Introduction

In regenerative medicine, tissue engineering has arisen as a promising approach for the restoration, repair and healing of organ and tissue functions, especially in tissues susceptible to disease, injury, and degeneration. At present, tissue engineering studies are focused on adult mesenchymal stem cells, which have become among the most frequently explored types of cells in this field1. Mesenchymal stem cells (MSCs) are undifferentiated multipotent cells that possess self-renewal capabilities and can differentiate into various mesoderm cell lineages, such as osteogenic, adipogenic and chondrogenic lines. Aside from bone marrow and adipose tissue, cells possessing stem cell characteristics have also been successfully isolated from different parts of the tooth. These isolated cells are known as dental stem cells and include dental pulp stem cells (DPSCs) 2, 3, stem cells from exfoliated deciduous teeth (SHED)4, periodontal ligament stem cells (PDLSCs)5, stem cells from apical papilla (SCAP)6, 7, dental follicle progenitor cells (DFCs)8 and gingival tissues derived from mesenchymal stem cells (GMSCs)9, 10.

The majority of dental-derived stem cells (DSCs) can be obtained from human exfoliated deciduous teeth and orthodontically extracted premolars and third molars. These sources of stem cells are considered biological waste in dentistry despite containing multipotent stem cells4, 11. Premolars are the most common teeth indicated for extraction in orthodontics and are often ideal for the relief of anterior and posterior crowding. However, the decision of extracting between the first or second premolars depends on several factors, including the anchorage requirement, the severity of crowding, and the amount of overbite and overjet12. Extraction of the third molar for orthodontic reasons is rare, as both orthodontists and oral surgeons must make appropriate decisions and consider the potential risks and benefits of the procedure specifically pertaining to the surgical removal of asymptomatic impacted third molars13. There are several orthodontic reasons for the extraction of third molars that aim to prevent late mandibular incisor crowding14, to allow molar distalization15 and to prepare for orthognathic surgery16.

Experimental research using stem cells showed that they produce reliable and effective tissue regeneration. However, there are several shortcomings in terms of the clinical application of therapy using MSCs17: (i) the host immune response might cause rejection of transplanted MSCs in the long term, (ii) there could be an imbalance and disturbance in the homeostasis of local tissue, (iii) the risk for tumor formation may increase due to long-term expansion and/or due to immunosuppression of the local tissue, and (iv) the chances for ectopic tissue formation by the transplanted MSCs become higher13.

To overcome these drawbacks of using MSCs, an increasing number of studies have reported the use of the MSC secretome18. Secretome are various cellular products, such as cytokines, growth factors and enzymes secreted by MSCs19, 20. These biologically active chemical products play significant roles in diverse aspects of tissue function, repair and homeostasis21, 22. The function of the secretome is to suppress immune responses23, reduce oxidative stress13, stimulate angiogenesis24, and induce the recruitment, proliferation and differentiation of endogenous stem cells25. Secretome are easily obtained via culture of various cells, where they are contained or extracted from the culture media, often referred to as conditioned media (CM). In various studies, CM from stem cells has shown promising results in regenerative medicine and tissue engineering26, including CM from SHED and CM from healthy and inflamed periodontal tissue27, 28.

When compared to stem cell therapy, secretome therapy offers potential benefits because it can reproduce most of the advantages of cell-based therapies with minimal side effects. Furthermore, using the secretome as therapy provides many practical advantages, such as ease of preservation, sterilization, and packaging1. Apart from that, they also have an extended duration of storage without losing their properties29, 30, ease in gauging proper dosages and can be produced in mass20 as well as a noninvasive extraction procedure that may save both time and cost of production24. Despite the potential benefits of the secretome, the effect of the dental-derived secretome on specific types of tissue has not been systematically reviewed. The focus question is to systematically review , and clinical studies on the effect of the dental-derived secretome in terms of tissue regeneration potential. Thus, we aim to identify the most prominent effect of using a dental-derived secretome on tissue regeneration using a systematic review approach.

Methods

The Preferred Reporting Items for Systematic Review (PRISMA) checklist was used as a guideline in conducting this review31. A comprehensive and systematic electronic search of MEDLINE via PubMed, Scopus and Wiley online library databases was conducted for studies published in English up to October 2020 (Figure 1).

Results

Discussion

Most of the studies reviewed here were preclinical and animal studies reporting possible mechanisms of secretome effects, which can be grouped into neurogenic, angiogenic, osteogenic and odontogenic. When compared to the list of proteins detected in the secretome, a correlation can be observed between the type of protein found and the effects seen. As an example, TGF-ß1 is a mediator of osteogenic differentiation and was repeatedly reported to be a component of secretome from various DSCs. Furthermore, NGF, NT-3 and BDNF are well-known neurotrophic factors70, 71, 72 that correlate with the neuroregenerative effects seen in a large proportion of the studies here.

While there are various sources of the DSC secretome, those obtained from the culture of DSCs acquired from permanent teeth (such as DPSCs and PDLSCs) as well as deciduous teeth (called SHEDs) were mostly reported. This is well explained by the fact that such cells are the most easily accessible for the isolation of stem cells73. However, not all studies confirmed their claim that the cells they used are stem cells, as only 19 studies reported performing stem cell marker analysis. Other studies that did not report performing this analysis referred to previous publications in their methodology sections. This step is crucial, as it confirmed the stemness of the cells obtained from the various intraoral sources74.

At present, clinical studies reporting the effectiveness of the DSC secretome in clinical tissue regeneration are still absent. Until June 2021, there were only three ongoing clinical trials using the MSC secretome registered with the US National Institute of Health (https://clinicaltrial.gov), with the secretome used in all three studies reported to be from bone marrow MSCs. This could be due to a number of reasons. First, the potential of the DSC secretome to instigate an adverse reaction may not have been well studied. This is reflected in an article search on Scopus using the terms “secretome OR (conditioned media)” AND “toxic*”, which returned a result set that was overwhelmingly positive in nature. While this is encouraging, the lack of reports on the propensity of the secretome to cause any adverse biological response will hinder their clinical translation, as the content of the secretome varies and is likely to be antigenic in nature when administered75.

Hence, the important nonclinical studies that need to be investigated further are on the inflammatory reaction at the local and systemic levels. Although the PDLSC secretome was found to be able to suppress the proinflammatory cytokine expression (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β) of the surrounding healing tissues after 5 days of implantation in an periodontal defect, the findings were not statistically significant compared to the control group45. In another study in which the PDLSC secretome was used on the spinal cord of a multiple sclerosis animal model, there was reduced expression of proinflammatory mediators (IL-17 and interferon gamma (IFN-γ))48. Other studies that showed immunosuppression of proinflammatory cytokines in animal models include the secretome originating from human SHEDs65, 40;43, human DPSCs52 and human GMSCs53. However, one study showed a contradictory finding that the DPSC secretome caused increased expression of proinflammatory cytokines such as IL-1α, IL-6 and IL-839. To advance knowledge regarding the secretome’s biocompatibility, more similar studies are needed to help with efforts to test the secretome in preclinical and clinical human studies.

Another possible reason for the lack of clinical studies would probably be the difficulty in determining the exact preparation of the secretome to be used. The preparation needs to ensure that the contents of the secretome are properly preserved with minimal protein degradation and that it has adequate shelf life. As mentioned in our results section, multiple lines of evidence were found with regard to the composition of the secretome, which includes a vast range of growth factors and cytokines76.

The results from the proteomic analysis in this review reveal that insulin growth factor-1 (IGF-1) was found only in rat DPSC-CM56, while no IGF-1 was detected in human DSC-CM. IGF-1 is a small peptide with 70 amino acids and has autocrine, paracrine and endocrine effects. The synthesis of IGF-1 mainly comes from the liver77, and it is an essential mediator of cell growth, differentiation and transformation78, 79. The findings from this review are in line with the results from Caseiro et al., who performed a study to compare the profiles of metabolomic and bioactive factors of the human umbilical cord and DPSC secretome80. The profiling revealed that no IGF-1 was found from the secretome analysis. The lack of IGF-1 expression in the human stem cell secretome, particularly DSC-CM, might contribute to the binding of this hormone with IGF-binding proteins (IGFBPs). IGFBP expression depends on the tissue site and developmental stage at different concentrations in different body compartments81. As shown by the results from this review, IGFBP-2, IGFBP-3, IGFBP-4 and IGFB-6 were expressed by different types of DSC-CM, such as human DPSCs, SHEDs and PDLSCs.

In addition, the difference in the expression and concentration of the factors may be due to the difference in the methods used to collect the conditioned medium. The current study also found that even when the CM is derived from the same type of cells, the expression of the growth factors varied. This result may be explained by the difference in cell numbers, culture medium and condition as well their CM processing method82.

Apart from various growth factors and cytokines produced by the DSC secretome, this review focused on the effect of the DSC secretome on tissue regeneration. The most abundant evidence was shown in neuroregeneration studies, which was supported by the release of numerous growth factors (NGF, BDNF, NT-3, NTF, GDNF and HGF). These neurotrophins promote the differentiation and growth of neurons and are paramount in treating neurodegenerative diseases as well as neural injuries51414843. Other than neuroregeneration, the DSC secretome has the potential to promote bone regeneration, probably due to the increase in the migration and mineralization potential of the surrounding osteoprogenitor cells by TGF-β183. The TGF-β1-BMP (bone morphogenic protein) signaling pathway has a pivotal role in osseous regeneration through the elevated expression of osteogenic genes in the targeted cells84, 85. This review also found the potential of DSC secretome in dentine formation for pulp protection, periodontal regeneration and other tissue regeneration, such as cartilage, salivary duct cells, liver and lung tissues, but the mechanism of action is still inconclusive.

Conclusion

In conclusion, evidence showing the effectiveness of the DSC secretome in neuroregeneration and bone regeneration is encouraging. However, data from human studies are still lacking, which could impede the translation of such works into the clinical perspective. Furthermore, the potential for the secretome to be used in the regeneration of other tissue types, such as smooth and skeletal muscle, needs to be explored, as secretome content analysis has shown the presence of the relevant factors.

Abbreviations

BDNF – brain derived neurotrophic factor

Acknowledgments

None

Author’s contributions

All authors were involved in the selection of the papers for this study. All authors read and approved the final manuscript.

Funding

The study was personally funded.

Availability of data and materials

Not applicable.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.