Oral tissues regeneration using intraoral mesenchymal stem cells

Background Oral pathologies or some treatments can cause facial and functional alterations, being fundament to retrieve those functions restoring the original anatomy of the lost tissues. On this purpose, various techniques have been studied, one of these was the tissue engineering. Mesenchymal stem cells (MSC) are multipotent adult stem cells. The MSC in the oral cavity have been striking for regenerative therapies by its high plasticity, good interaction with scaffolds and growth factors, good proliferation and differentiation, they are also easy to obtain. Objective: The objective of this study was to describe the current uses of the intraoral MSC for the regeneration of the tissues of the oral cavity. Material and Methods An electronic research was made in the databases PubMed, Cochrane Library, Google Scholar, Scopus and EBSCO between 2000 to 2018. Results 21 articles were included. 13 were studies in vivo and 8 were studies in humans. The site mostly used as a giver site was the dental pulp. Intraoral MSC are able to regenerate the pulp dentin complex, alveolar bone and periodontium. Conclusions Intraoral MSC come from easy access areas, less traumatic interventions and have high potential to regenerate intraoral tissues in comparison to MSC from other sites of the body which allows a more predictable oral tissues regeneration. Key words:Oral stem cells, oral cavity, regeneration, tissue engineering.


Introduction
Mesenchymal stem cells are multipotent adult stem cells. They were discovered by Friedenstein and his collaborators in the 70s, who conducted studies to determine the biological characteristics of mesenchymal stem cells derived from the bone marrow (1). One of its main functions is to maintain and repair cells in the tissue in which they are found, as well as maintain the cell population. Among its most important characteristics is that they have the ability to differentiate into adipocytes, chondrocytes and osteoblasts in in vitro conditions. In addition, mesenchymal stem cells have the ability to evade the immune system by being immunomodulatory, which allows them to be used with therapeutic roles (1,2).
The first site intervened to obtain mesenchymal stem cells was the bone marrow of adult patients. Although its characteristics were optimal, it was observed that the number of progenitor cells in adult tissue was quite low compared to the total number of cells extracted, in a ratio of 1 / 104-106. This is why it was necessary to carry out in vitro expansions to increase their number. In addition, the number of cells decreased as a function of increasing the patient's age (3). On the other hand, obtaining mesenchymal stem cells from the bone marrow proved to be a very invasive, painful procedure with infectious complications (1).This is why they started looking for new sites that had mesenchymal stem cells which would allow a minimum of discomfort for the patient and that were present in greater quantities (1,4). From this, it is that the oral cavity became one of the most accessible sites for obtaining mesenchymal stem cells (1), in which different sites have been identified that possess them such as the bone marrow of the alveolar bone (BMSCs), the oral mucosa (OMSCs), the periosteum (PSCs), the salivary glands (SGSCs), the adipose tissue (ASC), the dental pulp (DPSCs), the dental pulp of exfoliated teeth (SHEDs), the periodontal ligament (PLSCs), the dental follicle (DFSCs), the dental germ (GDSCs), the apical papilla (SCAP) and the inflamed periapical tissues (iPAPs) (5). Existing reviews describe mostly in vitro studies, with bone regeneration being the most performed action. Therefore, the aim of this review is to extend the search of the different regenerative uses that intra-oral mesenchymal stem cells present not only at the level of bone EBSCO "stem cells" AND ("periosteum" OR "apical papilla" OR "periodontal ligament" OR "dental follicle" OR "periodontium" OR "alveolar bone" OR "exfoliated deciduous teeth" OR "gingival tissue" OR "tooth germ" OR "dental pulp" OR "buccal fat pad" OR "salivary glands" OR "periapical inflamed tissue" OR "mouth") AND ("regenerative medicine" OR " tissue engineering") Scopus ("stem cells" AND "mouth" AND "tissue engineering ") Google Scholar "oral stem cells" and ("regenerative medicine" or "tissue engineering") regeneration, but also to describe their uses in other types of tissues of the oral cavity, including exclusively studies in vivo or in humans.

Material and Methods
A review of the literature was carried out between 2000 and 2018 in the databases PubMed, Cochrane Library, EBSCO, Scopus and Google Scholar, performing the search strategy detailed in Table 1.
All studies carried out in vivo and in humans, prospective and retrospective cohort, clinical trials, case/control studies, including articles in English, Spanish and French, where intraoral mesenchymal stem cells were used (regardless of the autologous intraoral donor site) to regenerate intraoral defects were included. We excluded all those in vitro studies, studies that did not specify the mesenchymal stem cell used, studies that included patients or animals with some underlying disease or that were under pharmacological therapy that affected the regeneration of tissues, immunocompromised or immunosuppressed, patients that had been irradiated in the craniofacial region in the last 6 months or were in treatment with intravenous bisphosphonates and in the case of orally bisphosphonates should not be more than 3 years.

Results
From the electronic search, a total of 1428 publications were found, selecting a total of 21 articles according to the inclusion and exclusion criteria (Fig. 1, Table 2  Both groups showed bone regeneration, the difference was not statistically significant. DPSCs are capable of regenerating alveolar bone suitable for implants. Complete regeneration of dental pulp regeneration of less mineralized dentin. Bone regeneration when using together mesenchymal cells of the adipose body of the cheek in conjunction with the iliac crest graft. -Periodontal defect is regenerated mesially of the first mandibular molar of each dog.
-Fill the defect with SC previously obtained from the granulation tissue.
Cells of alveolar granulation tissue promote periodontal regeneration (LP, cement and bone) -Incisions behind the upper incisor perpendicular to the bone crest -Periosteum within these incisions alone or with collagen sponge.
-Same procedure in the tibia of each mouse.
Little regeneration with tunneling technique, good regeneration when using collagen sponge.

DPSCs
Atelocollaen Dental pulp 6 Beagle female dogs, between 9 to 11 months of age.
-Sample of adipose tissue of the abdominal subcutaneous and bone marrow of the sternum.
-SC transplant inside the incisor duct.
Greater regeneration of the pulp matrix when using the SC from the bone marrow and fat cells compared to those of the dental pulp, but with the DPSCs, more vascularization and innervation was seen.  years.
-Case 1: Cells extracted from the immature apex.
-Case 2: Cells of the pulp of exfoliated teeth.
-Case 3: Cells of the apical papilla of a third molar -In all three cases, the SRC of the teeth was filled with the SCs next to the injection-shaped scaffolding.
-Regular clinical and radiographic controls and obturation with gutta-percha.
-Case 1: Apical closure, decrease in the radio apical and asymptomatic lucidity.
-1 year after the rest of the incisors and premolars were removed and 32 root implants were created with the teeth.
-Filling with the collagen scaffold with DPSCs and implanted in the alveoli after extraction of the jaw. -5 alveoli without cells with scaffolds were the negative controls.
Formation of a new dentin matrix within the canal. Both scaffolds worked the same. -Revascularization treatment.
-Third session the blood samples were taken in order to compare the mesenchymal cells and their maracadores in the apical blood and systemic blood.
Apical regeneration using the revascularization technique.
The apical bleeding contributes the mesenchymal cells that reach the canal and allow the apical closure.
Iohara et al. -Agmentation of the apical foramen 0.7 mm -Fill the ducts with the cells and the scaffolding.
The use of CD105 + MSC of the dental pulp with SDF-1 allows the complete regeneration of the dental pulp with vessels, nerves and dentine in addition to the apical closure. The fat cells showed low regenerative potential, dental pulp cells also regenerated, but in lesser quantity. -Extraction of the first molar and premolars extraction of DPSCs.
-Bone defect employment -Sample of the bone marrow of the iliac crest.
-Prepletion of defects in a random manner with PRP, PRP + pulpal mesenchymal cells and PRP + SC of the bone marrow.
Good bone regeneration with the cells of the dental pulpy of the bone marrow. There was no rejection in the SHEDsde puppy experiment.
Ito et al. DPSC greater osteogenic potential compared to bone marrow cells. Both were able to integrate the implant. The periosteal cells that were also evaluated did not manage to regenerate much bone or osseointegrate.
Yamada et al.
DPSCs PRP Alveolar bone y Adult dogs -Extraction of the first molar and premolars to dogs and DPSCs.
-Adjustment of the bony defect in the jaw.
-Sample of the bone marrow of the iliac crest of each dog.
-Fill the defects randomly with PRP, PRP + DPSCs and PRP + SC of the bone marrow.
Good bone regeneration with both DPSCs and bone marrow.
There was no rejection with SHEDs of puppies. The regenerated bone in all cases was suitable for implants.
Dentine formation when using the pellet with BMP2 in the middle and pulp mesenchymal cells.
13 (61.9%) corresponded in vivo studies and 8 (38,1%) corresponded humans studies. In relation to the mesenchymal stem cells, the mesenchymal stem cell of the dental pulp was the most used in a total of 14 articles (60.8%), followed by the cells of the apical papilla used in 2 articles (9.5%) (Fig. 2). In relation to the scaffolds the most used was atelocollagen, used in 5 (23.8%) articles followed by PRP (platelet rich plasma) used in 3 (14.2%) articles. 38% of the included articles had as main objective the use of mesenchymal cells is pulp regeneration, followed by alveolar bone regeneration with 37% (Fig. 3).

Discussion
Clinical application and regeneration of oral tissues -Regeneration of the dentin-pulp complex 38% of the included studies studied pulp regeneration, and 21% dentin. Of the studies that evaluated the pulpal regeneration, in 44.4% they also evaluated the dentinal regeneration; therefore, it is seen that there is a relation between pulp and dentine regeneration.  tometry which allowed them to obtain cells with surface markers CD31 -/ CD31+ and CD146-which would correspond to a sub fraction of these DPSCs. The results obtained by a histological analysis showed the neoformation of vascular and neural tissue, as well as dental pulp inside the teeth that obtained cell transplantation CD31-/ C146-and CD31+ / CD146-. But better and greater regeneration was obtained when using cells with the surface markers CD31-/ C146-.
Other studies have seen the potential of CD105+ surface markers found in DPSCS. Iohara et al. in 2011 (7) con-ducted a study where they managed to isolate this cellular sub fraction by using Stromal Cell Derived Factor 1 (SDF-1). Complete pulp regeneration in dog's teeth was also observed by histological analysis.
According to the previously studies, pulp regeneration seems to be more effective when using mesenchymal stem cells with CD31-or CD105+ compared to those where they only use pulp mesenchymal stem cells without determining the sub fraction (8,9). Other cell isolation techniques have been further investigated since the safety of cells with CD31-and CD105+ surface markers isolated by flow cytometry has not been established and the use of SDF-1 has not been approved for clinical use (9). This is why Murakami et al. in 2015 (9) used granulocyte colony stimulating factor (G-CSF) to induce mobilization with the aim of isolating the sub fractions of the mesenchymal pulp stem cells. The results obtained cells with a cell phenotype similar to cells that have CD105 + with high angiogenic and neurogenic potential (9). Iohara et al. in 2013 (10) managed to regenerate whole pulp with the combination of G-CSF and DPSCs. When compared with the control, it was observed that the combination of both achieved the highest regenerative potential (9,10). Similar results were observed by Murakami et al. (9) who also used G-CSF and DPSCs achieving complete pulp regeneration with high rate of angiogenesis and neurogenesis. In 2016, Iohara et al. (11) conducted a new clinical trial in dogs where they used the same technique used in 2013 by this same author (10) who this time treated teeth with pulpal diagnosis of irreversible pulpitis. Unlike the studies mentioned above (6,7,9,10) the evaluation of the animals was by magnetic resonance. The results of this study were also compared with histological methods and sensitivity test. The images made it possible to show that after 180 days there was regeneration of a more radiolucent dentinal tissue, which suggests that there is less mineralization of the dentin formed with the transplant of DPSCs (11). Mangione et al. (12)In 2017 they performed a Split Mouth randomized study in minipigs where, when comparing intervened and non-intervened teeth, pulp regeneration was not found when grafting DPSCs, an osteodentine with different characteristics was formed to a normal dentine. In the study by Iohara et al. in 2011 (7) they observed dentinal regeneration after transplantation of DPSCs. Therefore, they conclude that the DPSCs could allow the regeneration of the entire dentin-pulp complex. Kodonas (16) had already tested dentinal regeneration when using humans but in immunocompromised mice and performing transplantation of xenogenic type. It was in the investigation of Iohara et al. in 2004 (14) that dentin was regenerated with autologous transplantation and in healthy animals without the requirement of immunocompromise or immunosuppression. By joining the MSCs with a pellet with BMP-2 in vitro, the extracellular matrix of the pellet can be used as a scaffold to manipulate the growth of cells in odontoblasts prior to transplantation. By establishing and optimizing this technique, a treatment with relevance for endodontic and cavities treatment could be achieved (14). Studies in humans have also been conducted to achieve regeneration of the dentin tissue. In 2011, Lovelace et al. (17) conducted a clinical trial in which they performed a revascularization treatment in 8 patients with immature teeth and apical periodontitis. Both imaging and histology showed the apical closure of these immature teeth. This apical closure is explained by the presence of mesenchymal stem cells that reach the interior of the canal through apical bleeding. It is thought that said cells are SCAPs carried by the blood. However, a histological study conducted in 2010 by Wang et al. (18) showed that after a revascularization treatment in immature teeth diagnosed with pulpal necrosis or apical periodontitis, an apical closure was achieved but the root canals of the tooth were filled with ectopic bone tissue, fibrous tissue and cement apposition (11,18). More recent studies such as that of Nakashima et al. in 2017 (19) carried out a pilot study where they transplanted DPSCs obtained from teeth with irreversible pulpitis. Clinically, histologically and imaging, regeneration of the pulp and dentin could be seen with a sensitivity and vitality of the pulp almost normal in pulpectomized teeth. that the regenerated alveolar bone where DPSCs were implanted was more mature and of higher quality than in the control group after 60 days had elapsed. In addition, the bone regenerated with cells was apt to be rehabilitated based on IOI. However, in the study by Barbier et al. in 2018 (24) filled out post-extraction alveoli with DPSCs and a collagen scaffold and did not find a greater bone regeneration of the defect due to impacted third molar extraction when using DPSCs. Mesenchymal stem cells of the periosteum have been able to regenerate alveolar bone. Mouraret et al. in 2014 (25) conducted a clinical trial in animals where they created a vertical defect in mice at the palatal level which was filled with a collagen sponge with periosteum. It was compared to a tunneling technique without the use of a scaffold that was a collagen sponge. When the collagen sponge was not used, bone resorption was observed. Therefore, it was demonstrated that the periosteum has mesenchymal stem cells that participate in bone regeneration, but with the help of a scaffold such as collagen, for example. Shiehzadeh et al. 2014 (26) reported three cases in which they sought to repair bone defects of periapical lesions in teeth with apical peridontitis. For this, in three different patients, they carried out protocols with different mesenchymal stem cells. The first case used SCAPs, the second case used SHEDs and the third case used cells obtained with a new method in this study that obtained cells from the periapical tissues through the canal (iPAPs). With a radiographic method it was possible to see the repair of the lesions together with the apical closure of the immature teeth, clinically there were no complications or symptomatology after the treatment.  (22) also compared DPSCs and SHEDs with the BMSCs of the iliac crest but this time in order to regenerate bone alveolar. The results showed a good bone regeneration by the three types of cells, and bone apt to rehabilitate with implants. Therefore, DPSCs and SHEDs could be an alternative to regenerate bone, obtaining the same results as with BMSCs. In the study by Ito et al. in 2011 (20) they also compared the DPSCs with the BMSCs to regenerate the alveolar bone, they observed greater osteogenic potential from the DPSCs, although both cell types regenerated bone suitable for osseointegrating implants. Finally, there are intraoral studies with extra oral mesenchymal stem cells specifically of adipose tissue of the abdomen. In the study by Murakami et al. 2015 (9) they compared the DPSCs with the ASCs of the abdomen. The results were the same as with the BMSCs, that is, obtaining pulp matrix but with less vascularization and innervation compared to that obtained with DPSCs. Iohara et al. in their 2011 study (7) compared DPSCs with ASCs in order to regenerate dental pulp and dentin for apical closure. The histological results showed a low potential of the fat cells to regenerate the dentin-pulp complex.
In conclusion, although most of the studies were performed in vivo, it can be noted that PLSCs, DPSCs, ASCs, SCAPs, SHEDs were able to regenerate alveolar bone, DPSCs and SCAPs the dentin-pulp complex and the e277 TGSCs the periodontium. It should be noted that the studies used in this review are not of high quality, so there is a need in the literature to conduct more randomized controlled clinical trials with a larger sample size and homogeneity in follow-up times so that the results obtained are truly significant. and extrapolated to clinical use. It is necessary to mention that not only the cells influence the results, so the use of different scaffolds and biomolecules in the environments in the different studies means that these are not really comparable. This is why it is necessary to standardize the studies with the same scaffolds and biomolecules in order to effectively compare the results obtained using mesenchymal stem cells from different areas in the same environment. Although to date it can be concluded that the results obtained when regenerating using mesenchymal stem cells are positive, it is necessary to determine the best scaffolding and the best means to obtain the best results.