Biology, Preclinical and Clinical Uses of Mesenchymal Dental Pulp Stem Cells

Abstract

Dental pulp is a feasible source of stem cells that could be differentiated into osteoblast providing a therapeutic approach, which contribute to bone regeneration. Furthermore, as dental pulp stem cells originate from the neural crest, they have significant potential in regenerating neural tissues. To isolate dental pulp stem cells, it is not necessary to undergo an additional surgical procedure, they can be obtained from teeth that need to be extracted for specific reasons or naturally shed in children. Dental pulp stem cells have an expansive and clonogenic potential by culturing them in a high-security laboratory. As dental pulp stem cells do not express the Major Histocompatibility Complex, these cells can be used trough a universal donor in several clinical procedures. In this chapter, we present evidence about the role and the applications of DPSCs to regenerate bone as well as in clinic cases to treat neurological affections.

1. Introduction

Dental pulp MSCs (DPSCs) exhibit immunomodulatory, anti-inflammatory, and antifibrotic properties. Thus, DPSCs are used in cell therapies for conditions of chronic inflammation such as autoimmune diseases, as well as for regenerating or replacing damaged cells or tissues [1, 2, 3].

Stem cells have the capacity for self-renewal, generating exact copies that can remain in quiescence or follow the differentiation pathway marked by the signaling pathways to which they are exposed. Stem cells are classified according to their potential for differentiation into multipotent; although they are embryologically derived from the neural crest, they can induce the differentiation of neural progenitor cells into functional adult neurons through their secretome (e.g. Brain Derived Factor, Glial Cell Derived Factor) [1, 4]. DPSCs are located in the center of teeth called dental pulp. DPSCs can be differentiated into several cell types such as osteoblasts, chondrocytes, and muscle cells [5]. Thus, DPSCs are a promising source of stem cells that make then valuable for their use in regenerative medicine and tissue engineering applications [6].

2. Sources for obtaining MSCs from DPSCs

MSCs (MSCs) have been isolated from dental tissues such as dental pulp (DPSCs), periodontal ligament (PDLSC), deciduous teeth (SHED), apical papilla (SCAP), follicles (DFSC), and gingiva (GMSC) [3, 7, 8].

In 2000, Gronthos and cols. Carefully performed extractions from impacted third molars, reporting the first protocol to isolate, expand and cryopreserve DPSCs [9]. In this study, authors showed that adult dental pulps contain clonogenic cells, high proliferation, and tissue-regenerating capabilities. These properties allowed to define them as stem cells. Moreover, they demonstrated that a large number of cells can be obtained from a single tooth. It represented a potential clinical use for cell therapy in the future [9].

The main indications for third molar extraction are:

• Pericoronitis

• Orthodontic reasons

• Associated cystic or tumoral pathologies

• Damage due to resorption to the second molar [10].

Studies have shown that DPSCs can be isolated from human teeth without harming the tooth or the donor.

2.1 Obtaining from deciduous teeth

2.1.1 Exfoliation age

Planning is essential for the collection of dental pulp MSCs, because, it is necessary that the tooth have 1/3 of the root on the day of collection and that the root of the permanent tooth is partially formed and ready to be erupt.

3. Laboratory characteristics of INDEBIOC for manufacturing DPSCs

The MSCs expansion to perform the translation of preclinical into clinical-grade large-scale requires a precise standardization of the procedural parameters such as cell seeding density, culture medium, cultivation devices and laboratory infrastructure.

To ensure that MSCs are consistently produced and controlled according to high quality standards, the good manufacturing practice (GMP) should be employed to produce clinical-grade stem cell products [1, 10, 11, 12]. In this way, the company INDEBIOC has a security 2 laboratory to isolate, expand and cryopreserve MSCs.

INDEBIOC performs high quality control procedures to ensure the identity, potency, purity, and safety of the biological products produced. This is maintained from the arrival of materials to the release of the final product.

Noticeably, the laboratory performs microbiological analysis (aerobic and anaerobic bacteria, viruses, fungi), specially for Mycoplasma to ensure the sterility of the biological products (Figure 1). All cells used in patients are previously tested to ensure the absence of tumor markers. Furthermore, every batch of cells is shipped to specialized companies for a Genetic Test to prove that those cells have a normal karyotype.

Figure 1.

Mycoplasma detection by PCR. Lane 1–6 correspond to samples obtained from the INDEBIOC laboratory with negative results, lane 7 corresponds to the positive control and lane 8 to the negative control.

4. Isolation, expansion, culture, cryopreservation, thawing, and preparation of DPSCs for clinical using

To isolate cells from pulp tissue we collected molars by extraction or by waiting deciduous teeth exfoliation. We use the enzymatic dissociation (ED) method to digest the tissue and disaggregate the cells, which are then cultured in enriched medium. Cells placed in culture plates are maintained into the incubator at 37°C under saturated humidity, and 5% CO₂ conditions. At the moment that cells reach the 70% of confluence several passages are performed until we obtain the desired number of cells. Then, cells are cyoprotected using agents that maintain the integrity of the cells while the temperature is gradually reduced to be transferred to the liquid nitrogen tank, where they are stored until use (Figure 2) [13, 14, 15, 16].

Figure 2.

Isolation of MSCs from different tissues: (A) dental pulp, (B) adipose tissue, (C) olfactory bulb. Secretome INDEBIOCs study.

5. DPSCs characterization

To characterize mesenchymal cells it is needed that cells fulfill the following requirements conventionally established by global literature (Figure 3):

• Fusiform morphology.

• High clonogenicity and short duplication time.

• Positive (CD105, CD90, CD73) and negative (CD38 and HLA-DR) markers analyzed by flow cytometry.

• Differentiation potential to adipocytes, osteoblasts (alkaline phosphatase+ and osteocalcin+), and to chondrocytes.

Figure 3.

Key points for the characterization of DPSC. (A) Representative microphotograph of spindle shaped morphology. (B) Representative image of clonogenecity. (C) Dot plot for the positive markers CD90, CD73, CD105.

6. Mechanisms of action and therapeutic potential of DPSCs

6.1 Safety

Thompson and cols. Reviewed 7473 clinical studies. From those, 55 studies with a total of 2696 patients evaluated fulfill the inclusion criteria indicating the side effects of the intravenous infusion of MSCs.

MSCs as compared to controls were associated with an increased risk of fever, but not acute fever induced by infusion toxicity, infection, thrombotic/embolic events, tumor development nor death [17]. Unlike pluripotent stem cells, multipotent MSCs do not have a tumorigenic potential. However, considering Thompson’s date, we performed a DPSCs tumorigenesis study in immunocompromised mice. We aimed to evaluate the tumorigenic capacity of MSCs derived from dental pulp in 21 immunodeficient mice of the FoxN1nu strain. For this, each mouse was inoculated with 3 million cells in each side of the body (6 million cells per mouse).

Freshly thawed DPSCs were administered in the study group, while HeLa cells were injected as the positive control (Figure 4). Furthermore, mice injected with saline physiologic solution were the negative control was used.

Figure 4.

Developed tumor with HeLa cells 11 × 40 mm. All HeLa mice were sacrificed later. No tumor growth was developed in the mice DPSCs mice.

Malignant transformation of human MSCs has not been directly demonstrated, and attempts to induce a malignant phenotype by long-term ex vivo expansion have been unsuccessful [18, 19]. In contrast, the clinical applications that we have done using MSCs from our laboratory, we demonstrated that MSCs transplantations or infusions are safe.

6.2 Therapeutical mechanism of DPSCs

Years ago, biotechnologists and clinicians considered that the main therapeutic function of mesenchymal cells was to induce their differentiation into functional cells to regenerate organs. Today, we know that paracrine activity of the MSCs is a key mechanism in repairing mechanisms that involves the secretion of proteins/peptides and hormones that MSCs produce.

The secretome of MSCs include the secretion of growth factors, cytokines, and hormones. The MSCs secretome is involve in biological functions that include (1) immunomodulation, (2) anti-inflammation, (3) anti-fibrotic processes, (4) angiogenesis, and (5) induction of differentiation of progenitor cells from other organs (e.g. oligodendrocytes, cardiomyocytes, motor neurons).

Several reports show that MSCs are not constitutively inhibitory, but rather need to be activated by an inflammatory environment in the host for the induction of their immune-regulatory effect [20, 21].

Dra. Leblanc demonstrated for first time that MSCs have immunomodulatory properties [22]. To understand the immunomodulatory clinical potential, it is necessary to understand the dynamic interaction between MSCs and the innate and adaptative immune response [23].

To demostrare the pharmacodynamics of MSCs in humans is difficult, but animal studies have shown that once infused, these cells will not integrate into tissues and organs, but will go through the followed described mechanisms to carry out their therapeutic functions. The presence of living MSCs in the lungs is temporal, within the first 24 h most of them die in the lungs while their secreted molecules are distributed to other sites, mainly the liver. Apoptosis is the primary mechanism of death of MSCs.

Cells that undergo apoptosis release the known “find me signals” that are directly or indirectly produced through the executioner caspases.

Once phagocytized, the second step is the identification of the apoptotic cell through “eat-me signals” by exposing phosphatidylserine on the plasma membrane of the cell [24].

Within the first hours of establishing, the inflammatory response molecules expressed by damaged tissues are recognized by innate effector cells. This triggers phagocytosis promoting the release of inflammatory mediators that initiate the innate immune response, mainly through the activation of phagocytic cells, including type 1 polarized pro-inflammatory macrophages [25].

6.3 MSCs and innate immunity

MSCs promote the formation of anti-inflammatory M2 macrophages by both, cell contact and MSCs-soluble factors secreted such as Prostaglandin E2 (PGE2) and catabolites of tryptophan activity including kynurenine and cyclooxygenase 2 (COX-2) [26, 27]. The polarization resulting from the effect of MSCs on M2 macrophages is related to the ability of MSCs to promote the emergence of regulatory T cells (Tregs) [28]. Tregs are a subset of T cells that play an important role in regulating the immune system and preventing autoimmunity. MSCs induce the differentiation of Tregs from naive T cells through several mechanisms, including the secretion of immunomodulatory factors such as IL-10 [29].

Moreover, MSCs might suppress the proliferation of T lymphocytes through the secretion of β-Transforming Growth Factor, Hepatocyte Growth Factor, Prostaglandin E2, and Indoleamine 2,3-dioxygenase (IDO). The release of these suppressive factors increases after MSCs are stimulated with Tumor necrosis factor (TNF)-alpha and Interferon (IFN)-gamma [30, 31].

Importantly, apoptosis of the MSCs induces receptor mediated immunomodulation. Apoptosis of MSCs is actively induced trough Perforin Dependent Apoptosis by cytotoxic cells; this process is essential to initiate the immunomodulation induced by MSC themselves [32]. Thus, to explore the fate of MSCs could help predict clinical responses.

7. DPSCs for bone reconstruction

Several reports show good results in the application of DPSCs in repairing bone defects. Interestingly, there were no major side effects in animal or human studies, proposing the use of this cells as safe and harmless for health [7, 33].

Bone defects are the result of trauma, resection of tumors, or surgical correction of congenital defects. The use of autologous bone increases donor site morbidity and can cause deformity. In addition, allogeneic grafts also imply risk for infection, disease transmission, and immune rejection [34, 35].

The existing techniques used for the reconstruction of bone deficiencies reflect both, the inadequacies of each method and the significant need to develop novel and improved approaches for bone regeneration.

The use of MSCs, and particularly those derived from teeth, have attracted much clinical and research attention [36].

The ideal strategy to promote bone formation would be the combination of a biomaterial scaffold with a cellular and molecular component that responds to environmental signals or the environment in which they are found. In this way, bone regeneration in critical defects is unpredictable if cells with osteoinductive and/or osteogenic properties are not used.

Bone defects, specially maxillary and mandibular defects of critical size (which do not regenerate on their own) require for their adequate reconstruction to regenerate an adequate volume of bone, both in height and thickness. In addition, it is needed to maintain the regenerated bone as a metabolically active bone [37, 38].

Three key elements are required for bone tissue engineering [39]:

1. Scaffold/matrix

2. Cells

3. Cell modulators and/or regulators

Three types of reconstruction methods are typically used to achieve these goals:

1. Free autografts, a procedure that requires a donor area with a good volume of bone and a recipient area with adequate vascularity and local cell activity.

2. Microvascularized bone grafts, which are a very expensive and time-consuming procedure with a big donor side.

3. Recombinant human morphogenic protein 2 carried in collagen sponge.

We demonstrate that the use of MSCs of dental origin provide adequate results in repairing critical defects, without the morbidity presented by the donor bed of free grafts, and without the high economic cost of microvascularized grafts or morphogenic protein. This, by combined an adequate carrier and osteoconductive materials plus microporosity and small particles that are quickly absorbed after fulfilling their osteoconduction and angioconduction functions.

7.1 Bone regeneration

Bone regeneration requires 3 biological processes: osteoconduction, osteoinduction, and osteogenesis. The activation of tyrosine kinase membrane receptors induce the osteoinductive activity of morphogenetic protein 2 tyrosine kinase membrane receptors activate intranuclear SMADS proteins, which are translocated to the nucleus and activate the bone differentiation gene in the chromosome 3 to differentiate MSCs into osteoblasts that begin to secrete collagen 1 which will be mineralized. This event produce osteopontin, osteonectin, alkaline phosphatase, osteocalcin, which induce bone mineralization.

7.1.1 Preclinical studies

7.1.1.1 Surgical creation of bone defects and bone mandibular regeneration in pig

Here, we present some cases treated with DPSCs for reconstructions of mandibular critical size bone defects (CSBD) in domestic pig’s maxilla and mandible. We performed CSBD based in the extension of the defects, it is between 4 and 6 cm³. In all cases we took out the periosteum that surrounded the defect to avoid the presence and activation of MSCs and pre-osteoblastic cells from this surrounding periosteum [40].

We performed a Control Defect—1100 mm³ reconstructing it without cells (Figure 5). Furthermore, in each mandibular defect we used 6 × 10⁶ human DPSCs/cm³, cultivated in a collagen membrane as a scaffold in a clinical good manufacturing tissue practices settings (INDEBIOC’s laboratory) and covering the reconstructed defects with a titanium mesh to avoid the collapse of the soft tissues over the grafted defects (Figure 6).

Figure 5.

Control defect (left side). Upper molar extraction and alveolar created defect treated with a collagen membrane without DPSC (right side). No bone regeneration 4 months later.

7.1.1.2 Periodontal regeneration in pig’s periodontal induced disease

Figure 6.

New method of therapy for periodontal disease using stem cells in situ in pre-clinical studies. (1) Establishment of periodontal disease, (2) transplant human MSCs, (2a) human MSCs of dental origin, (3) regeneration (immature bone) after transplantation.

7.1.1.3 Critical size defects in pig’s mandibles

Critical defect in pig mandible of 6 cm3 without periosteum did not heal spontaneously. Transplantation of 6 million green fluorescent protein-labeled DPSCs cultured on bovine collagen membrane + demineralized human cadaver bone as osteoconduction, contained by titanium mesh or lyophilized bone were plate to avoid collapse of the tissue engineered graft (Figures 7–11).

7.1.1.4 Created bone defects in the pig mandible mandibular body

Figure 7.

Created critical bone size defect without periosteum and regenerated bone.

7.1.1.5 Created critical size bone defects in pig’s mandibular angle

Figure 8.

Critical bone defects in pig’s mandibular angle. (A) Complete size of the defect. (B) Periosteum removal from masseter muscle and periosteum from medial pterygoid muscle. (C) P full-thickness mandibular inferior alveolar artery and muscles without periosteum to avoid osteogenesis from these structures. (D) Critical size bone defect and pterygoid and masseter muscles without periosteum.

Figure 9.

Critical bone defects in pig’s mandibular. (A) Full size of critical size defect. (B) Placement of lingual (internal) part of the titanium mesh. (C) Placement of the previously mentioned graft based on dental pulp MSCs. (D) Placement of the external side of titanium mesh.

Figure 10.

(A) Re-approach to the grafted site 5 months after the bone and periosteum regeneration with DPSCs. (B) Cleaned sample obtained from the grafted mandible. Same size to the created defect. (C) Bone and periosteum thickness obtained from the DPSCs grafted site.

Figure 11.

(A) Titanium mesh impression which is observed in the periosteum of the DPSCs regenerated bone. (B) Normal macroscopic regenerated bone and periosteum with DPSCs. (C) and (D) Regenerated bone and periosteum histologic view that shows woven bone and some well-organized lamellas as mature bone.

8. DPSCs in human’s maxillary and mandible bone defects

8.1 Case #1

This is a 58-year-old female patient who underwent titanium dental implant placement in the anterior maxilla 2 years ago. Despite significant maxillary bone atrophy, without any bone regeneration procedure, 4 implants were placed, which were not covered by bone. Fourlane of them were inserted into the nasal cavities. The patient developed multiple infectious processes. Moreover, the implants were mobile due the lack of bone and because they supported a dental prosthesis that was too large and heavy (Figures 12 and 13).

Figure 12.

(A) and (B) Clinical view of implants in an atrophic maxilla, with chronic inflammation, infection, and mobility. (C) and (D) Panoramic X-ray and CT scan that shows 5 nonintegrated implants, 2 of them inside the nose and very atrophic bone in the maxilla.

Figure 13.

(A) Infected nonintegrated titanium dental implants. (B) Unaesthetic nonintegrated implant supported denture.

Two years later, the implants and 2 mobile teeth were removed, and a tissue-engineered graft was performed, as the patient refused the use of autologous grafts. We used 6 million DPSCs from dental pulp cultured in the INDEBIOC laboratory. Cells were cultured on collagen membranes + recombinant human bone morphogenetic 2 protein (RhBMP2) + platelet-rich plasma (PRP) + dried and frozen bone (FDB). DPSCs + RhBMP-2 + PRP + FDB were covered and spaced in suitable height and thickness by fixed lyophilized bone sheets using screws (Figures 14 and 15).

Figure 14.

(A) Uncovered vertical and vestibular-palatine critical bone defect. (B) Adapting, shaping height and thickness and securing the lyophilized bone sheet with 1.5 mm screws. (C) Grafting with DPSCs + RhBMP-2 + PRP + FDB into the space between the residual maxillary bone and the fixed bone sheet. (D) Fixed bone sheet covering the transplant of DPSCs + RhBMP-2 + PRP + FDB. (E) Suture covering the tissue-engineered graft while maintaining the height and thickness of the critical bone defect of the residual maxillary bone.

Figure 15.

(A) Frontal view. (B) Palatal view of the reconstructed maxilla with DPSCs + RhBMP-2 + PRP + FDB. (C) and (D) Comparative view of the height of the anterior maxillary bone reabsorbed by chronic infectious process before (C) and after (D) reconstruction with tissue-engineered. (E) Height and (F) thickness after transplant of DPSCs + RhBMP-2 + PRP + FDB covered with lyophilized bone sheets in a critical bone defect of the maxilla following the removal of chronically infected titanium dental implants.

8.2 CASE #2

In this case, a male 18 years old was underwent esthetic maxillomandibular osteotomy surgery. He exhibited post-surgical sequelae of maxillary necrosis. The entire anterior part of the maxilla became necrotic, resulting in the loss of bone, gum tissue, palatal mucosa, and 6 teeth. To face up this complex deformity with a critical size bone defect we combined 6 million cultured DPSCs × cm³ on a collagen membrane + 2.4 mg of RhBMP2 + PRP + FDB. 2 years later, he got 4 dental titanium implants. To restore the patient’s dental function and esthetic appearance for his age, an appropriate and esthetic fixed dental prosthesis was used (Figures 16–19).

Figure 16.

(A) Male, 18 years old. Underwent esthetic maxillomandibular osteotomy surgery. Post-surgical sequelae of maxillary necrosis. (B) Necrotic loss of maxillary bone and 7 anterior teeth. (C) and (D) In this complex deformity due to bone, dental, gingival, and palatal mucosa loss, we combined 6 million cultured DPSCs/cm³ on a collagen membrane + 2.4 mg of RhBMP2 + PRP + FDB.

Figure 17.

(A) Pre-surgical image of the defect. (B) Image of the defect before reconstruction with DPSCs. (C) Critical size bone defect in the anterior maxilla. (D) 3D digital planning where we created a titanium mesh to prevent collapse of the DPSCs graft.

Figure 18.

(A) and (B) Pre molded titanium mesh covering and avoiding collapse of the engineered bone graft done with DPSCs + RhBMP + PRP + FDB. Post-surgical CT scan of the mesh providing height and thickness to the reconstruction. (C) Sagittal view, (D) coronal view, (E) bone density measurements, at 6 months.

Figure 19.

(A) Post osteotomy maxillary necrosis defect. (B) Maxillary bone height and density obtained with DPSCs + Rh BMP-2 + PRP + FDB. Regenerated bone after 14 months. (C) Tissue engineered cortico-lamellar bone type. Excellent height and thickness for placement of titanium dental implants. (D) Implant surgical guide. 4 titanium dental implants in newly regenerated bone.

9. Case # 3 mandibular reconstruction with DPSCs

The following case is about a 18-year-old male who was diagnosed with mandibular ameloblastoma by incisional biopsy. We decided to perform an en bloc resection with 1 cm safety margins and placement of a reconstruction plate. The procedure was carried out under general anesthesia without complications. After one year without signs of recurrence, the planning for mandibular reconstruction under general anesthesia was initiated for future implant placement. At that time, it was decided to reconstruct using a block iliac crest graft, spongy iliac crest graft, and allograft as an osteoconductive material. The blocks were fixed to the preexisting reconstruction plate, and the aforementioned grafts were added.

The procedure was performed without complications. After this reconstruction, the COVID-19 pandemic favored that the patient stopped attending appointments but returned to us 2 years after the reconstruction surgery. Unfortunately, at that time, the graft had been resorbed by more than 50%, making it impossible to place dental implants on the new-formed bone. At this point, a second reconstruction was decided upon, in which BMP-2, allograft, and DPSCs were used. Under general anesthesia, the second reconstruction was successfully performed using the aforementioned materials. At 7 months post-reconstruction, guided dental implant placement is planned, which achieves an initial stability greater than 50 N. Currently, the patient is undergoing the necessary time for proper osteointegration to receive the implant-supported prostheses (Figures 20–22).

Figure 20.

(A) Mandibular 3D reconstruction with presence of mandibular ameloblastoma (green). (B) X-ray 2 years from iliac crest graft showing reabsorbed graft. (C) DPSCs in their vials for second reconstructive procedure. (D) Graft material with DPSCs (white scaffolds), allograft and BMP-2.

Figure 21.

(A) Newly formed bone 6 months after DPSC. (B) X-ray 6 months after reconstruction with DPSCs graft. (C) Grafted site with DPSCs. (D) Implant placement with adequate initial stability in new formed bone after DPSCs graft.

Figure 22.

Titanium dental implants placed on bone regenerated with DPSCs + Rh BMP-2 + PRP + FDB A, B and C.

10. Treatment of sequelae of cerebrovascular events in patients through different routes of administration

Considering that DPSCs have the same ectodermal origin as the central nervous system cells, we proposed to inject it into patients with spinal cord injury or stroke.

DPSCs have been also administrated in patients with neuropathic pain in the neck or lumbar region have been injected, from which any physical or organic injury has been ruled out. In this way, it leads us to treat the pain and being able to disguise any injury that compromises the patient’s life.

10.1 Spinal cord injury

The following data report the treatment of the administration of 100 million mesenchymal cells in 3 patients with traumatic spinal cord injury that were evaluated 3 months later.

10.1.1 Patient 1

A 56 years old male with spinal cord injury due to fall at L3 level presented chronic injury with paraplegia. The patient was presented with anesthesia at L3 level and the rest of scan was negative. We administrated 100 million of DPSCs intrathecally, using the same cerebrospinal fluid of the patient as vehicle, extracting 10 cc and processing the cells to administer them by the same route without complications. He did not show any type of reaction to the application. Three months later, the patient reported and improvement in his bowel movements.

10.1.2 Patient 2

A 54 years old male suffered an automobile accident resulting in chronic cervical spinal cord injury with quadriplegia predominantly in the legs, sensory level in C4, with lack of sphincter control and unable to move his upper extremities with limited flexion and extension of the fingers. We administrated 100 million of DPSCs intrathecally, using the same cerebrospinal fluid of the patient as vehicle, extracting 10 cc and processing the cells to administer them by the same route without complications. He did not show any type of reaction to the application. Three months later there was no improvement.

10.1.3 Patient 3

A 20 years old patient male with traumatic thoracic spinal cord injury due to a fall into a ravine. The patient presented paraplegia and had to undergo surgery to place bars and screws for spinal stability. The patient presented sensory level in T5 and lack of sphincter control. We administrated 100 million of DPSCs intrathecally, using the same patient cerebrospinal fluid as vehicle, extracting 10 cc and processing the cells to administer them by the same route without complications. He did not show any type of reaction to the application. Three months later the patient did not report evident improvement. Six months later, the patient exhibited greater sensitivity of the legs in the form of non-specific patches. Eight months later, the patient had a decrease in the sensory level from T5 to L1, and he had an erection and a little sphincter control, with slight movement of the feet and being able to feel the back, lower region and thus the incidence of bedsores was lower, since proprioception had improved. He missed his clinical evolution for a year.

10.2 Stroke

10.2.1 Patient 1

A 25-year-old male patient who, while in Australia for studies, presented a cerebral infarction without any type of recognized origin for it. The cerebral infraction leaving as a sequel right hemiparesis and language alterations. We administrated 25 million of DPSCs in saline solution intrathecally without complications. At follow-up there was no improvement. Three years later, 300 million cells were administered intracerebrally, administering the cells stereotaxically in the left posterior parietal region by a single application. There were no complications. The next day the movement of the right hand was significantly appreciated, less contracture and more movement. The walking was lighter and the patient was able to lift his leg higher. One month after treatment, the hand contracture returned. Despite the walking improved, language disturbances persisted. The patient has already been in office for four months and has been stable.

10.2.2 Patient 2

A 75-year-old male patient with left middle cerebral infarction with hemiplegia and language disorders was treated three months after his cerebral event. An angiography was performed and we administrated 100 million of DPSCs through the left middle cerebral artery and the overall increase in circulation was immediately appreciated in the control angiography at 15 minutes. Three months after its application, the patient has not had a noticeable improvement.

10.3 Subarachnoid hemorrhage

10.3.1 Patient 3

A 60-year-old female patient with subarachnoid hemorrhage in the frontal arteriovenous fistula, admitted to a coma, developed a subacute subdural hematoma during her hospitalization, which was operated on and drained adequately. Tracheostomy and gastrostomy were performed. It was totally dependent on the fan. At the third month, 100 million of DPSCs were administered intravenously without noticeable improvement. One week later, 100 million of DPSCs were administered intra-arterially via angiography through the left middle cerebral artery. In the control cerebral angiography performed 10 min later, vasodilatation was observed, removing the vasospasm that it presented. A week after the transplant she was able to go home (Table 1 and Figure 23).

Neurological commitment
Pre-transplant	Post-transplant
Patient who was already hospitalized for 3 months with tracheostomy and gastrostomy Two weeks before his discharge, 100 million DPSCs I.V. were applied. A week later, 100 millions MSC were applied by angiographic route in the posterior territory	No significant improvement in application I.V.
Not being able to speak	Started talking
Paralysis of the gaze (III, IV, and VI)	Onset with eye movements
Bifacial paralysis	Initiation with facial movements of oral predominance
Unable to eat or swallow (IX, X, XI and XII commited)	Began to eating and moving tongue
Paralysis of all four limbs	Started moving hands and legs
Dysautonomic imbalance	Began to normalizing
Other properties that the husband noticed	No hair loss, softer skin, more awake and other features more

Table 1.

Neurological compromise before and after transplantation of a 60-year-old female patient with subarachnoid hemorrhage from a frontal arteriovenous fistula treated with DPSC.

Figure 23.

Angiography before and after transplantation of a 60-year-old female patient with subarachnoid hemorrhage from a frontal arteriovenous fistula treated with DPSC.

10.3.2 Patient 4

This is the case of a 50-year-old male patient with subarachnoid hemorrhage due to bleeding that could not be identified angiographically. He was in a coma since admission development of hydrocephalus, performing referral. He presented a ventriculoperitoneal shunt, without being able to control exvacuo hydrocephalus due to neuronal damage. Three months later, he remained in a persistent vegetative state. We administrated 100 million of DPSCs by intraventricular route without complications, and without any type of rejection or dysautonomic reaction. He continued to depend on the ventilator and finally it was decided to withdraw the ventilator due to severe neurological damage and he died.

11. Conclusions

MSCs from dental pulp can be easily obtained and are highly clonogenic with multidifferentation and neuromuscular properties. Here, we presented a general view of the high quality methods used to isolate DPSCs and their subsequent culture and applications. In addition, we showed that DPSCs therapy for bone reconstruction.

Finally, we presented a series of clinical cases in which the administration of DPSCs was able to improve the quality life of patients who suffering of stroke or subarachnoid hemorrhage. All the present work is based in the fact that DPSCs and their secretome serve as critical immunomodulators that contribute with injury repair. Our clinical studies are promising in the use of DPSCs as a tool for treating bone disease, cerebrovascular events and spinal cord injury.

Acknowledgments

The authors want to especially thank Julián Enrique Valencia Guerson, Director of Innovations and Development in Cellular Biotechnology for being a fundamental part in the development of this project.

Conflict of interest

The authors declare no conflict of interest.

Acronyms and abbreviations

DPSCs	dental pulp MSCs
PDLSC	periodontal ligament MSCs
SHED	stem cells from human exfoliated deciduous teeth
SCAP	stem cells from apical papilla
DFSC	dental follicle stem cells
GMSC	gingival mesenchymal stem/progenitor cells
GMP	good manufacturing practice
INDEBIOC	Innovations and Development in Cellular Biotechnology
ED	enzymatic dissociation
MSCs	MSCs
PGE2	prostaglandin E2
COX-2	cyclooxygenase 2
Tregs	regulatory T cells
IDO	indoleamine 2,3-dioxygenase
TNF-alpha	tumor necrosis factor alpha
IL-10	Interleukin 10
SMADS	mothers against decapentaplegic protein
CSBD	critical size bone defects
rhBMP2	recombinant human bone morphogenetic 2 protein
PRP	platelet-rich plasma
FDB	dried and frozen bone
COVID-19	coronavirus disease
BMP-2	bone morphogenetic 2 protein