) Quantification of the diameter of MHC + /HLA + myofibers (µm) ( n = 3, 3 random fields per sample, * P < 0.05).

Article Snippet: The outcomes indicate the structural and functional skeletal muscle regeneration using the 3D biomimetic bioprinted muscle constructs, while the non-printed construct and cell-free construct (gel only) did not provide any therapeutic effects.

Techniques: Construct, Double Immunostaining, Muscles

Journal: Scientific Reports

Article Title: 3D Bioprinted Human Skeletal Muscle Constructs for Muscle Function Restoration

doi: 10.1038/s41598-018-29968-5

Figure Lengend Snippet: Immunofluorescence of vascularization and neural integration of the implanted bioprinted muscle constructs. ( A ) Immunofluorescent images of vWF (green)/α-SMA (red) of the regenerated TA muscles at 4 and 8 weeks after implantation. Quantification of ( B ) vessels/field and ( C ) area of vessels/field (µm 2 ) ( n = 3, * P < 0.05 compared with non-treated and gel only groups, ** P < 0.05 compared with other groups). ( D ) Immunofluorescent images of NF (green)/AChR (red)/MHC (white) and NF (green)/AChR (red)/HLA (white) of the regenerated TA muscles at 4 and 8 weeks after implantation. NF + /AChR + /MHC + neuromuscular junction (middle column, white arrow) was observed in bioprinted muscle constructs. NF + /AChR + /HLA + neuromuscular junction (right column) corresponding area NF + /AChR + /MHC + (middle column) indicates that bioprinted muscle constructs are integrated with host nervous system following implantation. The white arrow indicates neuromuscular junction on hMPC-myofibers. ( E ) Quantification of NMJ/field (×400) ( n = 3 per group, 3 random fields per sample, * P < 0.05 compared with other groups).

Article Snippet: The outcomes indicate the structural and functional skeletal muscle regeneration using the 3D biomimetic bioprinted muscle constructs, while the non-printed construct and cell-free construct (gel only) did not provide any therapeutic effects.

Techniques: Immunofluorescence, Construct, Muscles