Prospect of Exosome in Ligament Healing: A Systematical Review

Figures & data

Table 1 Overview of the Studies

Author	Type of MSC	Level of CM	Animal	Type of Controlled Laboratory Experiments	Animal /Cells Preparations	Intervention to the Main Group	Controls
Chamberlain et al^Citation3	BM-MSC	EV	Mice	In Vivo	27 male mature mice suffering unilateral surgical transection of Achilles tendon	Administering 20 µL of 1×10^6 BM-MSCs and 1×10^6 CD^14+MQS or 1×10^6 EEMS	Hank’s balanced saline solution administered within the Achilles tendon
				In Vitro	Monocytes isolated from human peripheral blood	EV isolated from MSCs were added to macrophages on day 7 and cultivated for 3 days to generate EEMS	Macrophages control were treated with PBS
Hsueh et al^Citation9	iPSC	EV	Rabbit	In Vivo	9 female New Zealand white rabbits suffering from ACL transection at their right knee joints to induce knee OA	A complete transection of the ACL and ACLT with added iPSC-EV at 3 weeks after ACLT administered once a week for 3 weeks	Similar open surgery without ACL transection and added injection PBS for 3 weeks
				In Vitro	Articular cartilage was collected from the femoral condyle of human patients	iPSC was added to the primary chondrocytes at the final concentration of 10microgram/mL medium	Using added PBS on equal volume to the primary chondrocytes
Ye et al^Citation10	iPSC (iMSC-LEVs)	CM	Rats	In Vivo	30 adults rats underwent tendinopathy modelling surgery	Rat were injected with 100µL PBS, sEVS or LEVs, one week after carrageenan injection and once per week for four weeks	Rat were injected with 100µL PBS around the right quadriceps tendon
				In Vitro	Tendon samples from patients from the surgery of iliopsoas tenotomy during hip arthroscopy	Incubated tendon samples with iMSC-LEVs for 72h	Incubated tendon samples without iMSC-LEVs for 72h
Cui et al^Citation15	BM-SC	CM	Mice	In vivo	Mice underwent complete transection and repair of the FDL tendon in the right hind paw	The FDL was surgically transected in the transverse plane at the myotendinous junction of the calf	The FDL tendon of the mice was left undamaged after isolating it
				In Vitro	Bone marrow derived macrophages (BMDMs) were isolated from the femur and tibias of adult mice	Fibroblast or tenocytes were transferred with Cy3-labeled-miR-21-5p mimic	Control mimic using lipofectamine 2000 transfection reagent
Tian et al^Citation16	DPSC (MSC)	CM	Rats	In vitro	DPSCs were isolated from pulpal tissue in the maxillary central incisors of rats	Purified SEV derived from DPSCs under hypoxic (Hypo-SEV) condition	Purified SEV derived from DPSCs under normoxic (Nor-SEV) condition
				In vivo	Mice received a subcutaneous injection of 25mg/kg LPS over the sagittal midline suture of the calvarium as mouse calvaria osteolysis model	100 µg of Hypo-SEV per mouse was simultaneously injected with LPS for 7 days	100µg of Nor-SEV per mouse was simultaneously injected with LPS for 7 days
Shen et al^Citation17	Ad-MSC (mouse)	EV	Mouse	In vivo	32 mice underwent partial transection and subsequent repair of Achilles tendon	Implantation of collagen sheet loaded with EVs from IFN-ɣ primed Ad-MSC (iEVs)	Collagen sheet loaded with EV from naive Ad-MSC (nEVs) Collagen sheet only
				In vitro	Macrophages were obtained from BM of femurs and tibiae of adult NGL or FVB mice	Macrophages were pretreated with iEV, nEV-free CM, or nEV-free CM, then treatment with IL-1β (6 hours, 5ng/mL)	Pretreatment with control the treatment with IL-1β (6 hours, 5ng/mL)
						Macrophages were pretreated with iEV, nEV-free CM, or nEV-free CM, then treatment with IL-1β (24 hours 10ng/mL)	Pretreatment with control the treatment with IL-1β (24hours, 10ng/mL)

Abbreviations: Ad-MSC, adipose-derived mesenchymal stem cells; DPSC, dental pulp stem cells; BM-SC, bone marrow mesenchymal stem cells; iPSC, induced pluripotent stem cell; iMSC-LEVs, induced mesenchymal stem cell large extracellular vesicula; CM, conditioned media; EV, extracellular vesicle.

Figure 1 Flowchart of study process selection.

Table 2 In vivo Study results

References	Assessment of the Main Outcome	Main Outcome Measure(s)	Scores /Result	Significant Difference from Baseline	Significant Difference Between Groups	Other Evaluation	Other Outcome Measure(s)
Chamberlain et al^Citation3	7 and 14 days	Mechanical testing	EEM treatment significantly increased ultimate stress (p< 0.01) and young modulus (p= 0.03). No significant difference was found in failure load (p= 0.260) and stiffness (p= 0.30)	Yes	Yes
		IHC	Total macrophages were not significantly different between treatment groups at day 7	No	No
		Fractal Analysis	sEV reduced type 1 collagen within the healing tendon 14 days after injury	Yes	No
Hsueh et al^Citation9	6 weeks	OARSI score	The average OARSI score in the ACLT group was significantly higher than that in the ACLT þ iPSC-EV group (p < 0.001)	N/A	Yes	IHC	Collagen II, MMP13, ADAMTS5, and TNFα expression
		MMP array assay	Chondrocytes co-cultured with M0^EV secreted lower MMP3 nd higher TIMP-1 levels than those exposed to M0
Ye et al^Citation10	4 weeks	Static pain-related behaviours	N/A	N/A	Yes	Haematoxylin- Eosin (HE) staining	Bonar score
Tian et al^Citation16	7 days	Haematoxylin & Eosin (H&E) staining and TRAP staining, RT-PCR, Luciferase Assay System	Expression levels of TSG101 and CD9 in Hypo-sEV were higher than in Nor-sEV	N/A	Yes
			Hypo-sEV significantly ameliorated LPS-induced inflammatory bone loss in vivo	Yes	Yes
			Hypo-sEV can inhibit LPS-induced macrophage inflammatory response and induce M2 macrophage polarization	Yes	Yes
			Hypo-sEV directly inhibit osteoclast genesis in vivo and in vitro	Yes	Yes
			miR-210-3p within Hypo-sEV induced M2 macrophages generation and inhibited osteoclast differentiation by targeting NF-kB1	Yes	Yes
Cui et al^Citation15	21 days	Histological scoring system	No difference was found between the two groups in tendon healing according to the histological tendon-healing score	No	No	Western Blotting Analysis	Levels of COL I, COL III, a-SMA, and TGF-b1 were significantly increased in the TI-PBS group compared with those in the SO-PBS group, but then significantly decreased after macrophage depletion in the TI-Clo group
		Gliding function testing	Higher adhesion grading score and lower MTP joint flexion ROM compared to the control	Yes	Yes
Shen et al^Citation17	1 day pre-treatment (baseline)	NF-ĸB, 1 day	iEVs: N/S nEVs: N/S	Yes No	No	PCR, histological examination	Proinflammatory genes expression gap-rupture rate
	1,3 and 7 days post-treatment	NF-ĸB, 3 day	iEVs: N/S nEVs: N/S	Yes Yes	No
		NF-ĸB, 7 day	iEVs: N/S nEVs: N/S	No Yes	Yes

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