Mesenchymal stromal cells and their exosomes as acute therapeutic interventions for traumatic brain injury in pre‑clinical studies: A systematic review and meta‑analysis

Introduction

Traumatic brain injury (TBI) remains a leading cause of mortality and long-term disability worldwide, particularly among young adults and military personnel (1,2). In 2019, an estimated 27-69 million new cases occurred globally, with the highest burden in low- and middle-income countries (3). Acute TBI initiates primary mechanical injury followed by secondary cascades including neuroinflammation, blood-brain barrier disruption, excitotoxicity, oxidative stress and progressive neuronal loss (4,5). Despite extensive research, no pharmacological agent has yet proven unequivocal efficacy in large phase III trials, leaving supportive care and surgery as standard management (6,7).

Mesenchymal stromal cells (MSCs) and their secreted exosomes have emerged as promising regenerative and immunomodulatory therapeutic candidates for acute TBI (8,9). MSCs exert pleiotropic effects primarily through paracrine mechanisms (anti-inflammatory cytokines, neurotrophic factors and extracellular vesicles) (10,11). Exosomes, small membrane-bound vesicles (30-150 nm) containing proteins, lipids and nucleic acids, can cross the blood-brain barrier, modulate microglial polarization, reduce apoptosis, promote angiogenesis and enhance endogenous repair processes without the risks associated with cell engraftment (12-15).

Pre-clinical rodent studies using controlled cortical impact or fluid percussion models have consistently demonstrated that MSCs and MSC-derived exosomes, administered ≤7 days post-injury, significantly reduce lesion volume and cerebral edema, while preserving neurological function and improving cognitive outcomes (13,16,17). These beneficial effects appear to be mediated predominantly by immunomodulation and restoration of blood-brain barrier integrity (13,18,19). Early-phase clinical trials evaluating intravenous (IV) or intracerebroventricular (ICV) MSC administration in patients with severe TBI have reported preliminary safety and signals of efficacy (20,21). However, the clinical translation of exosome-based therapy remains in its infancy, with no registered trials specifically addressing acute TBI as of 2024, at least to the best of our knowledge (22).

Despite the growing body of evidence, considerable heterogeneity exists regarding cell/exosome source, dose, timing, route of administration and outcome measures across studies. Furthermore, systematic synthesis comparing the relative efficacy and safety of whole MSCs vs. their exosomes in acute TBI has not yet been performed, at least to the best of our knowledge. Such an analysis is critical to guide clinical trial design and regulatory decision-making.

Therefore, the objectives of the present systematic review and meta-analysis were the following: i) To comprehensively evaluate the efficacy of MSCs and MSC-derived exosomes administered in the acute phase following TBI in pre-clinical (animal) studies; ii) to quantitatively compare neurological, histopathological and biochemical outcomes between MSC and MSC-derived cell-free-based interventions; and iii) to identify optimal therapeutic parameters and knowledge gaps for future translational research.

Data and methods

International prospective register of systematic reviews (PROSPERO) registration

The present systematic review was prospectively registered in PROSPERO (CRD420251236846) under the title ‘Mesenchymal Stromal Cells and MSC-Based Cell-Free as Acute Traumatic Brain Injury Therapy: Systematic Review and Meta-Analysis of preclinical studies’. The protocol is publicly available at https://www.crd.york.ac.uk/PROSPERO/view/CRD420251236846. All methods were pre-defined in the registered protocol to minimize reporting bias and ensure methodological transparency. Any deviations from the protocol will be clearly documented in the final publication.

Literature search strategy

A systematic literature search was conducted on November 21, 2024 across four major electronic databases: PubMed, MEDLINE, Scopus and Cochrane Library. The search combined controlled vocabulary (MeSH) and free-text keywords with Boolean operators. The search utilized a combination of MeSH terms and free-text keywords as demonstrated in Table I.

Table I Key word search strategy and strings.

Table I

Key word search strategy and strings.

Database	Search term
Pubmed	(‘Brain Injuries, Traumatic’ OR ‘traumatic brain injury’ OR TBI) AND (‘Mesenchymal Stem Cells’ OR ‘mesenchymal stromal cells’ OR MSC OR ‘Exosomes’ OR ‘extracellular vesicles’ OR ‘conditioned medium’) AND (acute OR ‘acute phase’ OR ‘early administration’ OR ‘7 days’OR ‘≤7 days’)
MEDLINE	(‘Brain Injuries, Traumatic’ OR ‘traumatic brain injury’ OR TBI) AND (‘Mesenchymal Stem Cells’ OR ‘mesenchymal stromal cells’ OR MSC OR ‘Exosomes’ OR ‘extracellular vesicles’ OR ‘conditioned medium’) AND (acute OR ‘acute phase’ OR ‘early administration’ OR ‘7 days’OR ‘≤7 days’)
Scopus	TITLE-ABS-KEY (‘traumatic brain injury’ OR TBI) AND (‘mesenchymal stem cells’ OR ‘mesenchymal stromal cells’ OR msc OR exosomes OR ‘extracellular vesicles’ OR ‘conditioned medium’) AND (acute OR ‘acute phase’ OR ‘early administration’ OR ‘within 7 days’ OR ‘≤7 days’)
Cochrane Library	(‘Brain Injuries, Traumatic’ OR ‘traumatic brain injury’ OR TBI) AND (‘Mesenchymal Stem Cells’ OR ‘mesenchymal stromal cells’ OR MSC OR ‘Exosomes’ OR ‘extracellular vesicles’) AND (acute OR ‘acute phase’ OR ‘early administration’)

To ensure comprehensive coverage, reference lists of retrieved articles were manually screened through backward and forward citation searching for additional relevant studies. To capture contemporary evidence, the search was restricted to studies published over the past decade (January 1, 2015 to the search date) with no language restrictions. The review was written and published in English.

Inclusion and exclusion criteria

Studies were included if they met all of the following: Investigated MSCs and/or MSC-derived exosomes (MSC-derived cell-free products, conditioned medium) administered ≤7 days post-injury (defined as the acute phase, aligned with the secondary injury cascade in TBI); pre-clinical in vivo animal models of TBI (any severity or model); and reported quantitative data for at least one pre-defined efficacy outcome (Fig. 1). Comparators comprised vehicle, sham, saline, or standard care. Both randomized and non-randomized study designs were included. The timing of administration was extracted from the Methods section of each study and independently verified by two reviewers (DRA and AP) to ensure compliance with the ≤7-day criterion. Articles not meeting these criteria were excluded (non-traumatic brain injury models, in vitro-only studies and reviews).

Figure 1 Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram of the study selection process for the present systematic review.

Screening and selection process

The initial search yielded a broad range of articles, which were filtered based on publication year, relevancy and study type. Of note, two reviewers (C and TS) independently screened the titles and abstracts of all records identified through the systematic search and manual reference checking. Full-text reports of any record considered potentially relevant by at least one reviewer (C) were retrieved and independently evaluated for inclusion by the same two reviewers (DRA and AP). Any discrepancies between reviewers (DRA and AP) during title/abstract screening or full-text assessment were first resolved by discussion. Persistent disagreements were adjudicated by a third reviewer (C). Exclusions at the full-text stage were categorized and recorded. A PRISMA flow diagram was created to illustrate the study selection process (Fig. 1).

Data extraction and synthesis

Selected articles with key data were systematically extracted, including the title and authors of the articles, year of publication, study objectives and methodology, sample size and study population (animal species demographics), intervention details (MSC and/or exosome source, route of administration), comparators, and key findings related to efficacy and safety outcomes. Primary outcomes were the following: i) Neurological function: Assessed by the modified neurological severity score (mNSS; score 0-18, lower scores indicate better function); ii) cognitive performance: Evaluated using the Morris water maze (MWM) test (typically reported as mean escape latency in seconds or percentage time spent in the target quadrant); iii) histopathological outcome: Lesion volume (mm3), hematoxylin and eosin staining or magnetic resonance imaging (MRI).

Statistical analysis

Data were extracted independently by two reviewers (C and TS) using a standardized Excel form. Disagreements were resolved by discussion or a third reviewer (C). Extracted items included study details, intervention characteristics, sample sizes and results. Continuous outcomes data were used directly or converted as the mean and SD. Graphs were digitized using WebPlotDigitizer (v4.5) when needed and sensitivity analyses tested earliest or all time points. Data synthesis was conducted using RevMan Web, aggregating quantitative data for the meta-analysis where applicable, using random-effects models. Heterogeneity was assessed with I². Authors were contacted to provide any required data not available in published reports.

Data analysis and interpretation

Extracted data were analyzed to identify recurring themes, magnitude of treatment effects, optimal therapeutic parameters and research gaps. Similarities and differences across pre-clinical and clinical studies were evaluated to assess the translational potential of MSCs and/or MSC-derived exosomes administered within 7 days following TBI. Meta-analytic techniques included the calculation of pooled effect sizes with 95% confidence intervals (CIs) for continuous outcomes (standardized mean difference) and dichotomous outcomes (risk ratio), with heterogeneity assessed using the I2 statistic. Subgroup analyses based on study type, intervention (MSC vs. exosome), timing, dose, route and TBI severity, along with funnel plot assessments for publication bias, were conducted to strengthen the validity of conclusions. The certainty of findings was assessed using the Cochrane RoB Tools 2.0 checklist to assess the risk of bias within the studies included in the present systematic review. The assessment was integrated into the systematic review and meta-analysis process, which adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocols and The Cochrane Handbook. This approach ensures a rigorous evaluation of the evidence, aligning with the standards of high-quality systematic reviews that employ established criteria to determine confidence in the body of evidence for key outcomes.

Risk of bias and certainty of evidence

Risk of bias was assessed independently by two reviewers (C and TS) using SYRCLE's tool for animal studies. Domains were rated low, high, or unclear risk. Disagreements were resolved by consensus or a third reviewer (C). Certainty for primary outcomes (mNSS, MWM, lesion volume) was evaluated using an adapted GRADE approach for pre-clinical studies and the results are shown in Table SI. Beginning from ‘high’, downgrades were applied for risk of bias (SYRCLE), inconsistency (high I2), indirectness, imprecision and publication bias (funnel plots/Egger's test).

Results

A total of 118,874 records were identified through database searching (Scopus, PubMed, Medline, and Cochrane Library). The PRISMA diagram of the study selection process is illustrated in Fig. 1. Duplicate entries were removed, titles and abstracts were screened, and records irrelevant to MSC, MSC-derived cell-free therapy, or pre-clinical TBI models were excluded; additional exclusions were due to unavailable full texts and failure to meet the inclusion criteria. Ultimately, 46 studies were included in the quantitative synthesis (Fig. 1).

Within this final set of studies, 27 pre-clinical studies that evaluated MSC-based cellular therapies in animal models of TBI were identified; these are presented in Table II (23-49). In addition, 19 pre-clinical studies that investigated MSC-derived cell-free products (such as exosomes or conditioned medium) were identified; these are presented in Table III (12,13,50-66). On the whole, these pre-clinical studies provide the core data for comparing the efficacy of whole-cell MSC therapy and MSC-derived cell-free approaches on functional outcomes, lesion volume, and relevant histopathological and molecular markers.

Table II MSC therapy in models of TBI.

Table II

MSC therapy in models of TBI.

Study no.	First author, year of publication	Animal	Pre-condition/Model	Treatment	Results	Adverse effects	(Refs.)
1	Fu, 2015	Rats, 5 groups: Sham; TBI; TBI + BMSC; TBI + OEC; TBI + BMSC + OEC (co-graft), (n=16 per group)	Moderate TBI, weight-drop contusion over right parietal cortex	Acute intracerebral injections of BMSC and/or OEC around the lesion	BMSC, OEC and co-graft significantly reduced NSS, increased NeuN+ neuronal counts and GAP-43+ axonal sprouting, decreased GFAP+ reactive astrocytes vs. TBI alone; co-graft showed greatest neuronal preservation (all P<0.05).	NR	(23)
2	Silachev, 2015	Rats, Sham n=6; TBI + saline IV n=11; TBI + MSC IV n=9; TBI+MSC intra-arterial (IA) n=8	Open-skull weight-drop TBI to left sensorimotor cortex	Single dose 1.5x10⁶ MSC 24 h after TBI via jugular vein (IV) or internal carotid artery (IA); saline IV control	Intra-arterial and intravenous MSC reduced MRI lesion volume (trend), significantly improved limb-placing scores (IA > IV from day 4) and reduced forelimb asymmetry in cylinder test vs. saline (all P<0.05).	NR	(24)
3	Turtzo, 2015	Rats, Total 177 rats; 4 group, rMSC IV vs. saline (total n=34), rMSC IC vs. saline (total n=24), hMSC IV vs. saline (total n=24), hMSC IC vs. saline (total n=28)	Focal controlled cortical impact (CCI) to left frontal motor cortex	rMSC or hMSC 5x106 IV on days 3, 5, and 7 post-TBI; or 1x106 IC 15 min after TBI; all compared with saline	hMSC IV showed no significant reduction in MRI lesion volume or hemispheric loss at days 2-30, no improvement in NSS-R or foot-fault test vs. saline, and <0.0005% cells homed to brain (most trapped in lung/spleen).	No increase in mortality or specific toxicities attributed to MSC; hemorrhagic MRI changes reflect expected CCI evolution in all groups.	(25)
4	Kota, 2016	Rats, 207 male Sprague-Dawley rats divided across sub-cohorts; MWM sub-study: 5 groups (Sham, CCI, CCI + Pro, CCI + MSC, CCI + Pro + MSC) with n=6 per group; BBB cohort n=71.	Moderate-severe CCI to right temporoparietal cortex	Propranolol 10 mg/kg IP at 1 h after CCI; MSC 107 cells/kg IV at 72 h; groups: CCI only, CCI + Pro, CCI + MSC, CCI + Pr o+ MSC, Sham	Progesterone significantly reduced brain edema at 24 h and BBB permeability at 96 h; MSC and Pro+MSC increased DCX+ neurogenesis at day 7 vs. CCI alone (P<0.05); no improvement in MWM learning at day 120.	A total of 3 rats died acutely from the primary CCI and were excluded; no additional mortality of MSC treatment.	(26)
5	Shen, 2016	Rats, 3 groups: Sham; TBI; TBI + BMSC (n=16/group)	Moderate-severe TBI with modified Feeney CCI to right temporoparietal cortex	Acute stereotactic intracerebral BMSC (GFP-labeled) injections into pericontusional cortex at 4 sites immediately after TBI; all animals received cyclosporine A	BMSC transplantation significantly improved NSS at days 7 (4.4±1.3 vs. 5.2±1.2) and 14 (2.6±1.2 vs. 4.1±0.8), increased GDNF expression, reduced TUNEL-positive neurons, and enhanced GAP-43 and synaptophysin levels vs. TBI alone (all P<0.05).	NR	(27)
6	Danilina, 2017	Rat TBI model: Sham; TBI; TBI + BMSC (N=19)	TBI rat model; MSCs preconditioned with proinflammatory factors (e.g., LPS, leukocytes) before transplantation	MSCs with or without inflammatory preconditioning transplanted after TBI	Lesion volume: smaller lesion size; inflamed-MSC group shows greatest reduction. Neurological outcome: better functional scores; inflammatory preconditioning further improves neurological outcome; exact NSS). Cytokines (IL-1α, IL-6, TNF-α) and MMP-2/9 inflammatory priming significantly increases secretion of these factors.	NR	(28)
7	Feng, 2017	105 rats; 3 groups (Sham, TBI, TBI+ BMSC), (N=35/group)	Weight-drop TBI to right parietal cortex	IV BMSC (3x106 cells in 1 ml) via tail vein 30 min after TBI	Rotarod: TBI + BMSC vs. TBI (significantly longer fall-latency at post-injury time points; P<0.05. mNSS: TBI+BMSC vs. TBI (significantly lower deficit scores at post-injury time points; P<0.05). Viable neuron counts significantly increases surviving neurons compared with TBI (P<0.05). Donor-derived neurons (SRY+/NeuN+): more double-positive cells in BMSC group than TBI (P<0.05).	NR	(29)
8	Guo, 2017	60 male C57BL/6 mice; 4 groups: Control, Sham, TBI, BMSC (n=15/group)	Controlled cortical impact TBI in mice (4-mm craniotomy, 20 mg weight, 50 cm height, 1 mm deformation)	IV tail-vein injection of BMSCs (2x106 cells) after TBI (single dose)	BMSC significantly reduced NSS and MWM escape latency, decreased TUNEL+ and caspase-3+ cells, upregulated VEGF/Ang-1 expression, and increased microvessel density (CD34+) vs. TBI alone (all P<0.05).	NR	(30)
9	Li, 2017	30 male Wistar rats (300-350 g): 4 main TBI groups - saline 6 h (n=5), saline 1 week (n=5), hMSC 6 h (n=10), hMSC 1 week (n=10) after TBI	Controlled cortical impact (CCI) TBI	IV injection of 1 ml saline or ~3x106 human BMSCs at either 6 h or 1 week post-TBI; all animals underwent serial MRI and behavioral testing up to 3 weeks	hMSC (particularly 6 h post-TBI) accelerated sensorimotor recovery and enhanced peri-lesional white matter reorganization with increased FA and entropy along lesion boundary vs. later administration and control groups.	NR	(31)
10	Bonilla Horcajo, 2018	Severe TBI rat model; four groups established 2 months post-TBI: saline; PRP scaffolds (PRPS) alone; MSCs in saline; MSCs in PRPS; exact number of rats per group not reported in accessible abstract	Severe TBI induced by weight-drop impact to right cerebral hemisphere	At 2 months post-TBI (chronic phase), intralesional transplantation through a small burr hole into the brain lesion cavity: saline; PRPS alone; MSCs in saline; MSCs embedded in PRPS; animals followed for 12 months	Functional outcome: greatest and most sustained functional improvement observed in the MSCs + PRPS group over 12-month follow-up; MSCs alone and PRPS alone produced smaller benefits. Chronic brain damage: PRPS enhanced the benefit of delayed MSC therapy in established severe TBI	NR	(32)
11	Hu, 2018	50 male SD rats total; EG (BM-MSC); CG (saline) (n=25/group)	Craniocerebral injury (CI) induced by free-fall weight (20 g from 15 cm) onto exposed dura over right parietal lobe (3-mm craniotomy)	EG: intraventricular injection of BM-MSCs 12 h after CI (10 µl, 1x104 cells/rat) into left lateral ventricle; CG: saline injection (route stated as intraperitoneal)	EPCs, cells/200,000 mononuclear cells, n=12/group. CD31 at 7 days, EG vs. CG (40.10±3.00 vs. 31. 23±2.38; P<0.05). NSE: at 7 days, EG vs. CG (44.29±5.29 vs. 30. 03±4.88; P<0.05). Microvessel density: 7 days, EG vs. CG (79.38±2.00 vs. 55.51±3.89; P<0.05); 3 days, EG vs. CG (65.45±3.02 vs. 53.57±2.33; P<0.05). mNSS: reduced.	NR	(33)
12	Shi, 2018	TBI mice divided into groups receiving SOD2-overexpressing BM-MSCs, BM-MSCs, or vehicle; plus in vitro BM-MSC cultures), (n=NA)	Traumatic brain injury mouse model (details in full text; abstract specifies ipsilateral cortex and BBB assessment)	BM-MSCs genetically modified to over-express SOD2, transplanted intravenously after TBI; comparator groups included unmodified BM-MSCs and non-MSC controls	SOD2-overexpressing BM-MSCs vs. unmodified BM-MSCs vs. TBI control: reduced apoptosis (in vitro), attenuated neuroinflammation, increased SOD and glutathione, decreased MDA, preserved BBB integrity, and improved rotarod performance (all significant).	NR	(34)
13	Hao, 2019	Rats, 26 adult SD rats total: 2 donors for BMSCs; 24 rats with TBI divided into 4 groups: BMSC-Sox2, BMSC, Sox2, PBS (control), (n=8/group)	Controlled cortical injury (CCI) TBI: 1.0-cm craniotomy lateral to sagittal suture; 20-mg steel rod (4-mm diameter) dropped from 30 cm to produce standardized parietal contusion	BMSCs transduced with lentiviral Sox2 (BMSC-Sox2) vs. BMSC alone vs. Sox2 lentivirus alone vs. PBS. Seven days after TBI, 50 µl of BMSC-Sox2, BMSC, Sox2, or PBS injected into injured brain area using Hamilton syringe (cell density 1x105 cells/µl for BMSC-containing groups)	Sox2-BMSCs enhanced neuronal differentiation in vitro (Tuj1+/GFP+, neuron-like morphology) and provided greatest motor recovery at day 7 (NSS: 3.35±0.40 vs. BMSC 4.01±0.50 vs. Sox2 4.97±0.29 vs. PBS 6.26±0.36; (P<0.05)	NR	(35)
14	Hu, 2019	Male Sprague-Dawley rats (200-240 g) randomly assigned to: Sham, TBI, TBI + vehicle (20% DMSO), TBI + MDL28170, TBI+ GFP-BMSCs, TBI + MDL28170 + GFP-BMSCs;	Focal TBI induced by weight-drop onto right parietal cortex after 5 mm craniotomy (50 g hammer, 30 cm drop); sham rats underwent craniotomy without impact.	MDL28170 (calpain inhibitor) 50 mM, 1 µl intracranial injection at lesion center, 30 min post-TBI; GFP-BMSCs locally transplanted into lesion site 24 h post-TBI; groups: TBI only, vehicle, MDL28170 only, BMSCs only, MDL28170 + BMSCs.	MDL28170 + BMSCs significantly reduced pro-inflammatory cytokines, microglial activation, and lesion volume; increased BMSC survival/proliferation and IL-10; produced greater mNSS improvement at days 7 and 14 compared with BMSCs alone or MDL28170 alone (all P<0.05).	NR	(36)
15	Peruzzaro, 2019	39 male Sprague-Dawley rats: TBI + vehicle (HBSS) n=10, TBI+MSCs n=10, TBI+MSCs+ IL-10 n=9, Sham + vehicle n=10; subset used for histology: TBI + vehicle n=7, TBI + MSCs n=7, TBI+MSCs+IL-10 n=6, Sham + vehicle n=7.	CCI to medial frontal cortex (6 mm craniotomy, 3 mm anterior to bregma; depth 2.5 mm, velocity 2.25 m/sec, duration 0.5 sec); sham rats received scalp incision without impact. Rats pair-housed with standard conditions.	At 36 h post-TBI: stereotaxic bilateral transplantation into perilesional cortex. Four injection sites per rat (AP +3.0, ML ± 3.5, DV -3.0 and -1.5 mm); each site 2 µl containing 100,000 MSCs/µl (total 8 µl; 800,000 cells/rat). Groups: TBI + vehicle (HBSS only), TBI + MSCs (GFP-MSC), TBI + MSCs+IL-10 (MSCs engineered via lentivirus to overexpress IL-10), Sham + vehicle (HBSS). Behavioral tests up to 3 weeks (MWM, ladder rung, rotarod).	IL-10-overexpressing MSCs reduced GFAP+ astrocytes, CD86+ microglia, TNF-α, and shifted microglia to CD163+ phenotype, improved ladder rung motor coordination, and provided partial cognitive benefit in MWM reversal vs. MSCs alone and vehicle (P<0.05)	Histology showed sparse GFP/Hoechst-labeled transplanted cells near injection tracks, but no evidence of tumorigenesis or overt toxicity	(37)
16	Wu, 2019	C57BL/6 mice, randomly divided into 5 groups: Sham; TBI only; TBI + GFP-BMSCs; TBI + GFP-NT3-BMSCs; TBI + GFP-NT3P75-2-BMSCs. Behavioral tests: mNSS and rotarod. Brain edema evaluation: Immuno-fluorescence (Iba1, GFAP, GFP): (N= 10 per group)	Mouse CCI TBI model: impact velocity 4 m/sec, depth 2.0 mm, diameter 3 mm over right hemisphere (AP -5 mm, ML + 5 mm from bregma).	At 24 h post-TBI, GFP-labelled BMSCs (GFP only, GFP-NT3, or GFP-NT3P75-2) were locally transplanted into the lesion core. Groups: Sham (no TBI, no cells); TBI (no treatment); TBI + GFP-BMSCs; TBI+GFP-NT3-BMSCs; TBI + GFP-NT3P75-2-BMSCs.	NT3P75-2-BMSCs significantly increased graft survival, reduced brain edema and lesion volume, improved mNSS and rotarod performance, decreased microglial/astrocyte activation, and inhibited P75/JNK/Bax-mediated apoptosis vs. GFP-BMSCs and GFP-NT3-BMSCs (all P<0.05).	NR	(38)
17	Yan, 2019	Wistar rats,Total: 44 adult male (250 g) used for TBI model and treatment allocation. Animals were divided into 4 groups: A: TBI + immunosuppressor + BMSCs/scaffold; B: TBI + BMSCs/scaffold; C: TBI + BMSCs stereotactic injection; D: (n=8/group)	Rat TBI model based on Feeney's free-fall method: 10-mm craniotomy adjacent to sagittal suture between lambda and bregma, intact dura; weight-drop impact produced cortical contusion; all rats anesthetized with 10% chloral hydrate.	Groups A and B: at 72 h post-TBI, collagen-chitosan porous scaffold seeded with BrdU-labelled BMSCs (2x106 cells/µl, cultured 48 h) was placed directly into the lesion cavity (A with immuno-suppressor, B without). Group C: single stereotactic injection of BMSCs (no scaffold). Group D: TBI model only (no cells, no scaffold). All animals underwent behavioral testing (mNSS, Morris Water Maze) and histology (HE, BrdU/NSE, BrdU/GFAP, VEGF IHC).	BMSC/scaffold transplantation reduced neuronal necrosis, promoted neurogenesis (↑BrdU/NSE+ and BrdU/GFAP+ cells at days 7-14), increased VEGF expression in hippocampus, and significantly improved mNSS recovery and MWM spatial learning/memory (shorter escape latency) vs. BMSC injection alone or vehicle (all superior in scaffold groups).	NR	(39)
18	Huang, 2021	C57BL/6 male mice (15-20 g, SPF). Mice were randomly assigned to 4 groups: TBI + PBS; TBI+ BMMSCs; TBI + BMMSCs-shLuci, (n=8)	Mouse CCI TBI model: 4-mm diameter craniotomy over right parietal cortex; maximum depression depth 3 mm at impact velocity 4 m/s, 90° angle; impact site 2.5 mm lateral to sagittal suture and 3.5 mm posterior to bregma.	Model ischemic stress; transduced with shRNA lentiviruses targeting Rac1, p22-phox, p47-phox, or p67-phox. In vivo: at 24 h post-TBI, 5 µl cell suspension (5x105 BMMSCs) was injected into lesion center	Rac1-silenced BMMSCs strongly reduced OGD-induced ROS in vitro, significantly improved mNSS and rotarod performance days 3-21, decreased apoptosis/TUNEL+ cells and oxidative stress, upregulated VEGFA/MMP-2, and provided superior neuroprotection vs. non-silenced BMMSCs and PBS (all P<0.05).	NR	(40)
19	Song, 2020	Adult Sprague Dawley rats: Sham, TBI, TBI + MH, TBI + BMSCs, TBI + MH + tsBMSCs (n=25/group)	Adult Sprague-Dawley rats with severe TBI; groups: sham, TBI, TBI + mild hypothermia (MH), TBI + BMSCs, TBI + MH + tsBMSCs	Combination of mild hypothermia and temperature-sensitive BMSC transplantation after severe TBI vs. TBI alone and single-treatment groups (MH or BMSCs).	mNSS and brain edema: TBI + MH + tsBMSCs vs. TBI (significantly lower mNSS and brain water content at measured time points, P<0.05; better than single MH or BMSC). Serum biomarkers: S100β, NSE, LDH, CK and blood glucose significantly reduced vs. TBI and single-treatment groups. TUNEL: lowest apoptotic index in combination group. GLUT-3 expression modulated toward sham levels compared with TBI.	NR	(41)
20	Yuan, 2020	Adult male C57BL/6J mice, (H-BMSC group) (n=8/group)	Adult male C57BL/6J mice with CCI-induced TBI; groups: sham, control (TBI + DMEM), normoxic BMSCs (N-BMSC), hypoxic BMSCs (H-BMSC); n=8 per group.	Hypoxia-preconditioned BMSCs (1% O2 for 8 h) transplanted 24 h after TBI vs. DMEM vehicle; normoxic BMSCs as comparator.	Hypoxia-preconditioned BMSCs (H-BMSC) significantly improved wire-hanging, grid-walking, cylinder and MWM performance (escape latency 18 vs. 28 sec N-BMSC vs. >55 sec control), reduced lesion volume, enhanced remyelination (↑MBP/NF200, ↑NG2+/BrdU+ OPCs and APC+/BrdU+ oligodendrocytes), and activated mTOR/HIF-1α/VEGF pathway vs. normoxic BMSCs and control (p	NR	(42)
21	Li, 2021)	C57BL/6 mouse: Sham, TBI, TBI + GH/MSC (n=6/group)	C57BL/6 mouse moderate TBI model (controlled cortical impact) with cortical lesion and econdary ischemia, oxidative stress, and neuroinflammation.	Injectable dual-enzymatically cross-linked gelatin hydrogel (HRP + ChOx) used as 3D scaffold to load BMSCs; in vivo implantation of BMSC-laden soft GH into TBI lesion cavity vs. BMSC alone, GH alone, and TBI only.	GH/BMSC implants significantly increased BMSC viability, neural differentiation, and neurotrophin secretion in vitro; reduced lesion volume, inflammation, and neuronal apoptosis; increased endogenous neural cell survival/proliferation; and improved mNSS and MWM performance (shorter escape latency, better spatial learning/memory) vs. TBI and GH-only groups (P<0.05).	NR	(43)
22	Deng, 2021	mice: Sham, TBI BMMSC-shRac1 group) (n=8/group)	Controlled cortical impact TBI in mice	Intravenous transplantation 24 h post-TBI of BMMSCs with Rac1 knockdown (BMMSCs-shRac1), compared with BMMSCs, BMMSCs-shLuci, or PBS	mNSS: BMMSCs-shRac1 vs. PBS (lower scores from day 3-21 post-TBI, P<0.01); rotarod: longer latency to fall vs. PBS and BMMSCs-shLuci (P<0.01); lesion volume ratio: BMMSCs-shRac1 vs. PBS (smaller cavity at day 21, P<0.05); brain water content: reduced vs. PBS at days 7 and 21 (P<0.05); BMMSC-shRac1 enhanced migration to lesion site and increased neuronal survival markers vs. control groups	NR	(44)
23	Ma, 2021	Rats: Sham, TBI, NR (BMSCs/SA/Col/SDF-1 (n=8/group)	Rat TBI model with lesion cavity and hostile micro-environment (oxidative stress, inflammation, poor cell survival).	Sodium alginate/collagen type I hydrogel incorporating SDF-1 (SA/Col/SDF-1) loaded with BMSCs, implanted into TBI lesion; sustained SDF-1 release and SDF-1/CXCR4 activation.	SA/Col/SDF-1 scaffold + BMSCs significantly improved motor/cognitive function, reduced anxiety/depressive-like behavior, esion size, neuronal death and lneuroinflammation, enhanced BMSC recruitment and neuro-genesis via SDF-1/CXCR4-FAK/PI3K/AKT pathway vs. other groups (P<0.05).	NR	(45)
24	Chen, 2020	Sprague-Dawley rats: Sham-operated control, TBI, TBI + HUCDMSC (n=10/group)	Acute traumatic brain injury in adult male Sprague-Dawley rats	Intravenous injection of HUCDMSC (1.2x106 cells) at 3 h post-TBI	Treatment group significantly improved corner/inclined plane tests, reduced ischemic volume, apoptosis (↓Bax/cleaved-caspase3/PARP), fibrosis (↓Smad3/TGF-β), oxidative stress (↓NOX-1/2/p22phox), brain edema/DNA-damage (↓AQP4/γ-H2AX), inflammation (↓CD14/GFAP/F4/80); increased angiogenesis (↑VEGF/SDF-1α/CXCR4) and neural integrity (↑NeuN/nestin/DCX) vs. TBI alone (all P<0.001-0.0001).	NR	(46)
25	Zhou, 2016	Sprague-Dawley rats: TBI, UC-MSCs, HBO, UC-MSCs + HBO (n=40/group)	Severe traumatic brain injury in Sprague-Dawley rats	UC-MSC intravenous injection via tail vein (1x106 cells/ml, 1 ml) combined with HBO therapy (0.2 MPa, 4 times/day for 4 days)	UC-MSC + HBO combination therapy most effectively reduced AQP4, improved neurological scores and MWM performance (shorter escape latency, more platform crosses), reduced lesion cavities/scarring, increased GAP-43 and grafted cell survival (54.21±14.52 vs. 30.53±9.43 in UC-MSC alone) vs. monotherapy and TBI groups.	NR	(47)
26	Li, 2019	Wistar rats: Saline, hMSCs (n=10/group)	Traumatic brain injury in Wistar rats	Human bone marrow stromal cells (hMSCs) intravenous injection (~3x106 cells) at 1 week post-TBI	hMSC treatment accelerated recovery of FA and AWF in corpus callosum/external capsule (significant from 3-5 weeks vs. saline), increased MK at 3 weeks, reversed RK decline earlier, and significantly improved spatial memory in mMWM vs. saline group (all P<0.05).	NR	(48)
27	Bayat Tork, 2025	Male Wistar rats: Sham, TBI, TBI + PBS, TBI + Nano-SDF, TBI + NSCs, TBI + Nano-SDF + NSCs (n=10/group)	Traumatic brain injury induced by controlled cortical impact in male Wistar rats	Implantation of nano-scaffold containing SDF-1 (PLLA-PCL with Young's modulus 3.21 kPa) loaded with NSCs into lesion cavity post-TBI	SDF-1 scaffold + NSC significantly increased proliferation (2.1-fold), migration (1.8-fold), neuronal differentiation (NeuN + 45% vs. 28%), graft survival (62 vs. 38%), neurite outgrowth, synaptogenesis (PSD-95/Syn 1.7-fold), mNSS (12→5 at 28 days), and MWM latency (↓35%) vs. control scaffold (all P<0.05-0.001).	NR	(49)

[i] MSC, mesenchymal stem cell; TBI, traumatic brain injury; BMSC, bone marrow-derived mesenchymal stem cell; OEC, olfactory ensheathing cell; MWM, Morris water maze; CCI, controlled cortical impact; Pro, propranolol; LPS, lipopolysaccharide; EPCs, endothelial progenitor cells; NA, not available; NR, not reported.

Table III MSC-derived cell-free therapy in models of TBI.

Table III

MSC-derived cell-free therapy in models of TBI.

Study no.	First author, year of publication	Animal	Pre-condition/model	Treatment	Results (condensed, with exact numbers where reported)	Adverse effects	(Refs.)
1	Zhang, 2015	Wistar rat: TBI + MSC-exosomes; TBI +vehicle; sham (n=8/group)	Wistar rat moderate TBI (controlled cortical injury/contusion model)	Single IV tail-vein injection of 100 µg MSC-derived exosomal protein at 24 h post-TBI; vehicle control	MSC-derived exosomes significantly shortened MWM escape latency, reduced mNSS and foot-fault errors days 14-35, decreased lesion volume, increased angiogenesis (BrdU+/EBA+) and neurogenesis(BrdU+/DCX+ & BrdU+/NeuN+) vs. vehicle (all P<0.05).	NR	(50)
2	Zhang, 2017	Wistar rats; exosomes in 2D or 3D, liposomes (n=8/group)	Controlled cortical impact (CCI) TBI in Wistar rats	Single IV tail-vein injection at 24 h post-TBI: exosomes from 2D-hMSC culture, exosomes from 3D collagen scaffold-grown hMSCs, or liposome control; behavioral follow-up to day 35	2D- and 3D-exosomes significantly improved MWM spatial learning, reduced NSS and foot-fault errors days 14-35, increased angiogenesis and neurogenesis, decreased neuroinflammation vs. liposome control (all P<0.05); 3D-exosomes superior to 2D in MWM performance; no difference in lesion volume.	NR	(51)
3	Ni, 2019	59 male C57BL/6 mice (12-14 weeks): Sham + PBS n=17, TBI + PBS n=21, TBI + exosomes; n=21.	Controlled cortical impact (CCI) to right hemisphere (velocity 4 m/sec, depth 1.0 mm, 150 msec duration) after 4 mm craniotomy; sham: same surgery without impact.	Single retro-orbital IV injection 15 min post-TBI: TBI + exosomes group received 30 µg total protein of BMSC-derived exosomes in 150 µl PBS; TBI + PBS and Sham + PBS received equal volume PBS.	MSC exosomes significantly improved mNSS and rotarod at days 7-14, reduced lesion area at day 14, decreased apoptosis (↑Bcl-2, ↓Bax), early inflammation (↓IL-1β/TNF-α), and shifted microglia to M2 phenotype (↓iNOS, ↑Arg1/CD206) vs. PBS (all P<0.05-0.01).	NR	(12)
4	Xu, 2020	Rats: sham group, PBS group, MSCs-Exo group, BDNF-induced MSCs-Exo group (n=12/group)	Adult rats with TBI induced by electric controlled cortical impact; groups: sham (n=12), PBS (TBI + PBS, n=12), MSCs-Exo (n=12), BDNF-induced MSCs-Exo (n=12).	Single tail-vein injection of BDNF-induced MSC exosomes (100 µg total protein in 0.5 ml PBS) 24 h post-TBI vs. PBS injection; standard MSC exosomes as additional comparator.	BDNF-engineered exosomes significantly reduced mNSS days 7-28, shortened MWM escape latency and increased target quadrant time days 31-35, decreased TUNEL+ cells, improved neuronal morphology and neuro-regeneration markers vs. standard MSC-Exo and PBS (all P<0.05; effects mediated by miR-216a-5p).	NR	(52)
5	Zhang, 2020	Adult male Wistar rats: Exo-100 µg, D1 group used here as Exp (n=8 /group)	Adult male Wistar rats with unilateral moderate CCI to left parietal cortex (6-mm tip, 4 m/s, 2.5-mm depth), causing cortical lesion, hippocampal damage and white-matter injury.	Single IV tail-vein injection of human MSC-derived exosomes (100 µg protein in 0.5 ml PBS) at 1 day post-TBI (Exo-100, D1) vs. PBS; study also tested 50/200 µg and 100 µg at days 4 or 7.	MSC exosomes (50-200 µg) dose-dependently improved mNSS, foot-fault and MWM performance days 7-35 (optimal at 100 µg), reduced hippocampal neuronal loss, increased vascular density/BrdU+/EBA+ cells, and decreased CD68+ microglia/macrophages and GFAP+ astrocytes vs. PBS (all P<0.05; best effects when given day 1).	NR	(13)
6	Zhang, 2021	Young male Wistar rats: TBI + Exo-17-92 (n=8/group)	Young male Wistar rats with unilateral moderate cortical contusion (CCI); 24 TBI animals + 8 sham; no mortality during 35-day follow-up.	Single IV injection of 100 µg exosomes (Exo-17-92 cluster-enriched) in 0.5 ml PBS at 24 h post-TBI; comparators: Exo-empty (100 µg) and Vehicle PBS.	miR-17-92-enriched exosomes significantly improved mNSS (77% vs. 63% reduction), foot-fault (65 vs. 48%), adhesive-removal (58 vs. 21%), and MWM performance; reduced DG/CA3 neuronal loss, increased angiogenesis/neurogenesis, and decreased CD68+/GFAP+ cells more effectively than empty exosomes and vehicle (all P<0.05)	NR	(53)
7	Wen, 2022	C57BL/6J male mice, (TBI + Exo, various timepoints) (n=5/group)	C57BL/6J male mice with lateral fluid percussion TBI; microgliamediated neuroinflammation and neuronal apoptosis present.	Tail-vein injection of BMSC-derived exosomes (200 µl, 6.3x1010 particles/ml) daily after TBI (1-7 days) vs. saline additional lentiviral modulation of miR-181b (up/down) in separate TBI cohorts.	BMSC-exosomes significantly shifted microglia to M2 phenotype (↑CD206+, ↓CD86+), reduced IL-1β/IL-6/TNF-α, increased IL-10/TGF-β in vitro; decreased cortical apoptosis, neuroinflammation and promoted M2 polarization in vivo via miR-181b-mediated IL-10/STAT3 pathway.	NR	(54)
8	Zhang, 2024	C57BL/6 wild-type mice: Sham, TBI, TBI + antagomir 9-5p (n=8/group)	Traumatic brain injury-induced acute lung injury in C57BL/6 wild-type mice	Injection of brain-derived exosomes (100 µg via tail vein) or antagomir 9-5p treatment	TBI induced pulmonary ferroptosis (↑ROS/MDA/Fe²+, ↑miR-9-5p, ↓Scd1); miR-9-5p antagomir significantly reduced ferroptosis markers, normalized ferroptosis-related proteins, decreased inflammatory cytokines/cells in BALF, and attenuated acute lung injury severity vs. TBI alone (all P<0.05).	NR	(55)
9	Wang, 2025	Mice: Sham; PBS; NExo; SeNExo (n=NA)	Traumatic brain injury and spinal cord injury in murine models	Intravenous administration of SeNExo (selenized neural stem cell-derived exosomes)	SeNExo significantly enhanced ROS scavenging and BBB penetration, reduced cerebral lesion size, alleviated neuronal apoptosis, restored glia homeostasis, remodeled glia-neuron networks, decreased neuronal loss, and improved neurological/locomotor function vs. control in TBI and SCI models (all P<0.01).	NR	(56)
10	Qian, 2024	Sprague Dawley rats: Sham, TBI, TBI + miR-148a-5p agomir, TBI + miR-148a-3p antagomir (n=20/group)	Traumatic brain injury in male Sprague Dawley rats	Intracerebroventricular infusion of miR-148a-3p agomir (5 nmol, 20 µl) or antagomir (5 nmol, 20 µl) 20 min after TBI	miR-148a-3p-enriched exosomes promoted M2 polarization (↑Arg-1/IL-4/IL-10, ↑CD206, ↓CD32/iNOS), reduced IL-1β/IL-6/TNF-α, inhibited p-ERK/NF-κB, decreased brain edema, mNSS and lesion area v.s TBI control (all P<0.05-0.001).	NR	(57)
11	Wang, 2024	Mice: Sham, rmTBI, rmTBI + EXO, rmTBI + EXO-124 (n=8/group)	Repetitive mild traumatic brain injury in adult male C57BL/6J mice	Intranasal delivery of microglia-derived exosomes overexpressing miR-124-3p (100 µg total, 3 doses at 24 h intervals) post-rmTBI	miR-124-3p-overexpressing microglial exosomes significantly suppressed ER stress markers (GRP78/p-IRE1α/XBP1s/CHOP/cleaved caspase-12/3) and neuronal apoptosis in vitro and in vivo via direct targeting of IRE1α vs. control exosomes (all P<0.0001).	NR	(58)
12	Liu, 2023	Sprague-Dawley rats: Sham, TBI, 3D-CC-NExos, 3D-CC-INExos (n=30/group)	Traumatic brain injury in male Sprague Dawley rats	Implantation of 3D-printed collagen/chitosan scaffolds (2 mm diameter, 2 mm height) loaded with IGF-1-pretreated NSC exosomes into lesion cavity post-TBI	3D-CC-INExos scaffold significantly shortened MWM escape latency, reduced mNSS at day 28, decreased lesion cavity, increased nestin/NF/MBP/NeuN/MAP2/SYP/CD31/α-SMA, reduced CD68/Iba-1/TUNEL, and improved neuronal/myelin ultrastructure vs. 3D-CC-NExos and TBI groups (all P<0.05-0.01).	NR	(59)
13	Liu, 2025	Mice: Injury (n=4-11); Injury + A-Exo (n=4-11); Injury + Y-Exo (n=11/group)	Repeated mild traumatic brain injury in C57BL/6 mice	Intranasal administration of brain-derived exosomes from aged mice (A-Exo) post-rmTBI	Y-Exo significantly improved Y-maze alternations, MWM crossings/latency/target time, reduced TUNEL+ apoptotic neurons, increased cell viability, and decreased apoptosis vs. A-Exo; Tnfrsf25 knockdown and desoxycortone/propantheline inhibitors reversed A-Exo-induced apoptosis (all P<0.05-0.0001).	NR	(60)
14	Kim, 2025	Mice: Sham, TBI, TBI + NI-Exo (1x104 particles/ml), TBI + NI Exo (1x105 particles/ml) (n=30/group)	Traumatic brain injury in C57BL/6 mice	Intracerebroventricular injection of NI-Exo (1x104 or 1x105 particles/ml) 1 h post-TBI	NI-Exo significantly reduced IL-6/IL-1β/TNF-α, increased IL-4/IL-10, improved rotarod/EBST/cylinder performance, enhanced cell viability/NeuN/MAP2, decreased p53/ROCK1/Bax/p-ERK/p-p38/p-NF-κB vs. TBI alone (all P<0.001).	NR	(61)
15	Moss, 2021	Mice: Sham (n=15); TBI (n=30); TBI + EXO (n=21); TBI + EXO-M (n=21)	Traumatic brain injury induced by controlled cortical impact in mice	Intranasal delivery of hASC-derived exosomes (hASCexo) containing MALAT1 at 48 hours post-TBI; exosomes depleted of MALAT1 as control	MALAT1-enriched exosomes significantly improved EBST motor recovery, RAWM cognitive performance, reduced cortical damage and MHCII+ microglia, and modulated inflammation/NRTK3-TrkC expression vs. TBI and MALAT1-depleted exosomes (all P<0.05).	NR	(62)
16	Zhang, 2021	Sprague-Dawley rats: Sham, Sham + AS-Exo, TBI, TBI + AS-Exo (n=15/group rats)	Traumatic brain injury in adult male Sprague-Dawley rats and C57BL/6 mice (Nrf2+/+ and Nrf2-KO)	Intravenous injection of astrocyte-derived exosomes (AS-Exos, 100 µg) via tail vein 30 min post-TBI	AS-Exo significantly reduced mNSS, brain edema, lesion volume, ROS/mitochondrial H2O2 and apoptosis (↓TUNEL/CC-3/Bax), improved forelimb placement, rotarod, MWM performance and increased viable neurons, SOD/CAT/GSH, Nrf2/HO-1 vs. TBI (all P<0.05-0.01); effects abolished in Nrf2-KO mice.	NR	(63)
17	Pischiutta, 2025	Mice: TBI saline, TBI NB, TBI CM, TBI SYNT (n=8/group)	Traumatic brain injury in mice	Intravenous administration of MSC-conditioned medium (CM) or synthetic cocktail (SYNT: prostaglandins and kynurenine) post-TBI	SYNT and CM significantly reduced in vitro neuronal death, improved sensorimotor function and memory up to 4-6 months, decreased microglia activation/astrogliosis at 6 months; CM additionally reduced contusion volume at 5 months vs. saline (all P<0.05).	NR	(64)
18	Chen, 2020	Sprague-Dawley rats, Sham-operated control; TBI; TBI + HUCDMSC (n=10/group)	Traumatic brain injury in male Sprague-Dawley rats (hydraulic injury model)	Intracerebroventricular injection of astrocytes-derived exosomes carrying GJA1-20k (10 µg) post-TBI	GJA1-20k exosomes significantly increased neuronal uptake, reduced apoptosis 22%, improved mitochondrial function (ATP ↑1.5-fold, ROS ↓40%) in vitro; reduced lesion volume 35%, increased neuronal survival 1.8-fold, decreased TUNEL+ apoptosis 28% and Bax/Bcl-2 ratio in vivo vs. control exosomes (all P<0.05-0.01).	NR	(65)
19	Zhuang, 2023	Male Sprague-Dawley rats: Sham, TBI + Vehicle, TBI + BMSCs-Exos, TBI + SB203580, TBI + BMSCs Exos miR-124-3p, TBI + BMSCs Exos miR-124-3p inhibitor (n=22/group)	Traumatic brain injury in male Sprague-Dawley rats	Intravenous injection of BMSCs-derived exosomes enriched with miR-124-3p (BMSCs-Exos^ miR-124-3p, 200 µg) at 24 h post-TBI	miR-124-3p was downregulated in TBI and directly targeted p38 MAPK. miR-124-3p-enriched BMSC-derived exosomes reduced apoptosis and p38 MAPK expression while increasing GLT-1 in vitro. In vivo, they decreased lesion volume and neuronal cell death, enhanced neuronal survival, and improved cognitive performance, with effects comparable to the p38 inhibitor SB203580.	NR	(66)

[i] MSC, mesenchymal stem cell; TBI, traumatic brain injury; Exo, exosome; BMSC, bone marrow-derived mesenchymal stem cell; OEC, olfactory ensheathing cell; MWM, Morris water maze; CCI, controlled cortical impact; Pro, propranolol; LPS, lipopolysaccharide; NA, not available; NR, not reported.

The total number of animals across all 46 included studies was 1,558; not all studies reported every primary outcome (Fig. S1): The mNSS was reported in studies contributing 403 animals; MWM outcomes were available from studies with 408 animals; and lesion volume data came from studies involving 403 animals. This discrepancy arises as some studies measured only one or two of the three primary outcomes. All analyses were restricted to studies providing the relevant outcome data, ensuring transparency.

Characteristics of the pre-clinical studies

The key characteristics of the included interventions are summarized in Fig. 2. Among the 46 pre-clinical studies, a greater proportion of experiments used MSC-based cell therapy than MSC-derived cell-free products (59 vs. 41%; Fig. 2A). As regards the delivery route, almost half of the interventions were administered IV (48%), followed by ICV injection in 40% of the studies (Fig. 2B). Only a small minority used alternative routes, such as intranasal delivery (6%) or other less frequently applied approaches (intra-arterial, intraperitoneal and intra-retrobulbar; ≤2% each), indicating that the current pre-clinical evidence is dominated by systemic IV and ICV administration strategies.

Figure 2 Characteristics of the 46 preclinical studies, with (A) MSC therapy vs. cell-free therapy; and (B) injection route from all studies. MCS, mesenchymal stem cell; IN, intranasally; IR, intra-retrobulbar; IP, intraperitoneal; IA, intra-arterial; IV, intravenous; ICV, intracerebroventricular.

Risk of bias

The SYRCLE risk-of-bias assessment across all 46 included pre-clinical studies is summarized in Fig. 3. Overall, the majority of studies had at least one domain with an unclear or high risk of bias. In total, 43% of the assessments were rated as low risk, 53.5% as unclear risk and 3.5% as high risk. Domains related to random sequence generation, allocation concealment, random housing and blinding of caregivers or investigators were predominantly judged as unclear risk due to insufficient reporting. By contrast, the domains of incomplete outcome data, selective reporting and other sources of bias were mostly assessed as low risk.

Figure 3 Risk of bias assessment with SYRCLE of (A) from all MSC and MSC-derived cell-free therapy studies; (B) percentage of high risk (red), unclear risk (yellow), low risk (green). MCS, mesenchymal stem cell.

Publication bias

The visual inspection of funnel plots for the three main outcomes is illustrated in Fig. 4. For mNSS (Fig. 4A), the funnel plot appeared mildly asymmetric, with a relative paucity of small, imprecise comparisons reporting null or detrimental effects and a cluster of small studies favoring MSC-based therapies, suggesting the presence of small-study effects and possible publication bias. A similar pattern of moderate asymmetry was observed for lesion volume (Fig. 4C), where several imprecise studies reported very large reductions in lesion size without a corresponding number of small studies showing neutral effects. By contrast, the funnel plot for MWM (Fig. 4B) was more symmetric around the pooled effect line, providing no strong visual evidence of publication bias. Nevertheless, given the high between-study heterogeneity and the experimental nature of the included studies, all pooled estimates should be interpreted with caution, particularly for mNSS and lesion volume.

Figure 4 Funnel plots for all groups for (A) the modified neurological severity score, (B) Morris water maze test, and (C) lesion volume. MD, mean difference.

Stratified meta-analysis

The meta-analysis was then stratified by outcome domain. For global neurological function, 92 comparisons reporting mNSS were pooled. Using a random-effects model with the Hartung-Knapp adjustment, MSC-based and MSC-derived cell-free therapies significantly reduced mNSS scores compared with the control (95% CI; P<0.0001; I2=100%), indicating improved neurological recovery. The corresponding pooled and stratified effects are presented in Fig. 5.

Figure 5 Forest plots demonstrating mean effect size and 95% CI values of mNSS for (A) all routes from MSC and cell-free groups; (B) MSC groups; (C) MSC ICV route groups, (D) MSC IV route groups, (E) cell-free groups, (F) cell-free ICV route groups, and (G) cell-free IV route groups. CI, confidence interval; mNSS, modified neurological severity score; MCS, mesenchymal stem cell; ICV, intracerebroventricular; IV, intravenous.

For cognitive performance, 51 comparisons reporting MWM outcomes were combined. The pooled analysis demonstrated substantially lower (improved) MWM scores in the treatment groups than in the controls [overall mean difference (MD), -16.72; 95% CI, -22.87 to -10.58; P<0.00001; I2=100%], these findings are presented in Fig. 6. For structural brain damage, 50 comparisons reporting lesion volume were analyzed and a robust reduction in lesion volume was observed in the treated animals compared with the controls (overall MD, -0.15; 95% CI, -0.17 to -0.14; P<0.00001; I2=98%) (Fig. 7). Overall, across neurological deficit (mNSS), cognition (MWM) and lesion volume, stratified analyses consistently favored MSC-based or MSC-derived cell-free therapies in pre-clinical TBI models, albeit with considerable between-study heterogeneity.

Figure 6 Forest plots demonstrating mean effect size and 95% CI values of MWM for (A) all routes from MSC and cell-free groups; (B) MSC groups; (C) MSC ICV route groups, (D) MSC IV route groups, (E) cell-free groups, and (F) cell-free IV route groups. CI, confidence interval; MWM, Morris water maze test; MCS, mesenchymal stem cell; ICV, intracerebroventricular; IV, intravenous.

Figure 7 Forest plots demonstrating mean effect size and 95% CI values of lesion volume for (A) all routes from MSC and cell-free groups; (B) MSC groups, (C) MSC ICV route groups, (D) MSC IV route groups, (E) cell-free groups, and (F) cell-free IV route groups. CI, confidence interval; MCS, mesenchymal stem cell; ICV, intracerebroventricular; IV, intravenous.

Subgroup analysis (cell-based vs. cell-free and route of administration)

Subgroup analyses were then performed according to product type (MSC vs. MSC-derived cell-free products) and route of administration (ICV, IV or other routes). For mNSS, subgroup analyses suggested that the magnitude of benefit varied across intervention types (test for subgroup differences: P=0.02; I2=60%). As shown in Fig. 5, larger pooled improvements for MSC-derived cell-free products were observed in mNSS (MD, ~-4.7) compared with MSC-based cellular therapies, while the ICV delivery of cell-free products yielded very imprecise estimates with wide confidence intervals due to the small number of experiments. Despite this variability, all subgroups favored treatment over the control.

For MWM, consistent cognitive benefits were observed across subgroups as shown in Fig. 6. Both MSC-based and cell-free interventions, delivered either ICV or IV, improved MWM performance to a similar extent, and the test for subgroup differences was not significant (P=0.78; I2=0%). This finding suggests that, for cognitive recovery, the beneficial effect is relatively robust to the choice of MSC product type and delivery route.

For lesion volume, subgroup analyses revealed clearer differences between modalities (test for subgroup differences: P<0.00001; I2=95.7%), As shown in Fig. 7, MSC-derived cell-free therapies produced the largest reductions in lesion volume, particularly when delivered intravenously (pooled MD, -0.21 to -0.24), whereas MSC-based cellular therapies exhibited, minimal, but still significant effects (pooled MD, -0.09). Nevertheless, each subgroup exhibited a shift in favor of treatment compared with the control, indicating that both MSC and MSC-derived cell-free approaches confer structural neuroprotection, with cell-free products, particularly via IV administration, tending to provide the greatest lesion-reducing effect.

Taken together, these subgroup findings support a consistent neuroprotective signal across different MSC-based strategies, while also suggesting that MSC-derived cell-free products and IV delivery may be particularly promising for optimizing functional and structural outcomes after experimental TBI.

Heterogeneity and interpretation

All primary outcomes exhibited high heterogeneity (I2=98-100% for mNSS, lesion volume and MWM), typical in pre-clinical TBI meta-analyses due to variations in animal species, TBI models, injury severity, MSC/exosome sources, isolation methods, doses, administration routes (IV vs. ICV) and follow-up durations. Sensitivity analyses (leave-one-out analysis) confirmed that pooled estimates remained directionally consistent and significant in favor of treatment, with no single outlier dominating the results (Figs. 5, 6 and 7). Pre-specified subgroup analyses by product type (MSC vs. cell-free) and route (IV vs. ICV vs. other) partially accounted for heterogeneity; notably, IV cell-free interventions showed larger effects on lesion-reducing effect (subgroup difference P<0.05 for lesion volume). Residual heterogeneity remained high within subgroups, likely due to unmeasured factors such as exosome characterization and preconditioning.

Meta-regression was not conducted owing to insufficient studies per covariate and a very high baseline heterogeneity. The most robust conclusion is the consistent directional benefit across diverse pre-clinical settings: MSC-based therapies and, particularly IV. MSC-derived (exosomes) demonstrate clear neuroprotective signals in neurological function, cognition, and lesion volume. These results underscore paracrine mechanisms and emphasize the urgent need for standardized protocols in future pre-clinical studies to facilitate clinical translation.

Discussion

MSC and cell-free efficacy on functional outcomes

In the present meta-analysis, 403 animals contributed mNSS data (194 experimental and 209 controls). Across all product types and routes, MSC-based interventions significantly improved global neurological function following experimental TBI, as shown by a lower mNSS in treated animals (P<0.0001). This indicates a consistent neuroprotective effect on composite motor, sensory, reflex and balance deficits, and aligns with pre-clinical studies in which systemic MSC administration upregulated neurotrophic factors [brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF) and nerve growth factor (NGF)] in the peri-lesional cortex, reduced apoptosis, and improved behavioral recovery (12).

The overall neuroprotective effect is largely attributable to paracrine mechanisms, whereby MSCs secrete anti-inflammatory cytokines, neurotrophic factors and extracellular vesicles that modulate the post-injury microenvironment without requiring long-term cellular engraftment. MSC-derived exosomes, as key paracrine mediators, carry bioactive cargo including proteins (BDNF and VEGF for neuroprotection and angiogenesis) and miRNAs (miR-133b for axonal growth and synaptic plasticity, miR-216a-5p for inhibiting neuroinflammation via BDNF pathways, miR-124-3p for suppressing Rela/Apolipoprotein E to mitigate neurodegeneration). Recent studies (2024-2025) confirm that these cargoes enable exosomes to cross the blood-brain barrier efficiently, deliver targeted payloads to neurons and glia, and regulate transcriptional/translational processes for repair (67,68).

Immunomodulation is central

Exosomes shift microglial polarization from pro-inflammatory M1 (iNOS, TNF-α, IL-1β and IL-6) to reparative M2 phenotype (Arg-1, CD206 and IL-10), downregulating NF-κB/MAPK pathways and reducing oxidative stress/apoptosis (68,69). The study by Liu et al (67) highlighted exosomal miR-133b and miR-22 upregulation under hypoxic conditions to facilitate nerve repair, while Xiong et al (68) emphasized neurorestoration via anti-apoptotic and anti-inflammatory cargo. These mechanisms explain greater mNSS improvements with cell-free products, particularly IV exosomes, as they provide concentrated, standardized delivery without cell survival issues (8,10-12). Beyond immunomodulation, both MSCs and MSC-derived exosomes attenuate neuronal apoptosis, promote angiogenesis, support neurogenesis and enhance synaptic plasticity, all of which contribute to the restoration of cortical and subcortical networks that mediate motor and sensory functions (11,12). These effects align with the longstanding view that MSCs exert their therapeutic effects predominantly through paracrine signaling rather than direct cell replacement, a concept supported by accumulating pre-clinical and early clinical evidence in TBI (13,70).

However, high heterogeneity (I2=100%) substantially limits the precise interpretation of pooled estimates. Sources include variability in TBI models controlled cortical impact (CCI) and fluid percussion injury (FPI), injury severity, MSC sources (bone marrow vs. umbilical cord), exosome isolation/characterization methods, doses, timing (≤7 days), routes (IV vs. ICV) and follow-up durations. This heterogeneity reflects real-world translational challenges, but also underscores the robustness of directional benefits across diverse settings (70). Future studies are required to standardize protocols Minimum Information for Studies of Extracellular Vesicles (MISEV) guidelines for exosome characterization, consistent dosing in particle number or protein content) to reduce variability and facilitate meta-regression or clinical trial design. Sensitivity analyses confirmed directional consistency, but residual heterogeneity within subgroups suggests unmeasured factors (preconditioning, miRNA cargo profiling) as key contributors.

Although almost all point estimates favored treatment over control, statistically significant gains were driven mainly by MSC-derived cell-free preparations, particularly when delivered intravenously, whereas MSC subgroups exhibited similar directions, but wider confidence intervals. This pattern suggests that IV cell-free products, particularly exosomes, may provide larger and more reliable benefits, likely due to better bioavailability and targeted immunomodulation.

MSC and cell-free efficacy on cognitive outcomes

For cognitive outcomes, 408 animals with MWM data were included. Cognitive recovery is a central target for TBI therapies, and tje pooled MWM analysis revealed that MSC-based interventions significantly improved spatial learning and memory compared with the controls, despite marked between-study heterogeneity (P<0.00001; I2=100%). This indicates that, at a global level, MSC-centered strategies do not only reduce gross neurological deficits, but also confer measurable benefits on higher-order functions that rely on hippocampal integrity and neuroplasticity.

Both MSCs and MSC-derived cell-free products act on multiple levels of hippocampal circuitry. Pre-clinical TBI research has demonstrated that intravenous MSC-derived exosomes enhance spatial learning by increasing dentate gyrus neurogenesis (BrdU+/DCX+ and BrdU+/NeuN+ cells), promoting synaptic plasticity (upregulation of GAP-43, synaptophysin and PSD-95), and reducing neuronal loss in CA1-CA3, changes that parallel improved MWM performance (50). BDNF-induced MSC exosomes further augment these effects by delivering miR-216a-5p and other neurotrophic cargos that support neuronal survival and dendritic complexity, thereby accelerating recovery of spatial learning following TBI (13). In parallel, hypoxia-preconditioned MSCs promote oligodendrogenesis and remyelination, restore white-matter integrity, and activate mTOR/HIF-1α/VEGF signaling, which together improve network connectivity and result in better cognitive outcomes in MWM testing (42).

Cell-free preparations appear particularly effective as they concentrate the paracrine signals that drive hippocampal plasticity. MSC-derived exosomes modulate microglial phenotypes in the hippocampus, shifting from pro-inflammatory M1 to anti-inflammatory M2 via miR-181b/IL-10-STAT3 signaling, leading to reduced IL-1β, IL-6 and TNF-α, less synaptic pruning, and improved MWM performance (13,52,54). Other exosomal cargos, such as the long non-coding RNA MALAT1 have been shown to activate pro-regenerative and synaptogenic pathways, and to attenuate chronic neuroinflammation when delivered intranasally following TBI, again translating into improved motor and cognitive scores (62). Together with broader evidence that MSC-based therapies enhance neurogenesis, angiogenesis and network-level plasticity across neurological models, these data provide a biological rationale for the finding of the present study that the majority of MSC-based and MSC-derived cell-free interventions improve spatial learning and memory, with the most robust and consistent cognitive gains arising from standardized exosome-based strategies (71).

Subgroup analyses revealed that almost all point estimates favored treatment, indicating a broadly consistent trend toward better MWM performance with both MSC and MSC-derived cell-free products. However, statistically significant improvements were largely driven by the cell-free subgroup (P<0.00001), whereas MSC subgroups (overall MSC, MSC-ICV and MSC-IV) exhibited effects in the same direction but failed to reach conventional significance (all P>0.05), reflecting wide confidence intervals and substantial heterogeneity. The test for subgroup differences was not significant (P=0.78, I2=0%), meaning that confidence intervals overlapped and formal interaction testing does not prove clear superiority of one modality over another. Practically, these findings suggest that the majority of MSC-based and cell-free strategies tend to improve cognitive performance, with the strongest and most statistically robust signal arising from cell-free interventions. This aligns with the concept that post-TBI cognitive recovery depends heavily on synaptic remodeling, neurogenesis and network-level plasticity driven predominantly by the paracrine cargo of MSC-derived secretome and extracellular vesicles, while the very high overall heterogeneity and variable methodological quality emphasize the need for more standardized MWM protocols, predefined treatment timing, and rigorous blinding in future pre-clinical studies.

MSC and cell-free efficacy on structural outcomes

For structural outcomes, 403 animals contributed lesion volume data, with 194 animals in the experimental groups and 209 in the control groups. Lesion volume provides a structural correlate of tissue preservation and is closely linked to long-term functional outcome following TBI. The meta-analysis demonstrated a very robust and highly significant reduction in lesion volume in animals treated with MSC-based interventions compared with controls (P<0.00001, I2=98%). This magnitude of effect indicates that MSC-centered strategies consistently limit the extent of brain tissue loss across diverse experimental models, injury severities, and treatment protocols.

At the subgroup level, a markedly coherent pattern was observed: All subgroups (lesion volume, MSC, MSC-ICV, MSC-IV, cell-free and cell-free IV) exhibited highly significant reductions in lesion size (P<0.001). Thus, in contrast to the more variable statistical significance observed in mNSS and MWM, the structural endpoint of lesion volume exhibited a uniformly strong treatment signal across both cell-based and cell-free approaches. Nevertheless, the test for subgroup differences was highly significant, with very high heterogeneity between subgroups (P<0.00001, I2=95.7%), indicating that the magnitude of neuroprotection is not identical for all modalities.

This structural protection is consistent with pre-clinical evidence that MSCs and their extracellular vesicles primarily act by limiting secondary injury cascades in the peri-lesional ‘penumbra’ rather than reversing the primary mechanical damage (72). MSC-derived exosomes rapidly reach the injured cortex and hippocampus, where they modulate microglia/macrophage polarization from a pro-inflammatory M1 phenotype to a reparative M2 state, downregulating iNOS and pro-inflammatory cytokines, while upregulating Arg-1 and CD206(12). By dampening neuroinflammation and oxidative stress, these vesicles reduce apoptotic cell death in neurons and oligodendrocytes and thereby preserve viable tissue, which translates into smaller cavitary lesions on histology and MRI (13,68).

In parallel, MSCs and MSC-derived extracellular vesicles enhance neurovascular remodeling and white-matter integrity, two additional processes that constrain lesion expansion. Hypoxia-preconditioned bone marrow-derived MSCs promote remyelination and reduce white-matter injury via mTOR/HIF-1α-dependent pathways, leading to reduced overall lesion volume in mice with TBI (42). MSC/EV therapies also stimulate angiogenesis and restore blood-brain barrier integrity, which decreases edema and secondary ischemic damage in the peri-contusional zone (73,74). Complementary data from broader extracellular vesicle research indicate that stem cell-derived exosomes can lower cortical and hippocampal water content and lesion volumes, while protecting against mitochondrial oxidative stress and apoptosis (75).

The findings of the present study are summarized by the illustration in Fig. 8 demonstrating different administration routes of MSCs and exosomes in rodent TBI models (IN, ICV and IV) and their effects on functional, cognitive and structural outcomes. Across all three panels, the bars indicate that MSC-derived cell-free products, particularly when administered IV, tend to produce the greatest improvement compared with whole-cell MSC therapy.

Figure 8 Illustration of the key findings of the present study demonstrating different administration routes of MSCs and exosomes, which affect: (A) Functional outcomes (mNSS), (B) cognitive outcomes, and (C) structural outcomes (lesion volume) in rodent models of traumatic brain injury. MCS, mesenchymal stem cell; mNSS, modified neurological severity score; MWM, Morris water maze test.

Although the present meta-analysis revealed consistent neuroprotective effects of MSC-based therapies and MSC-derived exosomes in acute pre-clinical TBI models, translation to clinical practice remains challenging, with no registered trials specifically for acute TBI using MSC-derived exosomes as of 2025. Key barriers include the lack of standardization in exosome production, characterization and cargo consistency (MISEV guidelines), undefined optimal dosing, therapeutic window (≤7 days in rodents vs. variable human timing), administration route (IV most promising), and long-term safety concerns (low immunogenicity). To advance translation, future efforts should prioritize large-animal models for better bridging to humans, standardized protocols, multicenter pre-clinical studies with long-term follow-up, and early-phase human trials focused on safety, biodistribution, and biomarkers (73).

In line with previous pre-clinical TBI meta-analyses, high statistical heterogeneity limits precise interpretation of pooled effect sizes, arising from variations in animal species, TBI models, injury severity, MSC/exosome sources, doses, routes, timing and follow-up durations; residual heterogeneity suggests unmeasured factors; thus, estimates should be viewed as directional summaries rather than protocol-specific predictions (73). The risk of bias (SYRCLE tool) was often unclear or high (43% low risk), particularly in randomization, blinding and allocation concealment, potentially inflating positive effects as indicated by funnel plot asymmetry. Translational limitations include fundamental differences between rodent models and human TBI (smaller brain size, distinct neurovascular/immune responses, absence of comorbidities such as age or polytrauma, and species-specific biodistribution/blood-brain barrier penetration), complicating direct extrapolation. Despite these constraints, the consistent directional benefit across settings supports the paracrine neuroprotective potential of MSC and exosome therapies, warranting standardized protocols for improved reproducibility and clinical relevance.

In conclusion, the present systematic review and meta-analysis demonstrates that MSC-centered interventions provide consistent neuroprotective effects in pre-clinical models of traumatic brain injury. Across the included experiments, treatment groups exhibited lower neurological deficit scores, improved performance on cognitive testing, and smaller lesion volumes than controls, indicating improvement at functional, cognitive and structural levels. Stratified and subgroup analyses further suggested that MSC-derived cell-free products, particularly when administered intravenously, tended to yield larger and more precisely estimated benefits than whole-cell MSC preparations, particularly for cognitive recovery and lesion volume reduction. Taken together, these findings support a predominant role of paracrine mechanisms in MSC-mediated neuroprotection and identify intravenously delivered cell-free MSC therapies as the most promising MSC-based strategy in current pre-clinical TBI research.

Supplementary Material

Diagram of animal selection. MCS, mesenchymal stem cell; mNSS, modified neurological severity score; MWM, Morris water maze test.

GRADE assessment.

Acknowledgements

The authors would like to acknowledge the Faculty of Medicine, Universitas Islam Sultan Agung, Semarang, Indonesia, for institutional support during the preparation of this manuscript.

Funding

Funding: No funding was received.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

DRA was involved in the conceptualization of the study, in the study methodology, formal analysis, data investigation, in the writing of the original draft, visualization and project administration. AP involved in the conceptualization of the study, in the study methodology, validation, in the writing of the draft, in the review and editing of the manuscript, and in study supervision. C was involved in data investigation, data curation and data validation. TS was involved in data validation, in the writing, reviewing and editing of the manuscript, and in study supervision. DRA and AP confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Use of artificial intelligence tools

During the preparation of this work, AI tools were used to improve the readability and language of the manuscript or to generate images, and subsequently, the authors revised and edited the content produced by the AI tools as necessary, taking full responsibility for the ultimate content of the present manuscript.

References