Can SHED or DPSCs be used to repair / regenerate non-dental tissues ? A systematic review of in vivo studies

Dental pulp has been identified as a novel and promising stem cell source. The following systematic review presents and summarises in vivo studies that have used stem cells from the dental pulp of permanent and deciduous teeth to repair or regenerate non-dental tissues. An electronic customised search was performed using 4 different databases (Entrez PubMed, Cab Abstracts, Scopus and Web of Science). Only full-text research manuscripts published in English between the years of 2000 and 2012 were included. The manuscripts were retrieved based on the following keywords and/or abbreviations: [Stem Cells from Human Exfoliated Deciduous teeth (SHED)] AND/OR [Dental Pulp Stem Cells (DPSC)] AND [tissue regeneration] AND [tissue repair]. Only manuscripts involving in vivo applications of SHED or DPSC for the repair and/or regeneration of non-dental tissues were included. The search strategy produced 2309 papers, from which 14 were eligible according to the predetermined inclusion and exclusion criteria. Although human tissue was the source of cells in half of the studies included in our review, all of the studies involved transplantation into animals of other species, such as pigs, rats and mice. Most of the manuscripts reported the successful use of DPSCs or SHED for non-dental tissue repair or regeneration. While these cell populations represent promising alternative sources of stem cells for tissue engineering and cell-based regenerative medicine therapies, it is not yet possible to guarantee the appropriate clinical management of this technique.


Introduction
Stem cells are a promising tool for the treatment of many different human diseases.An enormous variety of stem cells have been isolated and studied from different tissues, such as bone marrow, 1 adipose tissue, 2 skin, 3 and umbilical cord. 4Among them, mesenchymal stem cells (MSCs) are the most promising for clinical purposes. 5ecently, MSCs were identified in the dental pulp.These cells represent a novel and promising stem cell population with self-renewal and multilineage differentiation capacities that are similar to other MSC populations. 6,7ince the discovery of stem cells in the dental pulp of permanent 6 and deciduous teeth, 8 a large number of animal studies have evaluated the suitability of these cells for dental 9 as well as non-dental 10,11 tissue engineering applications.Here, we present a systematic review of in vivo studies investigat-ing the use of dental pulp stem cells for the repair or regeneration of non-dental tissues.

Methodology
Two independent researchers, following the PRISMA guidelines, 12 carried out an electronic customised search of scientific papers published between 2000 and 2012 using the Entrez PubMed, CAB Abstracts, Scopus and Web of Science databases.The following combination of keywords and abbreviations was used: [

Results
Our search yielded a total of 2309 papers.Based on the abstracts, 1072 papers were excluded according to the previously mentioned inclusion/exclusion cri-teria.An additional 1221 papers were determined to be duplicates and were also excluded.Finally, of the 16 selected manuscripts, 2 were excluded because the full-text was not available in English.Thus, 14 manuscripts formed the basis of this review (Figure 1).The included manuscripts are summarised in Table 1.
In 7 of the studies included in this review (50%), the cells were isolated from human tissue.In all 14 studies, the cells were transplanted into animals of other species, including pigs, rats and mice.
Eight manuscripts investigated bone tissue repair and/or regeneration.Liu et al., 10 Seo et al., 13 de Mendonça Costa et al. 14 and Riccio et al., 15 investigated new bone formation in critical-size calvarial bone defects, while Yamada et al., 16 and Zheng et al., 17   TCP), 13,18 recombinant human nanohydroxyapatite/ collagen/poly (L-lactide) (nHAC/PLA), 10 platelet-rich plasma (PRP), fibrin, 17 bone morphogenetic protein 2 (rhBMP-2) 10 and collagen membranes 16 were used.Three groups of researchers used cells from deciduous teeth in their experiments, 3 groups used cells from permanent teeth and the remaining 2 groups compared both types of cells.Notably, both sources of cells (i.e., the pulp of deciduous and permanent teeth) presented bone-formation capacity 13,14,15,16,18 and, in some cases, their osteogenic ability was superior to that of bone marrow-derived SCs. 14,15n addition, DPSCs were shown to promote the repair of facial nerve gaps in rats 11 and induce neuroplasticity within the recipient nervous system in chicks. 20In another nerve repair study, both SHED and DPSCs promoted the recovery of hind limb locomotor function in rats. 21DPSCs also exhibited therapeutic potential for the repair of a myocardial infarction (MI) in a MI mouse model.Although the cells were able to engraft into the infarcted heart, they did not differentiate into cardiac, endothelial or smooth muscle cells.Nevertheless, angiogenesis was increased compared to that observed in animals that received a control treatment. 22Angiogenesis was found to be improved when endothelial progenitor cells from swine deciduous and permanent teeth were used to treat ischemia in a hind limb ischemia mouse model, as the cells helped to increase the blood supply and contributed to the formation of a new capillary network. 23Lastly, SHED were reported 24 to help accelerate wound healing when associated with fibroblast growth factor (b-FGF).

Discussion
Regenerative medicine is defined as the use of a combination of cells, engineering materials and suitable biochemical factors to improve or replace biological function. 25Thus, regenerative medicine treatments have the potential to restore damaged tissues and organs.Importantly, the regenerative potential of DPSCs and SHED has been studied in a variety of non-dental tissues.In vivo studies have shown that these cells can form bone, blood vessels, nerves, myocardium and skin.However, the majority of the studies described in the literature have investigated bone formation. 10,13,14,15,16,17,18,19ur systematic review aimed to analyse in vivo studies that were performed in humans and animals.The keyword [animals] was not an inclusion criteria, nor was [human] an exclusion one.Nevertheless, we did not find any in vivo studies in humans.Dental stem cells were first isolated from the dental pulp 6 of permanent teeth and subsequently from the dental pulp of deciduous teeth. 8Because of the relative novelty of this research area, there is most likely insufficient research to enable the approval of human testing or clinical protocols.
Apparently, SHED, which can differentiate into neural cells, adipocytes, osteoblasts and odontoblasts 8 have higher plasticity than DPSCs.In vivo, SHED are capable of spontaneously generating robust amounts of bone, 8,20 while DPSCs can only form bone and dentine. 6This difference suggests that DPSCs are distinct from SHED with respect to odontogenic differentiation and osteogenic induction.Furthermore, both DPSCs and SHED have a higher proliferative capacity than other well-known MSCs, such as bone marrow stem cells. 6,7SHED, in particular, are considered an outstanding source of stem cells.Considering that humans typically exfoliate 20 deciduous teeth throughout life and quite often have impacted and unerupted third molars extracted for clinical or orthodontic purposes, 26,27 teeth could represent an important, easily accessible and non-invasive (particularly for the deciduous teeth) source of stem cells.
As previously mentioned, most of the selected manuscripts in this review studied the use of DPSCs and SHED for bone tissue repair or regeneration. 10,13,14,15,16,17,18,19The use of SHED or DPSCs may avoid the inconvenience and morbidity associated with the removal of autogenous grafts from other sites. 28oreover, in contrast to the use of autologous bone grafts, DPSCs or SHED can be expanded in vitro prior to their use in vivo to generate an adequate number of cells for the tissue being repaired.This approach could prevent or decrease problems associated with autogenous grafting techniques, such as the limited amount of tissue that can be removed from the donor site and the risk of infection.
In general, SHED and DPSC-based bone repair/ regeneration strategies were found, at least in animals, to significantly increase bone formation compared to the control groups studied. 13,14,15,16,17,19However, Liu et al., 10 found no significant differences in bone formation when DPSCs associated with nHAC/PLA and rhBMP-2 were used compared to autologous bone.In this study, the nHAC/PLA + DPSCs + rhBMP-2 composite did prove to be suitable for maxillary bone tissue repair, but DPSCs were apparently not required for successful regeneration.Similarly, Zhang et al., 19 did not observe bone formation when rat or human DPSCs associated with HA/TCP were transplanted subcutaneously in nude mice.However, it should be noted that, according to these authors, a high number of cell passages was needed to obtain enough viable cells for the experiments.Furthermore, in contrast to their in vivo experiments, all four types of cells used by Zhang et al., 19 (i.e., rat bone marrow, rat DPSCs, human bone marrow and human DPSCs) were able to form mineralised tissues in vitro.DPSCs have also attracted interest as an alternative to improve the outcome of dental implants, 18 and this cell population has been shown to possess higher osteogenic potential than bone marrow stem cells, which are still considered the gold standard for bone tissue formation.
The analysis of the neural repair capacity of DPSCs and SHED was ambiguous, in our opinion, due to the differences in the methodology used by the different authors. 11,20,21DPSCs are apparently able to stimulate the formation of blood vessels and myelinating tissue in the peripheral nerves of adult rats.However, the authors did not perform any functional assays to prove a gain of function. 11To investigate this limitation, a more recent study showed that engrafted SHED and DPSCs were able to differentiate toward mature oligodendrocytes and promote functional recovery of the peripheral nervous system in hind limb defects in a rat model.The rats that received SHED were able to walk without weight support, while those that received bone marrow stem cells or fibroblasts exhibited only subtle joint movements. 21PSCs have also been reported to have potential for use in cell-based therapies for systemic diseases, such as cardiac disease. 22,23When DPSCs were injected into mice with myocardial infarctions, they had a significant effect on recovery compared to the control group.Notably, none of the injected cells differentiated into cardiac, endothelial or smooth muscle cells.Rather, according to the authors, the fact that the treated mice had a significantly improved recovery suggests that DPSCs can contribute to cardiac repair by some other mechanism. 22This result agrees with previous reports that support the ability of MSCs to induce cardiac repair, despite extremely rare fusion/ differentiation events. 29The benefits resulting from DPSC transplantation were possibly due to the secretion of paracrine factors. 22ore recently, it has been shown that it is possible to isolate a sub-fraction of endothelial cells from dental pulp tissue based on the expression of cell surface markers. 23These cells have been shown to have a high proliferative and migratory capacity as well as multilineage differentiation potential, including angiogenic, chondrogenic, adipogenic, neurogenic and odontogenic-potential.When these cells were applied in a mouse model to reverse hind limb ischemia, the results demonstrated successful engraftment and an increase in blood supply due to the formation of new capillaries.Hence, the use of endothelial progenitor cells derived from dental pulp might represent an alternative approach for the transplantation of autologous endothelial progenitor cells in which cells are obtained by an invasive and potentially painful biopsy procedure. 30astly, Nishino Y et al., 24 showed that the use of SHED, in association with basic fibroblasowth factor (b-FGF), in a nude mouse full-thickness skin defect model significantly accelerated wound healing compared with other groups.This work highlights SHED as a promising stem cell population for future wound healing therapies as well.
The lack of a standardised type and pattern of cell types and scaffolds may influence cellular responses and alter their properties for clinical bioengineering.Indeed, the different types of cells and the different structures and porosities of scaffolds and/or delivery systems are some of the issues that complicated comparisons between the studies included in our review.In addition, without standardised protocols that include cell culture conditions, sources, numbers, passages and their adhesion properties on tissue surfaces, it is not possible to guarantee the appropriate clinical management of these techniques.

Conclusion
In summary, most of the retrieved in vivo studies using SHED or DPSCs focused on bone tissue repair/ regeneration, and the results of these studies indicated that the use of DPSCs and SHED seems to be effective for these applications.However, very few studies were found regarding the potential of these cells to promote functional recovery of neuronal tissue, blood vessels, muscle, cartilage or other tissues.As a result, the potential of DPSCs and SHED for the repair of other non-dental tissues remains unclear.

Figure 1 .
Figure 1.Flow diagram presenting the results of the literature search and the process used to select studies for the systematic review.
Stem Cells from Human Exfoliated Deciduous teeth (SHED)], [Dental Pulp Stem Cells (DPSC)] AND [tissue regeneration] AND [tissue repair].Only full-text research manuscripts that were written in English and described in vivo analysis of tissue repair and/or regeneration of non-dental tissues using SHED or DPSCs were included in this review.Duplicate papers were excluded.

Table 1 .
Studies that used SHED or DPSCs for the repair and/or regeneration of non-dental tissues.