Clinical Safety Evaluation and Preliminary Efficacy Study of Subcutaneous Myografts Transplantation
Phase: EARLY_PHASE1
Conditions: Metabolic Disease, Amyotrophy
Sponsor: National Clinical Research Center for Orthopedics, Sports Medicine and Rehabilitation, China

 
 
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Clinical Safety Evaluation and Preliminary Efficacy Study of 
Subcutaneous Myografts Transplantation 
Study Protocol 
Protocol Version: V1.0; Protocol Date: January 20, 2026 
Background 
With the acceleration of population aging and the increasing burden of chronic 
diseases, skeletal muscle atrophy and functional decline in patients with long-term bed 
rest have become major clinical challenges that require urgent attention(1). Prolonged 
bed rest leads to muscle disuse, reduced protein synthesis, and rapid loss of muscle fibers, 
resulting in decreased muscle strength, reduced exercise tolerance, and metabolic 
dysfunction. More importantly, skeletal muscle is not only the primary organ responsible 
for movement but is also recognized as a critical endocrine organ. Through the secretion 
of multiple myokines, skeletal muscle regulates systemic metabolism, inflammatory 
responses, and the function of various organs. When skeletal muscle atrophy occurs, the 
secretion of myokines with anti-inflammatory, metabolic regulatory, and multi-organ 
protective effects is markedly reduced. This loss of endocrine function further 
exacerbates insulin resistance, chronic inflammation, and multisystem degenerative 
changes. Current rehabilitation strategies, including passive exercise, physical therapy, 
and nutritional interventions, show limited efficacy in preventing muscle atrophy or 
improving muscle function in patients with prolonged immobilization. Moreover, these 
approaches are insufficient to maintain long-term muscle mass and function. Therefore, 
there is an urgent need to develop innovative intervention strategies that can effectively 
reverse or delay muscle atrophy and functional deterioration in patients with long-term 
bed rest, thereby reducing complication risk, improving quality of life, and extending 
health span. 
In recent years, cell-based therapies have gained increasing attention. Muscle stem 
cells (MuSCs) are the source cells responsible for skeletal muscle regeneration and 
possess the capacity for self-renewal and differentiation muscle injury, MuSCs 
proliferate as myoblasts, differentiate into myocytes, and ultimately fuse to form 
multinucleated muscle fibers. Myoblast transplantation has been explored as a 
therapeutic approach for muscle atrophy and other muscle-related disorders and can also 

 
 
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serve as a delivery platform for therapeutic proteins. However, a major limitation of 
myoblast transplantation is the poor survival of transplanted cells, with most cells dying 
within 48 hours after transplantation due to local ischemia, apoptosis, inflammation, or 
immune rejection. In contrast, mature myocytes represent terminally differentiated cells 
and provide a safe, efficient, and programmable cell therapy platform. In addition to their 
potential for local tissue repair, mature myocytes also show promise as long-term 
delivery vehicles for therapeutic proteins. Compared with other cell types, mature 
myocytes exhibit distinct advantages in terms of safety and biological function. On the 
one hand, muscle fiber growth primarily relies on multinucleated hypertrophy rather than 
cell division, thereby minimizing tumorigenic risk(5-8). On the other hand, mature 
myocytes possess strong angiogenic potential, which facilitates vascular reconstruction 
and long-term graft survival after transplantation. To address these clinical limitations, 
we previously developed a novel technique for subcutaneous transplantation of 
autologous differentiated myocytes, generating engineered muscle grafts with 
spontaneous contractile activity and favorable vascularization. In preclinical animal 
studies, these grafts demonstrated long-term survival in vivo and mimicked a state of 
"continuous exercise." In addition, they stably secreted myokines and systemically 
improved muscle quality, bone mineral density, energy metabolism, and inflammatory 
status. These effects were consistently observed in both aging mouse models and obesity 
models, with significant attenuation of degenerative changes.  
In humans, application of this autologous differentiated myocyte subcutaneous 
transplantation technique has the potential to overcome key limitations of conventional 
myocyte transplantation, including low graft survival and inadequate vascularization. By 
enabling long-term graft survival and functional maintenance, and through spontaneous 
sustained contraction and stable secretion of multiple myokines, this strategy may 
fundamentally improve skeletal muscle atrophy, sarcopenia, and systemic metabolic 
disorders in patients with long-term bed rest. The present study aims to translate 
autologous differentiated myocyte subcutaneous transplantation into clinical application 
in patients with muscle atrophy caused by long-term bed rest, and to explore its safety, 
feasibility, and preliminary efficacy. This study is intended to provide a solid foundation 
for the clinical translation of autologous differentiated myocyte subcutaneous 
transplantation as a therapeutic strategy.  
Reference： 

 
 
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1. 
Rittweger J, Felsenberg D. Recovery of muscle atrophy and bone loss from 90 
days bed rest: results from a one-year follow-up. Bone. 2009;44(2):214-24. 
2. 
Wang YX, Rudnicki MA. Satellite cells, the engines of muscle repair. Nature 
reviews Molecular cell biology. 2011;13(2):127-33. 
3. 
Schultz E, Gibson MC, Champion T. Satellite cells are mitotically quiescent in 
mature mouse muscle: an EM and radioautographic study. The Journal of experimental 
zoology. 1978;206(3):451-6. 
4. 
Mauro A. Satellite cell of skeletal muscle fibers. The Journal of biophysical and 
biochemical cytology. 1961;9(2):493-5. 
5. 
Pedersen BK, Febbraio MA. Muscles, exercise and obesity: skeletal muscle as a 
secretory organ. Nature reviews Endocrinology. 2012;8(8):457-65. 
6. 
Beckerman M, Harel C, Michael I, Klip A, Bilan PJ, Gallagher EJ, et al. GLUT4-
overexpressing engineered muscle constructs as a therapeutic platform to normalize 
glycemia in diabetic mice. Science advances. 2021;7(42):eabg3947. 
7. 
Giudice J, Taylor JM. Muscle as a paracrine and endocrine organ. Current opinion 
in pharmacology. 2017;34:49-55. 
8. 
Severinsen MCK, Pedersen BK. Muscle-Organ Crosstalk: The Emerging Roles of 
Myokines. Endocrine reviews. 2020;41(4):594-609. 
 
Study Objectives 
At present, there is a lack of effective clinical strategies for patients with muscle 
atrophy caused by long-term bed rest due to multiple underlying conditions, to reverse 
or delay muscle atrophy and functional degeneration. Conventional rehabilitation 
training and nutritional support have limited efficacy in mitigating muscle atrophy 
induced by prolonged immobilization, highlighting the urgent need for novel therapeutic 
interventions. Based on our previous experimental findings, we have developed an 
autologous differentiated myocyte subcutaneous transplantation technique that enables 
long-term graft survival in vivo, mimics a state of "continuous exercise," and ensures 
stable secretion of myokines. Through these mechanisms, this approach systemically 
improves muscle quality, bone mineral density, energy metabolism, and inflammatory 
status. Building on these preclinical results, the present study aims to innovatively 
translate autologous differentiated myocyte subcutaneous transplantation into human 
application, with the goal of constructing a sustainable, spontaneously contractile, and 
endocrine-functional "muscle graft." 
The specific objectives of this study are as follows: (1) To evaluate graft survival, 
vascularization, and immune tolerance following subcutaneous transplantation of 

 
 
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autologous differentiated myocytes in patients with long-term bed rest–related muscle 
atrophy, and to ensure the safety of clinical application. (2) To systematically assess the 
effects of the muscle graft on skeletal muscle mass, muscle strength, and exercise 
endurance, and to explore its potential in reversing muscle atrophy and preserving muscle 
function. (3) To analyze the regulatory effects of graft-derived myokines on systemic 
energy metabolism, bone mineral density, and chronic inflammatory status, and to 
evaluate their impact on aging-related degenerative conditions, including sarcopenia, 
osteoporosis, and metabolic disorders.  
Ultimately, this study aims to establish a subcutaneous transplantation strategy for 
autologous differentiated myocytes capable of generating long-term surviving, 
vascularized, and spontaneously contractile muscle grafts, and to explore further the 
therapeutic potential of mimicking the skeletal muscle secretory profile during physical 
activity for the treatment of muscle atrophy, sarcopenic obesity, and aging-related 
degenerative diseases. 
 
Study Content 
This study focuses on the safety and functional validation of autologous 
subcutaneous transplantation of differentiated myocytes. The main study components 
include muscle cell sourcing and engineering, subcutaneous implantation and long-term 
observation, safety evaluation, and functional assessment using disease-relevant 
outcome measures. (1) Muscle Cell Sourcing and Engineering: Recruited participants 
will undergo muscle biopsy (e.g., from the vastus lateralis or gluteus medius) to obtain a 
small amount of skeletal muscle tissue. Muscle satellite cells will be isolated, purified, 
expanded in vitro, and induced to differentiate into mature myocytes. Quality control of 
the differentiated myocytes will be performed, including assessments of cell purity, 
functional characteristics, and secretory profiles, to ensure suitability for clinical 
transplantation. (2) Subcutaneous Implantation and Long-Term Observation: Engineered 
myocytes will be mixed with collagen and injected subcutaneously into the abdominal 
region. Dynamic evaluations will be conducted to assess graft survival, vascular 
reconstruction, contractile function, and local tissue responses. Electromyography and 
imaging modalities (MRI and ultrasonography) will be used to monitor graft contractility 
and tissue integration. (3) Safety Evaluation: Local and systemic safety will be assessed 

 
 
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through continuous monitoring. Serum immunological parameters, including white 
blood cell counts and inflammatory cytokines, will be measured. Local histopathological 
examination will be performed using graft tissue biopsy when indicated. Ectopic 
transplantation, cell migration, abnormal differentiation, or pathological proliferation 
will be actively excluded. Adverse events will be recorded during long-term follow-up. 
(4) Functional Assessment: Functional outcomes will include measurements of skeletal 
muscle mass using dual-energy X-ray absorptiometry (DXA), bone metabolism 
indicators, serum creatine kinase levels, and systemic energy metabolism parameters, 
including respiratory exchange ratio (RER) and energy expenditure. 
 
Study Design 
1. Study Design and Control Strategy 
(1) Study Type: Prospective, single-arm, self-controlled pre–post interventional study. 
(2) Control Strategy: Within-subject control: Each participant will complete the same set 
of assessments before and after the intervention, serving as their own control. A 
within-subject pre – post comparison will be used to evaluate individualized 
responses and temporal changes induced by subcutaneous muscle graft 
transplantation. 
2. Sample Size 
(1) Total Sample Size: 6 participants. 
(2) This study is designed as a Phase I/IIa exploratory clinical trial. According to 
common practice in early-phase cell therapy studies, including CAR-T therapy, 
myoblast transplantation, and induced pluripotent stem cell (iPSC) – based 
interventions, an initial cohort of 3–6 participants per group is typically enrolled for 
preliminary safety and feasibility evaluation. Given that the primary objectives of this 
study are to assess the safety, feasibility, and preliminary efficacy of autologous 
differentiated myocyte subcutaneous transplantation in patients with long-term bed 
rest–induced muscle atrophy, a small-sample design with intensive follow-up has 
been adopted. This approach allows close monitoring of adverse events and 
biological responses while generating foundational data to support subsequent larger-
scale clinical studies. 

 
 
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3. Study Population and Grouping 
(1) Inclusion Criteria: 
1) Age: 18–80 years, with no restriction on sex. 
2) Long-Term Bed Rest: Continuous bed rest for ≥4 weeks. Causes may include 
neurological injuries (such as brain death, stroke, or spinal cord injury), recovery after 
major orthopedic surgery, intensive care unit stay, or activity limitation due to chronic 
diseases. 
3) Evidence of Muscle Atrophy: A significant reduction in muscle mass or muscle 
strength confirmed by clinical assessment and imaging studies. According to dual-
energy X-ray absorptiometry (DXA), appendicular skeletal muscle mass index 
(ASM/height²) < 7.0 kg/m² in men and < 5.4 kg/m² in women, in accordance with the 
EWGSOP2 criteria.  
4) Stable Underlying Conditions: Underlying medical conditions are stable, with no 
acute exacerbation, and an APACHE II score between 0 and 20. 
5) Surgical Eligibility: Absence of severe comorbidities that contraindicate surgical or 
transplantation interventions. 
6) Informed Consent: Written informed consent obtained from the participant, an 
immediate family member, or a legal guardian, agreeing to undergo muscle biopsy. 
General health status must permit subcutaneous transplantation.  
(2) Exclusion Criteria 
1) History of malignant tumors. 
2) Coagulation disorders or current use of anticoagulant therapy. 
3) Active infection or immunodeficiency. 
4) Severe cardiac or renal insufficiency (estimated glomerular filtration rate < 60 
mL/min/1.73 m²; New York Heart Association class III–IV heart failure). 
5) Muscle-related diseases, including hereditary myopathies (such as muscular 
dystrophy) or acquired myositis (creatine kinase > 3 times the upper limit of normal). 
6) Severe local skin lesions or a history of allergic reactions at the injection site. 
7) Use of muscle growth–modulating medications (such as testosterone or growth 

 
 
7
hormone) within the past 6 months. 
8) Long-term use of corticosteroids or immunosuppressive therapy, including anti-
rejection medications following organ transplantation. 
9) Participation in other interventional clinical trials (excluding observational studies). 
10) Any other medical or ethical conditions deemed by the investigator to make the 
participant unsuitable for enrollment. 
(3) Study Grouping:  
1) Intervention Group: Participants will receive subcutaneous transplantation of 
autologous differentiated myocytes. 
4. Intervention Procedures and Standardized Workflow 
(1) Principles of Study Design 
This study adopts a strategy of subcutaneous transplantation of autologous 
differentiated myocytes. Muscle satellite cells are isolated from autologous skeletal 
muscle tissue, expanded and induced to differentiate into myocytes in vitro, and combined 
with collagen to form an engineered muscle graft (myograft). The graft is then implanted 
via subcutaneous injection. The study aims to evaluate long-term graft survival, 
spontaneous contractile activity, and myokine secretion capacity in humans, and to 
explore the potential clinical value of this approach in enhancing muscle function, 
regulating metabolic status, and delaying aging-related degenerative changes. This study 
is intended to provide a technical foundation for the translational application of skeletal 
muscle–based cell therapy in patients with long-term bed rest–induced muscle atrophy 
and chronic diseases. 
(2) Timeline of Sample Collection and Intervention Procedures 
1） Preoperative Preparation and Assessment 
Before intervention, all participants will undergo a comprehensive physical 
examination and laboratory testing, including complete blood count, serum biochemistry, 
liver and renal function tests, infection screening, and coagulation assessment, to 
reconfirm eligibility and exclude contraindications. The study team will explain the study 
protocol in detail, and written informed consent will be obtained. The muscle biopsy site 
will be determined in advance, with the vastus lateralis selected as the preferred site. 

 
 
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Preoperative marking and scheduling will be completed accordingly. 
2） Timeline of Sample Collection and Intervention Procedures 
Procedure Setting: Muscle biopsy will be performed under sterile conditions in a 
minor procedure room or outpatient surgical suite. 
Anesthesia: Local anesthesia with 1% lidocaine will be administered to the skin and 
underlying fascia. 
Biopsy Technique: A Bergström biopsy needle will be used to obtain skeletal muscle 
tissue through a 0.5–1.0 cm skin incision. Approximately 0.5 × 0.5 × 0.5–1.0 cm of 
muscle tissue will be collected in a single attempt whenever possible, minimizing fascia 
contamination and avoiding repeated needle insertions. 
Postoperative Care: After the procedure, a compression dressing will be applied and 
covered with a sterile dressing. Participants may leave after 30 minutes of observation if 
no discomfort occurs. Strenuous activity, local compression, and washing of the biopsy 
site will be avoided within 24 hours. 
3） Cell Preparation and Injection Readiness 
Muscle tissue will be immediately transferred to a GMP-compliant laboratory for 
isolation, expansion, and differentiation of muscle satellite cells into myocytes, a process 
requiring approximately 10 – 14 days. All cell products must pass quality control 
assessments, including sterility testing, cell viability, and differentiation efficiency, before 
being approved for clinical use. Prepared cell products will be packaged by designated 
personnel and transported under cold-chain conditions to the injection site. Participant 
identity and batch codes will be verified again prior to injection. On the day of injection, 
each participant will receive a total dose of 1 × 10⁸ cells, mixed with 6 mL of collagen. 
4） Subcutaneous Injection of Muscle Grafts 
Injection Site: The abdominal subcutaneous region will be selected preferentially, 
avoiding bony prominences and areas with dense vascular distribution. Patient Position: 
Participants will be placed in a prone or seated position. Anesthesia: Local subcutaneous 
anesthesia with 1% lidocaine. 
Injection Procedure: 
I. 
Bilateral, multi-point injections will be performed. 

 
 
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II. Injection volume per side: 2.5–3.0 mL. 
III. Maximum volume per injection point: ≤1.0 mL. 
IV. A 21-G needle will be used to create subcutaneous channels. 
V. A total of 1 × 10⁸ cells will be injected bilaterally at multiple sites, with an 
injection depth of 1–2 cm. 
VI. The graft will be distributed in a planar pattern rather than as a compact mass to 
facilitate integration. 
VII. After injection, the graft will be allowed to solidify for 5–10 minutes. 
Hemostasis will be achieved by compression, followed by sterile dressing and 
pressure bandaging. Injection parameters, including batch number, time, operator, and 
injection sites, will be recorded. Participants will be observed for one hour post-procedure 
and discharged if no discomfort is noted. Strenuous activity and washing of the injection 
area will be avoided for 72 hours. 
5） Postoperative Observation and Follow-Up 
Immediate Observation (Within 24 Hours): Vital signs, including body temperature, 
blood pressure, and heart rate, will be closely monitored. Signs of allergic reactions or 
acute immune responses will be assessed. 
Short-Term Follow-Up: At Day 1, Day 7, and Day 14 post-injection, the injection 
site will be evaluated for redness, swelling, induration, and nodules. Complete blood count 
and C-reactive protein (CRP) levels will be measured to assess acute inflammation. 
Mid-Term Follow-Up: Preliminary therapeutic effects will be evaluated. Graft 
survival will be assessed using imaging modalities. Laboratory assessments will include 
complete blood count, CRP, inflammatory cytokines, liver enzymes, creatine kinase, 
DXA-derived ASM, muscle ultrasonography, myokines, bone mineral density, bone 
turnover markers, and metabolic parameters assessed by indirect calorimetry. 
Long-Term Follow-Up: Participants will be followed for up to 12 months. Long-term 
endpoints will include skeletal muscle mass (DXA), bone mineral density, serum calcium 
and phosphorus, metabolic indicators, and muscle enzyme profiles. 
Management of Abnormal Findings: Local redness, pain, or mild enzyme elevation 
will be recorded and observed. Persistent induration, abnormal growth, or redness lasting 

 
 
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more than one week will prompt ultrasonography or MRI, and biopsy will be performed 
if necessary to exclude ectopic growth or abnormal proliferation. 
Adverse Event Management: Any serious adverse events, including high fever, 
widespread rash, respiratory distress, allergic reactions, or marked elevation of muscle 
enzymes, will be immediately reported to the research team and the ethics committee. 
5. Outcome Measures 
(1) Primary Outcome Measures 
1） Vital Signs Monitoring: Measurement of blood pressure, heart rate, respiratory rate, 
and body temperature. 
2） Local Adverse Reactions: Visual inspection of local reactions, including redness, 
swelling, induration, and infection at the injection site. 
3） Evaluation of Myocyte Graft Survival: Gross visual assessment of changes in graft 
volume. Ultrasonographic evaluation of graft morphology, echo uniformity, and the 
presence of necrosis or fluid accumulation. Assessment of blood perfusion to 
determine vascular regeneration and graft vascularization. 
4） Inflammatory and Immune Status: Complete blood count (CBC): total white blood 
cell count and differential percentages, with calculation of the neutrophil-to-
lymphocyte ratio (NLR). Inflammatory cytokines: IL-6, TNF-α, IFN-γ, and IL-1
β, quantified by enzyme-linked immunosorbent assay (ELISA). C-reactive protein 
(CRP): assessment of systemic inflammatory status. Anti-inflammatory cytokine: IL-
10, reflecting immune regulatory status. 
5）  Liver Enzymes and Metabolic Safety: Serum alanine aminotransferase (ALT), 
aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) will be analyzed 
to assess metabolic safety of the intervention. 
6） Skeletal Muscle Mass (DXA Assessment): Appendicular skeletal muscle mass (ASM) 
will be measured using dual-energy X-ray absorptiometry (DXA), and the skeletal 
muscle mass index (ASMI, kg/m²) will be calculated. 
7） Muscle Thickness (Muscle Ultrasonography): High-frequency ultrasonography will 
be used to measure the thickness of representative muscle groups (e.g., rectus 
femoris), defined as the distance from skin to periosteum. Each site will be measured 

 
 
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three times, and the mean value will be recorded. 
8） Circulating Myokines: Serum levels of FGF21, irisin, IGF-1, and BDNF will be 
measured to evaluate myokine secretion from the muscle graft.  
(2) Secondary Outcome Measures 
1） Bone mineral density of the lumbar spine and femur will be assessed using DXA or 
quantitative computed tomography (QCT). T-scores and trabecular microarchitecture 
parameters will be analyzed to evaluate improvements in bone metabolism. 
2）  Bone Turnover Markers: Serum levels of procollagen type I N-terminal propeptide 
(P1NP), C-terminal telopeptide of type I collagen (CTX), and alkaline phosphatase 
(ALP) will be measured to assess bone formation, bone resorption, and overall bone 
metabolic activity. 
3） Lipid Profile: Serum triglycerides (TG), total cholesterol (TC), high-density 
lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) 
will be measured to evaluate lipid metabolism and cardiovascular risk. 
4） Glucose Metabolism and Insulin Resistance: Fasting blood glucose (FBG) will be 
measured using venous blood samples. Fasting insulin (FINS) levels will also be 
assessed, and insulin resistance will be evaluated using the homeostasis model 
assessment of insulin resistance (HOMA-IR = FBG × FINS / 22.5). 
5） Energy Expenditure: Energy metabolism will be assessed using indirect calorimetry 
to measure the respiratory exchange ratio (RER) and resting metabolic rate (RMR). 
6） Body Fat Assessment: Whole-body DXA scanning will be performed to evaluate total 
body fat mass and fat distribution.  

 
 
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6. Safety and Efficacy Evaluation 
(1) Safety Evaluation 
Safety assessment in this study focuses on the standardized implementation and 
ethical compliance of autologous differentiated myocyte preparation, subcutaneous 
transplantation procedures, and post-intervention follow-up. The main components 
include the following:  
1) Adverse Event Monitoring: All participants will undergo systematic safety 
evaluations before, during, and after the intervention. Local complications related to 
subcutaneous transplantation (such as redness, swelling, induration, infection, or 
abnormal tissue growth), rare systemic adverse reactions (such as allergic reactions, 
high fever, or elevated creatine kinase levels), and serious adverse events (SAEs) 
requiring hospitalization, surgical intervention, or meeting criteria for study 
termination will be recorded. All adverse events will be graded according to the 
Common Terminology Criteria for Adverse Events (CTCAE), version 5.0, with 
reference to established safety benchmarks from international cell therapy studies 
2) Ethical Compliance: All participants will voluntarily sign written informed consent 
forms approved by an independent ethics committee. The target informed consent 
signing rate is 100%. The study protocol will be conducted in strict accordance with 
ethical standards and regulatory requirements.  
3) Data and Sample Privacy Protection: All biological samples will be managed using 
de-identified coding, with unique anonymous identifiers replacing personal 
information. Clinical data and biological samples will be stored separately with 
controlled access. The collection, storage, and analysis of samples and clinical data 
will strictly comply with relevant national regulations, including the Personal 
Information Protection Law of the People's Republic of China and the Regulations 
on the Administration of Human Genetic Resources. 
(2) Efficacy Evaluation 
The therapeutic effects of autologous differentiated myocyte graft transplantation 
will be comprehensively evaluated across three dimensions: functional improvement, 
biomarker validation, and systemic metabolic and inflammatory modulation.  
1） Functional Outcomes: Improvements in skeletal muscle mass (ASMI) and handgrip 

 
 
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strength (in participants with preserved upper limb function) will be used as primary 
clinical efficacy indicators. An intervention will be considered effective if, at 3 
and/or 6 months post-intervention, these indicators show a relative improvement of 
≥10% or recovery beyond the diagnostic thresholds defined by the Asian Working 
Group for Sarcopenia (AWGS). 
2） Biomarker Validation: Muscle-derived biomarkers, including MyoG, Pax7, and 
Myh1/4, will be assessed at predefined time points after intervention. Statistically 
significant changes compared with baseline and reference values will be used to 
support biological efficacy. 
3） Metabolic and Inflammatory Improvement: Within-subject pre–post comparisons 
will be conducted to evaluate changes in metabolic indicators, including resting 
metabolic rate (RMR), respiratory exchange ratio (RER), body fat proportion 
(DXA), and insulin resistance index (HOMA-IR). Concurrently, reductions in pro-
inflammatory markers (CRP, IL-6, TNF-α) and increases in the anti-inflammatory 
cytokine IL-10 will be evaluated as indicators of systemic immune modulation. 
7. Statistical Analysis and Quality Control 
(1) Statistical Analysis 
1） Between-group differences will be analyzed according to data type and distribution. 
Comparisons between two groups will be performed using the independent-samples 
t test for normally distributed data or the Mann–Whitney U test for non-normally 
distributed data. Categorical variables will be compared using the chi-square test or 
Fisher's exact test. Paired pre–post intervention data will be analyzed using the 
paired t-test or the Wilcoxon signed-rank test. 
2） Longitudinal follow-up data collected at multiple time points will be analyzed using 
repeated-measures analysis of variance (RM-ANOVA) or linear mixed-effects 
models (LMMs) to account for time effects and intervention effects. 
3） For analyses involving multiple biomarkers, such as inflammatory cytokines, false 
discovery rate (FDR) correction will be applied using the Benjamini–Hochberg 
method. Spearman or Pearson correlation analyses will be conducted to explore 
associations between these biomarkers and clinical outcomes. 

 
 
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4） All statistical tests will be two-sided. A P value < 0.05 will be considered statistically 
significant, and P < 0.01 will be considered highly significant. 
(2) Quality Control 
1） Standardization of data collection: All clinical measurements will be performed using 
standardized equipment and operating procedures (SOPs). Personnel will be trained 
before measurements. Key endpoint assessments will be conducted by the same 
testing center and, whenever possible, by the same personnel to ensure batch 
consistency. 
2） Double data entry and verification: All paper case report forms (CRFs) will be 
independently entered by two researchers and cross-checked. Built-in logical 
validation procedures will automatically flag discrepancies to ensure data accuracy. 
3） Participant compliance management: Dedicated follow-up records will be established 
to ensure timely sample collection and data completeness. At each follow-up visit, 
physicians will confirm completion of the intervention. Reminder messages and 
manual follow-up calls will be implemented as needed. 
4） Sample and data traceability system: Biological samples will be labeled with 
anonymous identifiers and stored separately from personal identifying information. 
The entire process of sample transport, processing, and cryopreservation will be fully 
documented, including time points, personnel, and conditions, to ensure closed-loop 
traceability. 
5） Adverse event documentation and review: All adverse events (AEs) and serious 
adverse events (SAEs) will be recorded in dedicated AE/SAE forms, graded, and 
signed by the principal investigator. SAEs will be reported to the ethics committee 
and monitoring authorities within 24 hours, and expert evaluation procedures will be 
initiated promptly. 
 
Eligibility Criteria, Withdrawal, and Study Termination Criteria 
1. Inclusion Criteria 
(1)General Conditions: 
1） Written informed consent obtained from the participant's immediate family member 

 
 
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or legal guardian, agreeing to undergo muscle biopsy. 
2） General health status adequate to permit subcutaneous transplantation procedures. 
(2) Patients With Long-Term Bed Rest–Induced Muscle Atrophy 
1) Age 18–80 years, with no restriction on sex. 
2) History of long-term bed rest: continuous bed rest for ≥4 weeks, with causes 
including neurological injury (such as brain death, stroke, or spinal cord injury), 
recovery after major orthopedic surgery, intensive care unit stay, or activity limitation 
due to chronic diseases. 
3) Evidence of muscle atrophy: a marked reduction in muscle volume or muscle strength 
confirmed by clinical assessment and imaging studies. According to dual-energy X-
ray absorptiometry (DXA), appendicular skeletal muscle mass index (ASM/height²) 
< 7.0 kg/m² in men and < 5.4 kg/m² in women, based on EWGSOP2 criteria. 
4) Written informed consent obtained from the participant's immediate family member 
or legal guardian, agreeing to undergo muscle biopsy, with general health status 
adequate to permit subcutaneous transplantation. 
5) Stable underlying medical conditions (APACHE II score 0–20), with no acute 
exacerbation or severe comorbidities that would contraindicate surgical or 
transplantation interventions. 
2. Exclusion Criteria 
1) History of malignant tumors. 
2) Coagulation disorders or current use of anticoagulant therapy. 
3) Active infection or immunodeficiency. 
4) Severe cardiac or renal insufficiency (estimated glomerular filtration rate < 60 
mL/min/1.73 m²; New York Heart Association [NYHA] class III–IV heart failure). 
5) Muscle-related diseases, including hereditary myopathies (such as muscular 
dystrophy) or acquired myositis (creatine kinase > 3 times the upper limit of normal). 
6) Severe local skin lesions or a history of allergic reactions at the injection site. 
7) Use of muscle growth–modulating medications (such as testosterone or growth 

 
 
16 
hormone) within the past 6 months. 
8) Long-term use of corticosteroids or immunosuppressive therapy, including anti-
rejection medications following organ transplantation. 
9) Other exclusion criteria: participation in other interventional clinical trials (excluding 
observational studies). 
10) Any other medical or ethical conditions deemed by the investigator to make the 
participant unsuitable for enrollment. 
3. Withdrawal and Study Termination Criteria 
(1) Participant or Family-Initiated Withdrawal 
1) Withdrawal of informed consent at any time. 
2) Inability to continue follow-up due to participant- or family-related reasons (e.g., 
relocation or scheduling conflicts). 
(2) Study Termination Criteria 
1) Medical reasons: occurrence of serious adverse events (SAEs), such as uncontrollable 
bleeding requiring surgical intervention after biopsy; newly identified contraindications 
(e.g., diagnosis of malignant tumor after enrollment); failure to obtain an adequate 
muscle tissue sample during biopsy; or inability to culture and obtain enough 
transplantable cells. 
2) Protocol deviations: failure to process samples according to standardized procedures 
or discovery that the participant did not meet inclusion criteria at enrollment. 
3) Investigator decision: occurrence of severe safety events beyond acceptable control 
related to the intervention, or termination required by the ethics committee. 
 
Management Plan for Common Adverse Events and Serious Adverse 
Events 
1. Definition and Grading of Adverse Events 
1) Common adverse events (AEs): Grade 1–2 according to CTCAE version 5.0, 
including local pain, mild bleeding, and transient fever (≤38.5 °C). 

 
 
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2) Serious adverse events (SAEs): Grade ≥3 according to CTCAE version 5.0, 
including infections requiring hospitalization, anaphylactic shock, deep vein 
thrombosis, and other severe conditions. 
2. Management Plan for Common Adverse Events 
Type of Adverse 
Event 
Management Measures 
Responsible 
Personnel 
Local 
skin 
reactions 
1. Assess pain intensity (VAS score);2. Oral 
nonsteroidal 
anti-inflammatory 
drugs 
(ibuprofen 400 mg, q8h, ≤3 days);3. Local cold 
compress (15 min per session, every 2 hours). 
Surgeon 
Local hematoma 
1. Compression bandaging (elastic bandage for 
24 hours);2. Ultrasonographic evaluation of 
hematoma extent (if diameter >5 cm);3. 
Discontinue anticoagulant medications until 
hematoma resolution. 
Surgeon 
Superficial 
infection 
1. 
Bacterial 
culture 
and 
antimicrobial 
susceptibility 
testing;2. 
Empirical 
oral 
cefuroxime axetil 500 mg twice daily (in 
patients without allergy);3. Daily wound 
dressing until healing. 
Infectious 
disease 
consultation 
Low-grade fever 
(≤38.5 °C) 
1. Complete blood count and CRP testing;2. 
Physical cooling (tepid sponging);3. Monitor 
body temperature every 4 hours for 48 hours. 
Surgeon 
Mild 
allergic 
reaction 
(rash/pruritus) 
1. Discontinue suspected agents or materials;2. 
Oral antihistamines (e.g., loratadine 10 mg once 
daily for 3 days);3. Continuous observation of 
symptom progression. 
Surgeon 
Injection-site 
induration 
1. Evaluate the nature of induration; 2. Local 
warm compress to promote absorption; 3. 
Weekly 
reassessment; 
ultrasonography 
if 
necessary. 
Surgeon 
 

 
 
18 
3. Management Plan for Serious Adverse Events 
Type of SAE 
Emergency Management Procedures 
Reporting 
Timeline 
Active bleeding 
1. Immediate local compression for 
hemostasis (≥15 min);2. Intravenous 
tranexamic acid 1 g (within 30 min);3. 
Vascular interventional hemostasis is 
ineffective. 
Immediate 
(report 
to 
ethics 
committee 
within 
one 
hour) 
Anaphylactic shock 
1. Intramuscular epinephrine 0.3 mg 
(lateral thigh);2. Establish intravenous 
access (rapid infusion of 500 mL normal 
saline);3. Cardiac monitoring and transfer 
to the ICU. 
Immediate 
(activate 
in-
hospital 
emergency 
code) 
Deep vein thrombosis 
(DVT) 
1. Confirm diagnosis by lower-limb 
vascular 
ultrasonography;2. 
Low-
molecular-weight heparin (enoxaparin 1 
mg/kg q12h);3. Avoid massage of the 
affected limb. 
Within 
24 
hours 
Sepsis 
1. Blood cultures and broad-spectrum 
antibiotics (meropenem 1 g q8h);2. Fluid 
resuscitation (30 mL/kg crystalloid);3. 
Transfer to infectious disease intensive 
care. 
Immediate 
(written 
report within 
6 hours) 
Abnormal growth 
1. Ultrasonography or soft-tissue MRI to 
define structure;2. If abnormal nodular 
structure, active proliferation, or recurrent 
growth is observed, classify as an adverse 
outcome endpoint;3. Surgical excision 
with immunohistochemistry and genetic 
testing to exclude tumorigenic risk. 
Immediate 
(written 
report within 
6 hours) 
Muscle 
necrosis/rhabdomyolysis 
1. Discontinue intervention;2. Intravenous 
fluid resuscitation (0.9% saline or lactated 
Ringer's solution) to maintain urine output 
≥200 mL/h;3. Monitor renal function to 
prevent acute kidney injury (AKI). 
Immediate 
(written 
report within 
6 hours) 
Autoimmune 
response 
activation 
1. 
Rheumatology 
consultation;2. 
Serological testing;3. Evaluate the need 
for immunomodulatory intervention. 
Within 
24 
hours 

 
 
19 
Type of SAE 
Emergency Management Procedures 
Reporting 
Timeline 
Multifocal injection-site 
infection 
1. Culture and broad-spectrum antibiotics 
(meropenem 1 g q8h);2. Debridement and 
culture;3. 
Evaluate 
suspension 
of 
subsequent injections. 
Immediate 
(written 
report within 
6 hours) 
4. Insurance of Plan Implementation 
(1) Emergency Resource Allocation: Emergency kits will be readily available in the 
biopsy procedure room, including epinephrine, tranexamic acid, and sterile dressings.  A 
24-hour on-call emergency response team will be maintained. 
(2) Personnel Training: Quarterly simulation drills for emergency scenarios (e.g., 
bleeding and allergic reactions). All study personnel will hold valid Basic Life Support 
(BLS) certification.) 
(3) Process Coordination: Upon occurrence of an SAE, the principal investigator will 
complete the Serious Adverse Event Report Form and simultaneously submit it to the 
ethics committee and regulatory authorities. Follow-up reports will be submitted every 
72 hours until the event is resolved or stabilized. 
5. Documentation and Traceability 
(1) AE/SAE documentation: Records will include time of occurrence, management 
measures, and outcomes (recovery, sequelae, or death). 
(2) Sample-related causality analysis: If an SAE is deemed possibly or more likely 
related to the study procedures, similar interventions will be suspended until completion 
of a root cause analysis (RCA). 
(3) Ethical archiving: All SAE-related documents will be retained for five years after 
study completion. AE/SAE.