Volumetric muscle loss (VML), characterized by a loss of muscle tissue greater than 20%, poses a significant clinical challenge due to limited natural regenerative capacity. Unlike minor muscle injuries, which typically recover via satellite cell activation and subsequent myogenesis, VML leads to extensive fibrosis, persistent functional deficits, and impaired quality of life. Existing therapeutic strategies, including autologous tissue grafts and muscle flap transfers, face limitations such as donor site morbidity, insufficient tissue availability, and inadequate functional recovery. Recent advances in tissue engineering and 3D bioprinting have provided promising platforms for generating implantable muscle constructs. However, current bioengineered muscle constructs rarely replicate the critical viscoelastic properties inherent in native muscle tissue, essential for cellular dynamics, vascular integration, and neuromuscular functionality. Although hydrogel stiffness has been extensively studied, the influence of hydrogel viscoelasticity on myogenesis, vascularization, and innervation remains underexplored. This study addresses these critical gaps by developing and evaluating a novel 3D bioprinted viscoelastic hydrogel-based muscle construct aimed at improving VML repair outcomes.
Methods:
Initially, we investigated the impact of viscoelastic hydrogel properties on myotube formation in primary human skeletal myoblasts (HSMMs). A comparative analysis was conducted between two experimental groups: one utilizing viscoelastic hydrogels and another employing elastic hydrogels, both matched for storage modulus. Myogenic differentiation was quantitatively assessed through immunostaining for myosin heavy chain (MHC), and gene expression profiles for myogenic markers, including MYOD, MYOG, MYH1, MYH2, MYH4, MYH7, MYH8, Desmin, and ACTN2, were compared between the two hydrogel types.
Subsequently, to promote comprehensive regeneration, vascularization, and innervation within the engineered muscle constructs, we incorporated primary human umbilical vein endothelial cells (HUVECs) and motor neurons derived from induced pluripotent stem cells (iPSC-MNs). The effectiveness of these cellular inclusions was evaluated through immunostaining of markers indicative of myofiber regeneration (MHC), neural integration (βIII-tubulin), and vascularization (VE-cadherin).
Results:
In preliminary studies, we successfully characterized and matched the storage modulus and loss tangent of human and rat muscle tissues, subsequently formulating an extrusion-based, 3D-printable viscoelastic hydrogel composed of FDA-approved gelatin and alginate. This viscoelastic hydrogel demonstrated a storage modulus comparable to native muscle tissues but differed significantly in its loss tangent, highlighting its unique viscoelastic characteristics. HSMMs cultured within viscoelastic hydrogels exhibited superior elongation, spreading, and enhanced myotube formation compared to those cultured in elastic hydrogels, indicating the pivotal role of viscoelasticity in promoting muscle regeneration.
Further evaluation revealed that constructs containing HUVECs exhibited pronounced endothelial sprouting and vessel-like network formation, essential for effective vascular integration. Similarly, constructs integrating iPSC-MNs demonstrated robust neurite extension, indicative of enhanced innervation. Importantly, constructs based on elastic hydrogels showed significantly reduced expression of myogenic and neurogenic markers compared to those employing viscoelastic hydrogels, underscoring the critical importance of viscoelastic mechanical cues in supporting comprehensive muscle regeneration.
Implications:
This study introduces an innovative bioprinting strategy leveraging viscoelastic hydrogel properties to fabricate engineered muscle constructs with enhanced vascularization and innervation capabilities. By closely mimicking the native biomechanical environment, this approach holds transformative potential for significantly improving regenerative outcomes in VML treatment. These findings provide foundational insights that can accelerate the translation of advanced regenerative therapies, addressing clinical challenges associated with traumatic muscle injuries, degenerative muscle diseases, and reconstructive surgery.
Furthermore, the capability to finely tune viscoelastic properties in bioengineered constructs highlights new possibilities for optimizing cellular interactions, facilitating rapid vascularization, and ensuring efficient neuromuscular integration. Future investigations will focus on refining hydrogel formulations with additional biochemical and biophysical cues, leveraging patient-specific iPSC models, and conducting extended implantation studies to evaluate long-term integration, immune compatibility, and functional persistence. Ultimately, this innovative approach aims to enable personalized, implantable muscle grafts tailored to individual patient needs, significantly enhancing therapeutic outcomes and broadening the applicability of bioprinted constructs in regenerative medicine.