Page 69 - OR-1-3

reassembly of undifferentiated endothelial cells (ECs) into policy support such as the Food and Drug Administration
capillaries or through organ-on-a-chip (OoC) technology, (FDA) Modernization Act 2.0, which permits bypassing
with the latter representing the prevailing approach in animal testing in new drug development. 181
current research. 176,177 For instance, Wang et al. cultured Compared to traditional animal models, organoids
178
skeletal muscle organoids in an OoC system, monitoring offer significant advantages, including reduced culture
their responses to both perfusion stimuli and electrical timelines, enhanced capacity for high-throughput drug
stimulation in real time.
screening, and markedly lower animal experimentation
The role of nerves must also be considered in the costs. More importantly, these 3D in vitro models faithfully
construction of MSK organoids. In the field of neurogenic recapitulate human physiological and pathological
muscle organoid research, co-culture and assembly processes by maintaining native tissue architecture and
techniques combine independently differentiated cell microenvironmental conditions, thereby providing
types, utilizing intercellular interactions and positioning to a more clinically relevant platform. Beyond disease
guide the formation of NMJs. Moreover, by employing research and therapeutic development, organoids
179
microfluidic chip technology, researchers can precisely demonstrate remarkable versatility in tissue regeneration
control the co-culture conditions of nerve and muscle cells, and regenerative medicine. This technology, through
further optimizing the construction of neurogenic muscle humanized modeling, precise microenvironment control,
organoids (Figure 4A-E). 165,179 For example, researchers have and high-throughput screening capabilities, bridges the
successfully constructed functional NMJs by co-culturing gap between traditional animal experiments and clinical
motor neurons with skeletal muscle cells. In another applications, propelling biomedical research from animal
180
study, the first self-organized hNMSOs were constructed validation to human prediction.
using human PSCs (hPSCs), providing a human in vitro
model for studying the human neuromusculoskeletal axis 4.1. The functions of MSK organoids
and related diseases (Figure 4F-K). Although extensive MSK organoids primarily model MSK development or
121
research has been conducted on the interaction between study the pathogenesis of specific diseases by investigating
nerves and bones, the construction of neurogenic bone intercellular interactions, cellular signaling pathways, and
organoids remains an emerging field. Future research biomechanical responses. Compared to in vivo animal
needs to further explore the mechanisms and strategies experiments, organoids demonstrate unique advantages.
of neurogenic bone tissue engineering to achieve more At the signaling pathway level, organoids can effectively
efficient and physiologically relevant bone regeneration isolate the interference from the variable systemic
effects. environments within animals (encompassing endocrine,
4. Attempts to replace in vivo experiments immune, and metabolic factors). This capability enables the
precise localization, simulation, and targeted intervention
with organoids of key molecular signaling pathways (Wingless-related
Organoid technology represents a transformative integration site [Wnt], nuclear factor-kappa B [NF-κB],
advancement in biomedical research, offering significant TGF-β, etc.) that regulate developmental processes in
improvements in precision, controllability, and ethical tissues, such as muscle, bone, and joints, all under highly
compliance compared to traditional animal models. By controllable conditions. For instance, neuromuscular
employing patient-derived cells or iPSCs to generate organoids (NMOs) generated from amyotrophic lateral
miniaturized tissue constructs, this approach recapitulates sclerosis (ALS) patient cells not only enable precise
human organ functionality while overcoming the inherent delineation of C9orf72 mutation-induced disruption of
limitations of interspecies variation that plague animal the Wnt/β-catenin pathway but also serve as platforms to
studies. The organoids enable unprecedented experimental validate the efficacy of R-like endoplasmic reticulum kinase
control through the establishment of precise growth factor inhibitor, GSK2606414, in ameliorating ALS phenotypes
gradients through microfluidic systems, optimization of through restoration of Wnt downstream signaling.
182
nutrient diffusion using 3D-printed biomimetic scaffolds Furthermore, the MSK OoC model enables investigation
with tunable porosity, and real-time monitoring of cellular of hypoxia’s impact on signaling pathways within the MSK
dynamics through advanced live imaging techniques. These system. Intermittent hypoxia (IH) downregulates muscle
capabilities provide researchers with direct observational mitochondrial sirtuin 3 (SIRT3), activating the NF-κB
access to developmental and pathological processes that pathway and promoting secretion of the myokine C-X-C
were previously only inferable through terminal animal motif chemokine ligand 5 (CXCL5), thereby suppressing
experiments. Ethically, organoid technology significantly osteogenic differentiation while enhancing osteoclast
reduces the use of animals, aligning with the replacement, activity. In addition, organoids derived from Duchenne
183
reduction, and refinement (“3R principles”) and gaining muscular dystrophy (DMD) patients not only permit

Volume 1 Issue 3 (2025) 11 doi: 10.36922/OR025280024