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International Journal of Bioprinting Biomaterials for vascularized and innervated tissue regeneration

tissue-derived decellularized extracellular matrix the ratio of hNSCs and hMPCs at 1:300 was optimal for the
(vdECM) bioinks were distributed in the core and shell formation of myotube, the neural differentiation of hNSCs
of the filaments through extrusion from a coaxial nozzle and the NMJs formation. Then, the hNSCs were integrated
in order to simulate the complex structure of muscle into the bioengineered skeletal muscle constructs via a
fibers coupled by blood vessels. Immunofluorescence multichannel 3D bioprinting technique. According to the
staining results indicated that coaxial 3D-bioprinted results, hNSCs-integrated constructs showed significant
muscle construct possessed the capacity of promoting improvement of myofiber formation, neural differentiation,
endothelial network formation and muscle maturation. and NMJs formation in vitro. Moreover, the in vivo results
Encouraged by the satisfactory in vitro outcomes, the further demonstrated the rapid integration with host
in vivo performance of prevascularized 3D-bioprinted neural networks and the vascularization of implanted
muscle constructs was evaluated in a volumetric muscle hNSCs-integrated constructs, leading to enhanced
loss model. As a result, 3D-bioprinted muscles constructs function restoration of muscle tissues. Taken together,
had the maximum recovery of muscle tissue weight and tissue engineering-based strategies possess great potential
minimum fibrosis as compared to other groups. Besides, in promoting functional muscle regeneration.
functional blood vessels with lumen structures, formation
of NMJs and integration with host neural system could 6. Conclusions and perspectives
be observed in the implanted area. Taken together, this In this review paper, we highlighted the essential role
study demonstrated that 3D bioprinting of biomimetic of vascular system and nervous system in functional
prevascularized muscle construct is an effective strategy tissue regeneration and summarized recent advances of
for treating volumetric muscle loss. 3D-printed biomaterials for vascularized and innervated
5.3. 3D-printed biomaterials for innervated skeletal tissue regeneration. In general, vascularization can
muscle regeneration accelerate the process of tissue regeneration through
Similar to the aforementioned vascularization strategies, providing sufficient oxygen and nutrients. Meanwhile,
promoting host neural infiltration and fabricating innervation actively participates in the process of tissue
preinnervated constructs contribute to enhanced regeneration and is indispensable for the functional
innervation . In the first strategy, tissue-engineered recovery of damaged tissues. Furthermore, blood vessels
[6]
muscle constructs functionalized with biochemical signals and nerve fibers closely distributed with each other and
and micro-topographical cues are capable to promote host have synergistic effect on tissue regeneration. However,
neural infiltration and formation of NMJs after implanted there are very few reports about biomaterials that can
into muscle defects [135] . For example, Lee et al. fabricated simultaneously induce vascularization and innervation,
self-aligned 3D skeletal muscle constructs through in situ which are mainly attributed to the difficulty of regulating
creating aligned surface topological microstructures of multiple cells.
3D-printed muscle constructs [136] . The fibrillation and As previously described, several strategies have been
leaching process of poly (vinyl alcohol) induced the proven to be beneficial to vascularization, such as fabricating
formation of aligned topographical structures, which macroporous/channel structures and integrating pro-
further promoted the directional arrangement of muscle angiogenic factors and cells. However, innervations have
progenitor cells. The self-aligned constructs obviously always been overlooked in the past few decades when
accelerated the integration with host neural networks, it comes to designing tissue regenerative scaffolds. The
leading to rapid functional muscle recovery. development of biomaterials for promoting innervated

Taking the advantages of 3D bioprinting technology, it tissue regeneration is still in its infancy stage. Hence, more
is practical to fabricate a preinnervated tissue engineering biomaterials that are capable of inducing innervation
muscle construct with the integration of neural progenitor should be developed and the underlying mechanism
cells or differentiated neurons [137,138] . The coculture of should be explored. The design criterion of pro-innervation
neural cells and myoblast in 3D constructs enable the biomaterials can be considered from the following aspects:
formation of NMJs in vitro, which is beneficial to the (i) Given the physiological properties of nerve fibers,
survival, differentiation, and maturation of myoblast. In biomaterials that have been extensively applied in
a recent study, Kim et al. developed neural stem cells- neural tissue engineering field such as electrical
containing 3D-bioprinted muscle construct to promote stimulation and electroactive materials may be
muscle regeneration and functional recovery (Figure 9) [139] . potentially useful for innervation [130] . As previously
2D co-culture assay was firstly performed to explore the mentioned, several studies have confirmed that the
cross-talking of human neural stem cells (hNSCs) on application of electrical stimulation or electroactive
human muscle progenitor cells (hMPCs). It was found that materials have positive effect on promoting

Volume 9 Issue 3 (2023) 228 https://doi.org/10.18063/ijb.706