Research ArticleExperimental Studies
Open Access

Characterization of Dental Pulp Stem Cell Populations in the Teeth of Patients With Neurofibromatosis Type 1 - Therapeutic Potential for Bone Tissue Engineering

MING YAN, WANG WANG, ULRIKE SPETH, LAN KLUWE, SANDRA FUEST, MARTIN GOSAU, RALF SMEETS, HONG-CHAO FENG and REINHARD E. FRIEDRICH
In Vivo March 2023, 37 (2) 548-558; DOI: https://doi.org/10.21873/invivo.13113

Abstract

Background/Aim: Neurofibromas (NF) are the most common benign nerve sheath tumors in the tongue, gingiva, major salivary glands, and jaw bones. Nowadays, tissue engineering is a revolutionary technique for reconstructing tissues. To explore the feasibility of using stem cells derived from NF teeth to treat orofacial bone defects, the differences in cell biological properties between an NF teeth group and Normal teeth group. Patients and Methods: The intra-dental pulp tissues from each tooth were extracted. The cell survival rates, morphology, proliferation rates, cell activity, and differentiation abilities were contrastively analyzed between the NF teeth group and Normal teeth group. Results: Between the two groups, there were no differences in the primary generation (P0) cells (p>0.05), the cell yield, and the time required for the cells to grow out of the pulp tissue and attach to the culture plate. Furthermore, no differences were found at the first generation (passage) between the two groups in colony formation rate and cell survival rate. The proliferation capacity, cell growth curve, and surface marker expression of dental pulp cells was not altered in the third generation (p>0.05). Conclusion: Dental pulp stem cells from NF teeth were successfully obtained and were not different from normal dental pulp stem cells. Although, clinical research using tissue-engineered bone to repair bone defects is still in its infancy, it will eventually enter the clinic and become a routine means of bone defect reconstruction treatment as related disciplines and technologies develop.

Key Words:

Peripheral nerve sheath tumors (PNST) can develop sporadically or be an indicator of a syndrome. In syndromic PNST, neurofibromas are the hallmark of neurofibromatosis type 1 (NF1), an autosomal dominant tumor suppressor gene disease (1). Numerous neurofibromas may occur in the skin of NF1 patients (cutaneous neurofibromas), which can be very noticeable and aesthetically detrimental. Another PNST observed almost exclusively in NF1 is the plexiform neurofibroma (PNF), a neoplasm that probably develops during the embryologic phase of life. PNF frequently arises from larger nerves and can infiltrate into adjacent organs. PNF is considered as a precancerous condition, i.e., a precursor to malignant peripheral nerve sheath tumors (MPNST). An estimated 50% of all patients with MPNST have NF1 mutations, and the diagnosis of MPNST is a major factor in the reduced life expectancy of these patients compared to the general population (1, 2).

Schwann cells or their precursors are the tumor cells of PNST in NF1 (2). Schwann cells derive from the neural crest (NC) (3). Both neurogenic tumors and many other findings in the NF1 patient can be interpreted as NC cell (NCC) differentiation disorders (4). Indeed, differentiation disorders of the NCCs explain numerous diseases and the relatively common syndrome NF1 is classified as a group of disorders of the NC (5). Therefore, NF1 is addressed as an important representative of the so-called neurocristopathies (6). The assignment implies that the NF1 gene product neurofibromin has many more functions in addition to the known tumor suppressor gene function. It was previously pointed out that NF1 gene is a histogenesis control gene (7, 8). Considering this characteristic of the NF1 gene, targeting of histogenesis and cell repair of NF1 patients in certain regions can be a suitable treatment for tumors or specific dysplastic conditions of the syndrome, for example pseudarthrosis of long bones (9, 10).

Due to the multipotent capacities of NCCs, some areas of NF1 research focus on the differentiation ability of adult stem cells (SC), which originate from NC. SC isolated from different tissues are used to analyze the differentiation potential of NF1-mutated cells (11-15). A well-known source of SC is the dental pulp (DPSC) (16). DP is source of SC, for which intact teeth (especially wisdom teeth) are extracted and used for research purposes after medical evaluation of the indications to extract the teeth. DPSC can differentiate into several cell types in cell culture and provide a valuable tool for understanding cellular differentiation (17-21) and the consequences of differentiation disruption (22, 23). The first studies on DPSC from NF1 patients showed their differentiation capacity (24-26). The aim of this study was to examine the differentiation capacity of DPSC from NF1 patients depending on a syndrome-specific oral environment.

Materials and Methods

Cultivation of human dental pulp cells.

Extraction of teeth. Eight teeth were extracted from two male NF1 patients. One of the teeth was extracted from the site of a facial plexiform neurofibroma. In order to assess the differentiation potential of the DPSC from NF1 patients, ten wisdom teeth of healthy teenagers (19-21 years old) were collected from November of 2020 to June of 2021 in our hospital. The teeth were removed and immersed in Dulbecco’s Modified Eagle medium (DMEM, Cat. No. 41965-049, Gibco, Leicestershire, UK) with 10% fetal bovine serum (FBS, Cat. No. 10500-064, Gibco, Carlsbad, CA, USA) and penicillin (100 U/ml)/streptomycin (100 μg/ml) (Cat. No. 15140-148, Gibco) at 4°C to keep the pulp alive because of the time and travel required to transfer it from the clinic to the laboratory. Experiments were conducted within a range of 4 h. Teeth were immersed in 4°C Dulbecco’s phosphate buffered saline (DPBS, Cat. No. 14190-094, Gibco) solution (including 2×105 U/l penicillin and streptomycin) for 30-60 min to disinfect the teeth.

Teeth derived from NF1 patients served as the experimental group (NF teeth group) whereas teeth from healthy individuals served as the normal control (Normal teeth group). In the following step, the intra-dental pulp tissues were divided into 0.5 mm3 pieces and emerged in 10% FBS/DMEM medium.

Cultivation of human dental pulp cells. Tissues from each group were distributed in 24-well plates and incubated at 37°C in a 5% CO2 humidified incubator. When the cells reached confluency, 0.05% trypsin- ethylenediaminetetraacetic acid (EDTA, Cat. No. 25300-054, Thermo Fisher, Waltham, MA, USA) was added and the cells were passaged at a density of 5×103/cm2. For each group, the time required for cellular outgrowth and attachment after the insertion of tissue blocks was recorded.

Calculation of cell yield/harvest. The primary dental pulp cells were digested with 0.05% trypsin at 37°C for 3 min after reaching 80-90% confluency. The dental pulp cells were then put into 50 ml tubes and centrifuged (Eppendorf 5810R, Hamburg, Germany) for 10 min at room temperature at 241×g. The cells were resuspended in DMEM after the supernatant was discarded. Cell counting and the following equation were used to determine the number of cells: Primary cell yield=Total cell counted/4×dilution factor×104×volume of cell suspension.

Colony-forming efficiency. A culture dish with a diameter of 10 cm was seeded with 1×103 first generation (passage) dental pulp cells from each group. Every three days, the culture media was replaced with fresh media. Two weeks later, the medium was discarded, and the dental pulp colonies were fixed with 95% methanol for 15 min, rinsed with DPBS, stained with Giemsa solution (Cat. No. 48900, Sigma-Aldrich, Buchs, Switzerland) for 10 min, and then washed three times with DPBS to remove any remaining Giemsa staining solution. More than 50 dental pulp cell colonies were noted. Three aliquots of cells were analyzed in parallel. The following equation was used to calculate the colony-forming efficiency: Colony-forming efficiency=number of successfully formed colonies/number of seeded cells×100%.

Cell survival rate after trypan blue staining. After being stained for two min with 0.4% trypan blue (Cat. No. 15250-061, Gibco) first generation dental pulp cells of each group were examined under an inverted microscope. From a total of 500 dental pulp cells, the number of living cells that remained unstained after five min was counted. The cell survival rate was then determined using the equation shown below:

Cell survival rate=number of unstained surviving cells/500×100%.

Cell survival rate after live-dead staining. First generation dental pulp cells were taken from each group and seeded on tissue culture coverslips (TCC, Cat. No. 83.1840.002, Sarstedt, Nümbrecht, Germany) at a density of 8×104/ml in 12-well plates that equals to 8×104 cells per well and maintained at 37°C in a 5% CO2 incubator for 3 h. Five hundred μl of fluorescein diacetate working solution (FDA) and 60 μl of propidium iodide (PI) (50 g/ml in PBS) were added to each well. Samples were examined under a fluorescent microscope (Nikon ECLIPSE Ti-S/L100, Düsseldorf, Germany) following 3 min of incubation at room temperature and a DPBS rinse. The cell survival rate was determined using the following equation: Cell survival rate=number of green-stained cells/number of total cells×100%

Evaluation of proliferation with the MTS assay. Third generation dental pulp cells were seeded into 96-well plates at a density of 2×104/ml, or 2×103 cells per well, and then cultured at 37°C in a 5% CO2 incubator. The proliferation of cells was assessed every day for eight days using the MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay (Cat. No. G1111, Promega, Madison, WI, USA). In each group, cells from three wells were submitted to MTS colorimetric examination. After a 3-h incubation, the absorbance was measured using a microplate reader (Thermo Fisher Scientific) at a wave-length of 490 nm. Each well received 20 μl of MTS reagent.

Multi-differentiation potential. Adipogenic differentiation. Dental pulp cells at the third generation (P3) were seeded at a density of 2×104/ml, corresponding to 4×104 cells per 35 mm dish. Adipogenesis induction medium (DMEM containing 10% FCS, 5 g/ml insulin, 0.5 mmol/l 3 isobutyl-1 methylxanthine, and 10 mol/l dexamethasone) was used to cultivate the cells for 21 days until they attained 60-70% confluency. The medium was changed every 3 days. On the 22nd day, the cells were fixed with paraformaldehyde (PFA; Electron Microscopy Sciences, Fort Washington, PA, USA) for 15 min and washed twice with DPBS. Then, 0.5% Oil Red O working solution (Cat. No. O1391-250ML, Sigma-Aldrich, St. Louis, MO, USA) was used to stain the induced cells at room temperature for 10 min. After staining, lipid droplets in the cytoplasm were observed and photographed under an inverted microscope after the cells had been washed twice with DPBS.

Quantitative analysis of adipogenic differentiation. To remove the remaining staining solution, stained cells on dishes were washed three times with DPBS. The lipid droplets were then dissolved in each dish using 2 ml of isopropanol (Cat. No. 34965-1L, Honeywell, Hamburg, Germany), and the dishes were gently shaken until the solution was evenly colored. A microplate reader (Thermo Fisher Scientific) was used to measure the absorbance of the staining solution at a wavelength of 540 nm after being placed into 96-well plates at a rate of 100 μl per well. Each sample was analyzed in triplicates.

Osteogenic differentiation. Third generation dental pulp cells were seeded at a density of 2×104/ml in 3.5 cm diameter culture dishes. After the cells reached 60-70% confluency, they were grown in osteogenic induction medium (DMEM containing 10% FCS, 10 mmol/l glycerophosphate, 5 mmol/ml ascorbic acid, and 1 mol/l dexamethasone) for 21 days. The medium was changed every three days. On the 22nd day, the cells were fixed with paraformaldehyde for 15 min before being thrice rinsed with DPBS. The induced cells were then stained at room temperature for 15 min using a 0.1% Alizarin red S solution (Cat. No. GT6383, Glentham, Carsham, UK). After staining, the cells were washed twice with DPBS before the stained calcium nodules were viewed and captured on camera using an inverted microscope (Olympus IX71, Olympus Corp., Tokyo, Japan).

Quantitative analysis of osteogenic differentiation. To get rid of the remaining staining solution, stained cells on dishes were washed three times with DPBS. Once the colored calcium nodules were completely dissolved, 1 ml of 10% acetic acid (Cat. No. 2289.1000, Geyer GmbH, Hamburg, Germany) was added to each dish. The dishes were then gently shaken. To neutralize the acetic acid, an equal volume (1 ml) of 10% ammonium hydroxide was added. The solution was transferred into 96-well plates, and a microplate reader (ELx800 Absorbance Microplate Reader, BioTek, Bad Friedrichshall, Germany) was used to measure absorbance at a 405 nm wavelength.

Adipogenic and osteogenic-induced gene expression. Total RNA from differentiated dental pulp cells was extracted using TRIzol reagent (Cat. No. 15596026, Ambion, Austin, TX, USA), and the quantity was determined using a spectrophotometer (model V 530, UV/Vis Spectrophotometer, Jasco, Japan) and 1% agarose gel electrophoresis. Using the GoScriptTM RT reagent Kit (Cat. No. A5001, Promega) following the manufacturer’s instructions. The extracted total RNA was reverse transcribed into cDNA and analyzed by reverse transcription PCR. We chose type I collagen, osteocalcin, and lipoprotein lipase (LPL) as adipogenic genes. Type I collagen and PPAR- were chosen as osteogenic genes. GAPDH was used as an internal control. Primer sequences and product lengths are shown in Table I.

View this table:
Table I.

Primer sequences of adipogenic and osteogenic induced gene expression.

Ethics approval and consent to participate. The investigations of anonymized data were performed in accordance with Hamburgisches Gesundheitsdienstgesetz (Hamburg Healthcare Act).

Statistical analysis. To examine differences in mean values between the two groups, a student t-test was employed. Values were considered significant at p<0.05. For statistical analysis, SPSS 22.0 software (SPSS Inc., Chicago, IL, USA) was utilized.

Results

Morphological evaluation of human dental pulp stem cells (hDPSCs). Morphological evaluation of hDPSCs: hDPSCs have a fibroblast-like morphology and seem as triangular or spindle-shaped cells under the microscope. In terms of cell morphology, there was no discernible difference between the two groups (the NF teeth group and Normal teeth group) (Figure 1A). In approximately 7 days of cultivation, the primary dental pulp cells were radically migrated out, forming a “growth halo” around the tissue block. In addition, no differences were observed between two groups in the time required to migrate out of the tissue block (Figure 1B) (p>0.05).

Figure 1.
Figure 1.

Electron microscopy of primary cells. A) Morphology of dental pulp tissue and cells at the 1st and 3rd generations. I-III: Normal teeth group - Primary cells grow out from tissue blocks (×40; ×100) and the cells at the 3rd generation after passage (×100). IV-VI: NF teeth group - Primary cells grow out from tissue blocks (×40; ×100) and the cells at the 3rd generation after passage (×100). B) The time of first appearance of primary cells in different groups: There is no significant difference between the two groups. p>0.05.

Cell yield. There was no difference in the number of harvested cells (p>0.05). The number of primary dental pulp cells harvested in the normal teeth group was (11.725±1.601)×105, whereas that of the NF1 teeth group was (11.333±1.341)×105 (Figure 2).

Figure 2.
Figure 2.

Number of harvested cells from (black) normal teeth and (red) NF teeth. Number of harvested viable cells (gain) did not significantly differ (p>0.05).

Comparison of proliferative rates. The MTS method was used to examine the proliferation rates of hDPSCs produced from normal teeth or NF teeth from day 1 to day 8 of culture of the pulp tissue. Between the two groups, there was no discernible difference in the ability of cells to proliferate, no evidence of obvious differences in Giemsa staining was identified by visual observation (Figure 3A), including cell cloning formation and growth curve (p>0.05) (Figure 4B and C).

Figure 3.
Figure 3.

Comparison of cell survival rate: A) Live-dead staining results of dental pulp cells; I: normal teeth group (×40); II: NF teeth group (×40); III: normal teeth group (×100); IV: NF teeth group (×100). B) The survival rate assayed using live-dead staining in each group. C) The survival rate assayed using trypan blue staining in each group.

Figure 4.
Figure 4.

Proliferation ability of primary dental pulp cells in each group. A) Giemsa staining results of dental pulp cells; I: normal teeth group; II: NF teeth group. B) Colony-forming efficiency. C) Cell growth curve (MTS assay).

Survival rate. There was no discernible difference in the cell survival rate between the two groups after trypan blue and live-dead staining (p>0.05) (Figure 3A, B and C).

Evaluation of the multipotent differentiation capacity. After osteogenic and adipogenic induction for 3 weeks, third generation cells were stained (Figure 5A), and the corresponding absorbance was determined at various wavelengths (osteogenic—405 nm, adipogenic—540 nm)using a spectrophotometer. The results showed that there was no difference in the capacities for osteogenic and adipogenic differentiation between the two groups (Figure 5B), corresponding to the results of PCR gel electrophoresis (Figure 5C).

Figure 5.
Figure 5.

Differentiation potential of dental pulp cells. A) Representative microscopic images from adipogenic and osteogenic differentiation assays. I-IV: Osteogenic differentiation. I: Normal teeth group (×40); II: Normal teeth group (×100); III: NF teeth group (×40); IV: NF teeth group (×100); V-VIII: Adipogenic differentiation. V: Normal teeth group (×40); VI: Normal teeth group (×100); VII: NF teeth group (×40); VIII: NF teeth group (×100). B) The absorbance value at 450 nm of the dissolved solution after Alizarin Red S staining in the two groups. C: The absorbance value at 540 nm of the dissolved solution after Oil Red O staining in the two groups. D: Gel electrophoresis results. I: RNA electrophoresis in the two groups: 1. Normal teeth group; 2: NF teeth group. II: Electrophoresis results regarding the expression of osteogenic and reference genes in each group. 1: Normal teeth group; 2: NF teeth group; 3: Negative control group; III: Electrophoresis results regarding the expression of adipogenic and reference genes in each group. 1: Normal teeth group; 2: NF teeth group; 3: Negative control group.

Discussion

This study reveals the adipogenic and osteogenic differentiation potential of DPSC of NF1 patients is similar to those healthy controls. Obtaining and further processing of DPSCs was not influenced by topographical peculiarities. Analogous to the apparently lack of influence of PNF in NF1 on the shape (27) and number of teeth (28, 29), and the capacity of oral diffuse-plexiform neurofibroma to differentiate extraosseous displaced odontogenic tissue to a tooth crown (30), DPSCs derived from teeth embedded in an oral PNF can be differentiated in the same way as teeth without any apparent contact to a PNST. In both situations, NF1 derived DPSCs differentiate identical to controls.

The area of oral and maxillofacial medicine urgently demands innovative treatment techniques to restore damaged tissues functionally and aesthetically. In recent years, the disciplines of SC research and regenerative medicine have seen tremendous development and groundbreaking discoveries. It has recently been reported that dental tissue-derived stem cells stand out as a significant stem cell source for bone regeneration in oral and maxillofacial surgery, craniofacial abnormalities, and orthopedics (31, 32). Furthermore, SCs can be collected intraorally from tooth pulp and periodontal ligaments. Our previous research has shown that dental SCs have the advantages in myogenic differentiation, odontogenic/osteogenic differentiation, and chondrogenic differentiation (33, 34).

PNF of the oral cavity often affects the trigeminal and upper cervical nerves (35). PNF has been documented to develop in the oral cavity on the tongue, lip, palate, gingiva, major salivary glands, and maxillary bones. PNF of the trigeminal nerve often leads to characteristic deformations of the jaw. The enlarged mandibular foramen is a radiologically conspicuous sign of a dorsal differentiation disorder through an adjacent PNF (36), as is the relatively frequently reported enlargement of the mandibular canal (37). However, the development of teeth in the orofacial PNF region is often disturbed in that the mesial migration of certain permanent teeth is incomplete or completely absent and the roots of the teeth are deformed due to the lack of bony space, but the visible structure of the tooth is not impaired (27).

NF-1 is significant because of the differences in clinical presentation, therapy, and prognosis. The current therapy for neurofibroma is total excision. These tumors are not radio-sensitive and have limited benefit from surgical treatment (38).

However, is it possible that stem cells existing at these sites have somewhat different properties from those in normal teeth. In the presented study, we compared multilineage potentials of dental pulp-derived mesenchymal stem cells isolated from dental pulps of two types of teeth (normal and PNF-associated).

We succeeded in culturing and separating original DPSCs from the tissue block using the limiting dilution method. It has been shown that, as is the case normal pulp tissues, stem cells are also present in NF teeth.

The ability to maintain self-renewal and differentiation is critical for the growth and effective use of stem cells in therapeutic settings (39). There was a concordance between the two kind of stem cells in their morphological and biological characteristics. MTS assay and colony-forming efficiency assay was used to assess the proliferation ability and colony forming potential; the intrinsic ability to display self-organizing morphogenetic properties in ex vivo culture may represent a general property of pulp tissue stem cells.

Traditionally, the potential of mesenchymal stem cells to develop into three lineages (osteogenic, chondrogenic, and adipogenic) has been used to assess their suitability for orthopedic and aesthetic regeneration applications (40). Collagen type I (COL I) is an early matrix mineralization marker, whereas Osteocalcin (OSC) is an osteogenic maturation marker (41); they indicate the differentiation and maturation of the bone, respectively. We have previously shown that dental pulp cells express bone-related genes (ALP, osteocalcin, collagen I) and can form mineralized nodules in vitro (42). The current cells derived from NF teeth were shown to possess a series of characteristics such as the ability to develop mineralized nodules in vitro and express the bone-associated markers OSC and COL I. The osteogenic differentiation capacity of these cells was commensurable with the bone-forming activity of normal pulp stem cells. Both mesenchymal stem cells were examined for their ability to undergo adipogenic differentiation. The most assessed genes αρε peroxisome proliferator activated receptor γ (PPAR-γ) and lipoprotein lipase (LPL). Like osteogenic differentiation ability, the adipogenic one showed same lipid vacuoles (Oil Red staining) in mesenchymal stem cells from NF and normal teeth.

DPSCs have been successfully studied in NF1 patients (24, 25). DPSCs obtained from NF1 patients can be differentiated into several specifications, for example chondrogenic, osteogenic or adipogenic (24, 25). In principle, this means that autogenous sources of cell and tissue regeneration are available for NF1 patients (11). This and previous studies show that the constitutive loss of the NF1 gene does not affect the ability of DPSCs to differentiate according to the chosen conditioning of the environment (43-45). Until now, it was unknown whether DPSCs from teeth in this tumor area have the same differentiation capacity as DPSCs from NF1 patients who have not developed this facial tumor.

NF1 is a disease with a plethora of signs and symptoms. For example, bony changes (36) are just as much a part of the spectrum of the syndrome as the often-plentiful accumulation of fat cells in body regions affected by diffuse neurofibromas (46). Basic research is of great importance for understanding the cellular and molecular basis of the disease and develop therapeutic strategies, for example in skeletal regeneration.

The presented initial results indicate that the differentiation capacity of DPSCs is the same between the two groups. Beyond the experimental evidence, this finding is interesting for the qualification of the NF1 gene as a histogenesis control gene (7). Even under the artificial conditions of cell culture, alteration of the genetic status by NF1 mutation does not prevent the differentiation of the DPSCs. This result may provide an opportunity for further basic research on the rehabilitation of these often severely disfigured patients by regenerative cell culture techniques. This can be achieved, for example, by the selective generation of SCs from DPSCs or other sources of SCs, which are used to stabilize the connective tissue. Especially diffuse PNF are often characterized by a significant lack of elasticity of the affected body region. Surgical treatment options do not guarantee stable results in terms of body shape and volume of the reduced tumors (38). Supportive measures are highly desirable. With the potentially unlimited differentiation of SCs derived from NF1 patients, there is a wide field of SC application in the reconstructive surgery of NF1 patients.

Conclusion

In this study, dental pulp stem cells derived from NF teeth were successfully obtained and found not to be different from normal dental pulp stem cells for comparison. However, the research in constructing tissue-engineered bone to reconstruct bone defects in the clinical setting is still in the exploratory stage, and there are still many challenges to realize its clinical application, such as how to obtain sufficient amount of high quality and high purity stem cells, how to improve the vascularization and physical and mechanical properties of tissue-engineered bone, and how to promote the effective integration of tissue-engineered bone tissue with host bone tissue. Along with the continuous development of related disciplines and technologies, the existing challenges will be gradually overcome, and tissue-engineered bone will eventually enter the clinic and become a routine means of bone defect reconstruction treatment.

Footnotes

  • Authors’ Contributions

    MY: conceived the study, supervised the experiments, and drafted the manuscript. WW: conceived the study, supervised the experiments, and drafted the manuscript. SF: data evaluation and manuscript preparation. US: data evaluation and manuscript preparation. MG: analyzed the data and revised the manuscript. REF: conceived the study, supervised the experiments, and revised the manuscript. RS: performed the data collection. HF: conceived the study, designed the data evaluation, and revised the manuscript. All Authors read and approved the final manuscript.

  • Funding

    M.Y. was supported by the Merit Scholarship for International Students (No. 7238065).

  • Conflicts of Interest

    The Authors declare that they have no competing interests in relation to this study.

  • Received January 6, 2023.
  • Revision received January 22, 2023.
  • Accepted January 30, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Characterization of Dental Pulp Stem Cell Populations in the Teeth of Patients With Neurofibromatosis Type 1 - Therapeutic Potential for Bone Tissue Engineering
MING YAN, WANG WANG, ULRIKE SPETH, LAN KLUWE, SANDRA FUEST, MARTIN GOSAU, RALF SMEETS, HONG-CHAO FENG, REINHARD E. FRIEDRICH
In Vivo Mar 2023, 37 (2) 548-558; DOI: 10.21873/invivo.13113
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