Efficacy and safety of small extracellular vesicle interventions in wound healing and skin regeneration: A systematic review and meta-analysis of animal studies

Abstract

Small extracellular vesicles (sEVs) have been proposed as a possible solution to the current lack of therapeutic interventions for endogenous skin regeneration. We conducted a systematic review of the available evidence to assess sEV therapeutic efficacy and safety in wound healing and skin regeneration in animal models. 68 studies were identified in Web of Science, Scopus, and PubMed that satisfied a set of prespecified inclusion criteria. We critically analyzed the quality of studies that satisfied our inclusion criteria, with an emphasis on methodology, reporting, and adherence to relevant guidelines (including MISEV2018 and ISCT criteria). Overall, our systematic review and meta-analysis indicated that sEV interventions promoted skin regeneration in diabetic and non-diabetic animal models and influenced various facets of the healing process regardless of cell source, production protocol and disease model. The EV source, isolation methods, dosing regimen, and wound size varied among the studies. Modification of sEVs was achieved mainly by manipulating source cells via preconditioning, nanoparticle loading, genetic manipulation, and biomaterial incorporation to enhance sEV therapeutic potential. Evaluation of potential adverse effects received only minimal attention, although none of the studies reported harmful events. Risk of bias as assessed by the SYRCLE's ROB tool was uncertain for most studies due to insufficient reporting, and adherence to guidelines was limited. In summary, sEV therapy has enormous potential for wound healing and skin regeneration. However, reproducibility and comprehensive evaluation of evidence are challenged by a general lack of transparency in reporting and adherence to guidelines. Methodological rigor, standardization, and risk analysis at all stages of research are needed to promote translation to clinical practice.

Keywords: animal models; exosome; extracellular vesicle; skin regeneration; wound healing.

Figure 1

PRISMA flow chart summarizing study screening and selection procedure. Web of Science, PubMed and Scopus were searched for relevant articles from inception to March 1^st, 2021.

Figure 2

The distribution of the reviewed studies by year (2A) and region according to the corresponding author's affiliation (2B).

Figure 3

An overview of study characteristics, including distribution of A) animal models B) disease models C) immuno-biocompatibility of the sEV source and host and D) administration route.

Figure 4

Sources of sEVs used to promote wound healing and skin regeneration. sEVs were isolated from cells, biofluids and tissues.

Figure 5

Distribution of A) separation methods, B) combined separation techniques, C) characterization techniques, and D) protein markers of sEVs across the 68 reviewed studies. AFM: atomic force microscope; DG: density gradient ultracentrifugation; DLS: dynamic light scattering; NTA: nanoparticle tracking analysis, precKit: precipitation-based isolation kits; SEC: size exclusion chromatography; SEM: scanning electron microscope, TRPS: tunable resistive pulse sensing; TEM: transmission electron microscope; UF: ultrafiltration; UC: ultracentrifugation.

Figure 6

Risk of bias assessment of the 68 reviewed studies based on SYRCLE's ROB tool represented by RevMan 5.4.1. (1) Randomization (selection bias); (2) Random sequence generation (selection bias); (3) Baseline characteristics (selection bias); (4) Allocation concealment; (5) Random housing (performance bias); (6) Blinding of personnel (performance bias); (7) Random outcome assessment (detection bias); (8) Blinding of outcome assessment (detection bias); (9) Incomplete outcome data (attrition bias); (10) Selective reporting (reporting bias). A domain concerning the declaration of the randomization method was added (domain 1), while the domain of “other sources of bias” was not covered in this review. Symbols used: : low risk; : unclear risk; : high risk.

Figure 7

Forest plot of mean difference of wound closure rate of 26 studies following sEV interventions in diabetic or non-diabetic skin wound models in comparison to placebo controls. The diamond represents the pooled SMD. sEV interventions were effective in promoting wound closure, (pooled SMD = 4.25, 95% CI: 3.39 to 5.11, p < 0.00001).

Figure 8

Forest plot of mean difference of scar width (in µm) of nine studies following sEV interventions in diabetic and non-diabetic wound models in comparison to placebo controls. The diamond represents the pooled SMD. sEV interventions were effective in inhibiting scar formation, (pooled SMD = -5.69, 95% CI: -7.79 to -3.58, p < 0.00001).

Figure 9

Forest plot of mean difference of blood vessel density (number of blood vessels/mm²) of nine studies following sEV interventions in diabetic and non-diabetic wound models in comparison to placebo controls. The diamond represents the pooled SMD. sEV interventions were effective in promoting new blood vessel formation, (pooled SMD = 5.03, 95% CI: 3.17 to 6.88, p < 0.00001).

Figure 10

A) Forest plot of mean difference of wound closure rate in diabetic and non-diabetic wound models of studies that characterized sEV based on MISEV 2018. The diamond represents the pooled SMD. A) sEV interventions were effective in promoting wound closure, (pooled SMD = 3.50, 95% CI: 2.61 to 4.38, p < 0.00001). B) A sensitivity analysis resulted in excluding one study , causing considerable heterogeneity in the meta-analysis (Figure 10A, without the study, and Figure 10B with the study, I² = 41% vs 79%).

Figure 11

Funnel plot for the assessment of publication bias. a) Funnel plot of the studies included in wound closure rate meta-analysis. b) Funnel plot of the studies included in scar width meta-analysis. c) Funnel plot of the studies included in angiogenesis meta-analysis d) Funnel plot of the studies included in wound closure rate meta-analysis for studies that characterized sEV based on MISEV 2018. The funnel plots for the four meta-analyses performed showed no evidence of publication bias.