Prospect of stem cells conditioned medium (secretome) in ligament and tendon healing: A systematic review

Abstract Background Tendon or ligament tears can decrease patients' quality of life. Many therapeutic interventions are available to treat such injuries. Mesenchymal stem cells (MSCs) have been shown to be effective in treating tendon or ligament tears; however, the use of stem cell‐conditioned medium (CM) requires further investigation. This review focused on the use of stem cell CM as treatment for tendon or ligament tears. Methods A systematic literature search was performed on PubMed (MEDLINE), OVID, EMBASE, the Cochrane Library, Scopus, Web of Science, and Science Direct with the terms conditioned media or conditioned medium or secretome or microvesicle or extracellular vesicle or exosome, and tendon or ligament as the search keywords. A total of 852 articles were reviewed. Five articles were identified as relevant for this systematic review. Results Meta‐analysis could not be performed because of the high heterogeneity of the reviewed studies; however, the results of this study support a positive effect of conditioned media in tendon and ligament treatment. Conclusion This review provides evidence of improvement in the tendon and ligament healing process with stem cell CM therapy in preclinical studies.


| INTRODUCTION
Tendon or ligament tears are common injuries that can diminish patients' quality of life. The healing process after injury may not be adequate, thus leading to the formation of scar tissue. Many therapeutic interventions are available for treating such injuries, including physiotherapy, oral pain medication, steroid injection, and surgical correction; however, some treatments do not provide a satisfactory result. Nonoperative treatments often have an unsatisfactory result and only serve for pain control. 1,2 Reconstruction surgery may be performed to reattach a tendon; however, the functional outcome may be compromised by tendon and muscle strength deterioration because the tendon was harvested. 1 Stem cell-based therapy has shown promise as an adjunct treatment due to its self-renewal properties, differentiation potentials, and immunomodulatory activities. 3 A systematic review by Ahmad et al 4 concluded that stem cell treatment was also applicable for tendon healing in animal and human subjects. Among stem cells, mesenchymal stem cells (MSCs) are particularly useful for tendon and ligament healing. 1 Extensive studies have shown the therapeutic effect of stem cells in many types of tissue injury; however, the survival ability of MSCs was too short to have an effective and long-term impact. The main effect of stem cells is probably mediated by the paracrine mechanism, which is known as stem cells conditioned medium (CM) or secretome. 5 CM or secretome is a medium where the stem cells are cultured and composed of soluble proteins, lipids, nucleic acids, and extracellular vesicles (EV) or microvesicles (MVs). 6,7 Those vesicles are further categorized into exosomes and shedding vesicles. Meticulous studies on EVs and exosomes have also demonstrated potential regenerative effects, including an anti-inflammatory effect and tendon healing. 5,8 There is some clinical benefit of CM (secretome) usage. It could resolve safety considerations associated with the transplantation of stem cells, such as tumorigenicity, transmission of infections, and immune incompatibility. 5 CM (secretome) could be produced in large amounts and stored for a long period of time without loss of product potency and without toxic cryo-preservative agents. It may be also modified for desired effects and can reduce cost and time for production and maintenance of cell-based therapy. 5 Previous in vitro studies of stem cells CM or secretome usage showed promising outcomes. Studies by Chen et al 9 and Shimode et al 10 reported cell rat tenocytes proliferation; both studies showed significant improvement for the bone marrow (BM) MSC CM groups compared with the control groups. These studies found that cell viability was increased significantly in the CM-treated groups compared with controls. These studies also mentioned significant reduction of inflammatory markers with CM treatment. A study by Wang et al showed significant increase of tissue inhibitor of metalloproteinase (TIMP), an anti-inflammatory protein, that could improve biomechanical properties. 11 Tenogenic differentiation was also enhanced significantly in cells treated with CM, as has been shown by a significant increase of Col-I expression and other tenogenic markers in some studies. [9][10][11] Studies by Lange-Consiglio et al 12 and Shen et al 13 showed significant reduction in peripheral blood mononuclear cell (PBMC) proliferation in CM treatment compared with control. Interestingly, the study by Lange-Consiglio et al found that EV did not significantly inhibit PBMC proliferation, compared with control. 12 The study by Shen et al found that nuclear factor κB activity of macrophages was inhibited significantly by EV, compared with control. 13 Stem cell therapy is an emerging therapeutic modality, and a systematic review of existing preclinical studies is needed to determine safety and efficacy and, ultimately, to guide future studies. The main purpose of this review was to systematically summarize the best available evidence in vivo studies regarding the use stem cells CM or secretome for the treatment of any tendon or ligament injury. It was hypothesized that the application of stem cell CM would promote tendon and ligament healing in animal models.

| Eligibility criteria
The inclusion criteria for this review consisted of the following: • Study design: controlled animal (in vivo study).
• Study group: animals with any tendon or ligament injuries (by surgery or primary injury).
• Interventions: any application of stem cell-conditioned media (CM), EVs/MVs, or exosomes to the study groups.
• Outcomes: main outcomes were any functional, biomechanical, and safety outcomes. The data collection of in vivo study outcomes are shown in Table 1. Meta-analysis could not be generated due to the high heterogeneity of the data (ie, source of MSCs, subject animals, outcome measures, and follow-up duration).

| Study selection
A PRISMA flow diagram (Figure 1) summarizes the process of study selection. A total of 852 studies were identified from the literature.
After screening of the titles and abstracts, 101 articles were eligible for further evaluation. After full-text assessment, five studies were included in this systematic review.

Study characteristics
An overview of the study is presented in Table 1, and the specific explanation about the study is shown in Table 2, including study design, type of MSC used and their sources, isolation of the CM and its subcomponents, animal injury models for in vivo study, and the interventions.
Most of the studies utilized small animals (three in rats, one in mice). A large animal (horse) was used in only one study. Four studies involved the tendon, and one study involved the anterior cruciate ligament (ACL). Injury models were established by resection, partial resection, collagenase injection, and unintentional tear.
The most commonly used MSC type was from BM (two studies), followed by tendon (one study), adipose tissue (one study), and amnion (one study). Two were from human sources, two from rats, and one from a horse. Conditioned media of the MSCs were isolated groups. 1 Shen et al compared scaffolds loaded with iEVs vs with nEVs, and with scaffold-only groups. 13 Timing of the interventions ranged from immediate to 16 weeks after injury.

In vivo study outcomes
In vivo study outcomes are summarized in Table 2, including the main outcomes measures, the scores or results, statements of statistical significance, and a list of other outcome measures.
Among the five studies, functional outcomes were reported in only one study. 12 In this study, 2 horses that received the nonCM injection were reinjured, whereas 11 horses improved. The differences from the baseline or the control groups were not stated.
Regarding safety, Lange-Consiglio et al reported that there was no adverse reaction after injections of the conditioned media. 12 5,18 Therapeutic effects of MSCs are largely attributed to paracrine pathways. The EVs, exosomes, and even its microRNAs, the subcomponent of CM, have also been found to contribute to their therapeutic and anti-inflammatory effects, but a detailed mechanism of action needs to be elucidated. 6 Scaffolds alone may be beneficial for cell health and attachment because of its structural approximation of the original tissue. 19,20 These effects are further enhanced by the addition of MSCs or its CM. 13,21,22 The included in vitro studies support the positive effect of CM in

| CONCLUSION
Utilization of stem cells CM (secretome) was promising in enhancing the tendon and ligament healing process, as shown by the available preclinical studies; however, more preclinical studies are needed to further confirm the benefits and to produce the most suitable CM for future clinical studies.

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.