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The combination of odontogenic stem cells and mandibular advancement promotes the length of the mandible in adult rats by facilitating the development of condylar cartilage

Stem Cell Research & Therapy volume 15, Article number: 441 (2024) Cite this article

Abstract

Background

Mandibular retraction is a prevalent dental and maxillofacial deformity that negatively affects patients' functional health and facial aesthetics. It has been challenging to achieve optimal outcomes for patients who have passed the peak of growth and development using only functional orthopedic treatment. There is a pressing need to explore innovative methods to promote the adaptive remodeling of adult condylar cartilage and the mandible in response to external stimuli. This study aimed to investigate the impact of varying injection frequencies of stem cells from the apical papilla (SCAPs) on the growth and development of condylar cartilage and the mandible, as well as their potential for adaptive remodeling.

Methods

The study was conducted on 8-week-old adult male Sprague–Dawley rats. The effects of SCAPs injection and different durations of mandibular advancement (MA) on the adaptive remodeling of condylar cartilage and the mandible were assessed. After the initial experimental findings, various injection frequencies of SCAPs were applied to determine the most effective conditions for promoting the growth and adaptive remodeling of condylar cartilage and the mandible during an 8-week period of mandibular advancement.

Results

The study found that rats with extended mandibular lead times (8 weeks) or an appropriately increased frequency of mandibular leading time (once every 2 weeks or once every 1 week) exhibited increased lengths of the mandibular body and ascending branch, and a thickened full layer of condylar cartilage. The highest proportions of the proliferative layer, mature layer, and hypertrophic layer were observed in these rats. Additionally, there was a significant increase in the expression levels of SOX9 and COL2A1.

Conclusion

The data from this study suggest that adult rats, even after missing their peak growth period, retain the potential for continued growth and development of their condylar cartilage. By prolonging the duration of mandibular advancement and administering injections of stem cells from the apical papilla (SCAPs), it is possible to stimulate the growth and development of the mandibular condyle.

Introduction

Angle Class II malocclusion is one of the a common malocclusion in clinical practice, and its pathogenesis involves many factors [1, 2], among which mandibular underdevelopment is particularly prominent [3, 4]. Clinical manifestations include mandibular retraction, anterior tooth protrusion, labial muscle tension during lip closure, shallow or flat mentolabial fold, shorter lower 1/3 of the face, etc. [5, 6]. In severe cases, Angle Class II malocclusion may lead to disharmony of occlusal relationship, misaligned teeth, temporomandibular joint disorders (TMD) and other lesions. Furthermore, it might affect the facial appearance, chewing ability, temporomandibular joint (TMJ) health, and even psychological disorders.

Regarding the currently corresponding treatment measures, many orthodontists advocate that the treatment must be performed before patients peak growth [7]. For example, mandibular advancement (MA) can be achieved through functional devices such as various activators (Forsus, Herbst, Twin-block, etc.), which would change the facial muscle environment and promote endochondral osteogenesis of condylar process, thus enabling adaptive remodeling of mandible after original malocclusion deformity was removed [8, 9]. Unfortunately, for the patients who miss the growth peak when they see a doctor, the likelihood of treatment with functional orthopedics alone is substantially reduced since their growth potential gradually declines with age.

Therefore, evaluating new and effective strategies for cartilage regeneration is a necessary condition for establishing feasible treatments for patients who have missed their peak growth period. Regeneration of articular cartilage tissue based on mesenchymal stem cells (MSCs) is considered a promising method for the treatment of cartilage injuries and has received significant scientific validation [10,11,12]. Studies have found that a novel type of MSC isolated from dental tissue, including Dental Pulp Stem Cells (DPSCs), Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs), Periodontal Ligament Stem Cells (PDLSCs), and Stem Cells from the Apical Papilla (SCAPs) [13], share the same characteristic mesenchymal stem cell surface markers as those derived from Bone Marrow (BM-MSCs). As undifferentiated stem cells within normal dental tissue, Dental Mesenchymal Stem Cells (DMSCs) represent an ideal source of autologous stem cells [14]. In 2006, Sonoyama et al. firstly obtained SCAPs from apical papilla tissue, which are derived from the root tip of a young third molar [15]. SCAPs not only play a crucial role in tissue repair and regeneration (Fig. 1A), but also, compared to several other types of dental-derived stem cells, SCAPs have superior osteogenic potential and growth rate [16]. Other studies have reported that SCAPs also possess significant chondrogenic differentiation potential [17, 18], making them a promising seed cell for comprehensive tissue regeneration.

Fig. 1
figure 1

Summary of SCAP potential applications in rat models and related disorders from the review. A Dental papilla stem cells have the potential of tissue regeneration and repair.(Images created with Biorender.com) B Treatments to promote mandibular growth and development include physical therapy, chemotherapy, and biological therapy. (Images created with Biorender.com). C Human age table corresponding to experimental SD rats of different ages.(Images created with Biorender.com)

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At present, the treatment for patients with Angle Class II mandibular hypoplasia mainly focuses on three aspects: physical, chemical, and biological.With the advancement of stem cell therapy technology, its application in the regeneration of oral and maxillofacial tissues has become a hotspot. Nonetheless, the effectiveness of using readily accessible dental-derived stem cells to treat adult mandibular developmental insufficiency still requires further research and exploration(Fig. 1B). To address this gap, we have reviewed the relevant literature to correlate the age stages of rats with those of humans(Fig. 1C) and have integrated stem cell therapy with traditional functional orthopedic techniques, with the aim of developing a novel method for the treatment of Angle Class II malocclusion. Through this study, we hope to provide more effective therapeutic approaches and strategies for adult patients with clinical mandibular developmental insufficiency.

Materials and methods

The work has been reported in line with the ARRIVE guidelines 2.0

Experimental animals

This animal experiment was approved by the Experimental Animal Ethics Committee of Jinan University (no. 20230522–19, 20,230,928–10). Forty-eight 6-week-old SPF male Sprague–Dawley rats (Beijing Weitong Lihua Experimental Animal Technology Co, LTD.) were selected. Under the conditions of 50 ~ 60% humidity, temperature 22 ± 2℃, light 12 h and good ventilation, feeding the standard soft food with the same amount of food and water. All animal experiments were conducted in strict accordance with international animal welfare standards, and the animals were quarantined for two weeks by the Experimental Animal Center of Jinan University for follow-up experiments.

This experiment was divided into two parts, with 24 rats in each part. The first part investigated the impact of a single injection of SCAPs on the growth of condylar cartilage and the mandible in adult rats over periods of 4 and 8 weeks. Rats were divided into two major groups for the 4-week and 8-week durations, with each group further divided into the following groups: Control, MA, and MA + SCAPs (N = 4 per group).

The second part investigated the effects of changing the injection frequency of SCAPs on the growth and development of the condylar cartilage and mandible under the same conditions of mandibular advancement for 8 weeks. Rats were selected and randomly divided into six groups (N = 4 per group): a control group (Control), an MA group (MA), and four groups receiving SCAPs injections at different frequencies—once at 8 weeks (MA + SCAPs 1), once every 4 weeks (MA + SCAPs 2), once every 2 weeks (MA + SCAPs 3), and once every week (MA + SCAPs 4). Samples were collected for analysis at the end of the 8-week period.

In accordance with previous methods12, the method of grinding the lower front teeth of rats was selected to establish a standardized rat MA model, and C-Sailor PRO graphic dental implant (Foshan Yusen Medical Equipment Co, LTD.) was used to grind 3 mm of both lower front teeth of the rats (with a standard slow cell phone and slow cell phone head; The torque was adjusted to 30 Ncm and the speed to 2500 rpm (Fig. 4A), and both lower front teeth were ground 1 mm every 3 days after the model was established until the end. The rats were checked daily after surgery. Administer an overdose of pentobarbital sodium to the animals intraperitoneally for euthanasia. Once the rats are unconscious and unresponsive to pain stimuli, dissect and collect their mandibles for subsequent analysis. During the experiment, the cages were housed in the same room and on shelves to minimize mixing.

Local injection of SCAPs into TMJ

After intraperitoneal injection of the anesthetic pentobarbital sodium (25 mg/kg), the mandibular movement of the rats was artificially simulated, and the injection site was determined again. Prepare a cell suspension of SCAPs at a concentration of 1 × 105 cells/mL with PBS for injection in subsequent animal experiments. 0.2 ml SCAPs cell suspension (approximately 2 × 105 cells) was injected into both sides.

Measurement indices of mandibular anatomy

The distance from the alveolar margin to the mandibular angle was measured with a vernier caliper, representing the mandibular body length (Go-Id), and the distance from the condylar peak to the mandibular angle, representing the mandibular ascending ramus length (Co-Go). Each side was measured independently three times, and then the average of both sides was taken.

Histological and immunohistochemical staining

After the anatomic measurement was completed, the fixed specimen was dehydrated by decalcification and paraffin embedded in paraffin. Continuous sections along the sagittal direction, and the thickness was approximately 5 um. The condylar cartilage was evaluated by hematoxylin–eosin (HE) staining, Safranin-Fast green staining and immunohistochemistry. Images were collected at 100 × magnification via an intelligent fluorescent inverted microscope (Leica, Germany) for analysis. Image J software was used to analyze and measure the average optical density of each image.

Cell isolation and culture

Approved by the Medical Ethics Committee of the First Affiliated Hospital of Jinan University, with the knowledge and consent of the patients and their families, the mandibular third molars of adolescent patients aged 16 years and below was collected. Place the tissue into a centrifuge tube containing 10% double antibody and PBS solution and put it in an ice box, transfer to a super clean workbench within 30 min. Separate and discard hard tissues such as dental tissue and bone tissue using a sterile nickel forceps, remove the PBS, and leave the apical papilla tissue near the apex of the tooth germ. Then, use sterile ophthalmic scissors to cut the apical papilla tissue into tissue pieces of about 1 mm3 in size at the mouth of a 50 mL centrifuge tube. Add 10% double antibody (penicillin, streptomycin) and PBS solution for repeated shock, rinse, and transfer to a T25 cell culture bottle evenly paved and inverted. Next, 2 ml of complete culture medium containing 10% FBS, 50 μg/mL L-ascorbate 2-phosphate, 100 U/mL penicillin and 100 μg/mL streptomycin was added and cultured in a cell incubator (temperature 37℃, air mass fraction 95%, carbon dioxide mass fraction 5%), and the bottle was turned over 4 h later. The medium was changed every three days until clusters of cells were observed. When the cell density was approximately 80%, the passage was carried out at a ratio of 1:2, and then the cells in the P3 generation were collected for further application.

Alcian blue staining

Chondrogenic induction differentiation medium was added to the centrifuge tube, the medium was changed every 3 days, the bottom of the tube was gently tapped until 1.5–2 mm of chondrocytes are formed. After 21 days, induction was stopped, proceed with paraffin embedding, sectioning, and Alcian blue staining were performed, and the secretion of acidic glycosaminoglycans by cells stained blue under a microscope was visualized.

Alizarin red staining

After 21 days of induced osteogenic differentiation, the preosteoblasts were stained with Alizarin Red S (ARS) dye. After being fixed with 4% PFA for 15 min, the cells were stained with 1% alizarin red S (pH 4.2) for 5 min. The mineralized substrate is stained in deep red.

Oil red O staining

Lipogenesis was induced by "liquid A for 3 days, liquid B for 1 day" and incubated for 21 days, The samples were fixed at room temperature with 4% PFA for 30 min, stained with Oil red O dye at room temperature for 30 min, and the formation of red spherical lipid droplets was confirmed under a microscope.

Flow cytometry

SCAPs from the P3 generation were collected after digestion with trypsin. The cells were centrifuged, the supernatant was discarded, the cells were washed with PBS, and cell suspension was prepared after cell counting. The fluorescently labelled antibodies (CD73, CD90, CD105, CD34, CD45, HLA-DR) were diluted in accordance with the instructions of the antibody instructions and added into the cell suspension. The supernatant was incubated on ice for 30 min in the dark, and then the supernatant was discarded after centrifugation. SCAPs were suspended in 500 μL of PBS and eventually analyzed with a flow cytometer.

Statistical analysis

The data collected in this study were statistically analyzed with GraphPad Prism 10.1.2 software, and the quantitative results are expressed as mean ± standard deviations (x ± s). Normality and homogeneity of variance tests were conducted for all the data first, and two-factor RM ANOVA analysis and Tukey’s multiple comparison test were performed for the first part of the experiment. In the second part of the experiment, one-way ANOVA and Tukey’s multiple comparison test were used. 0.05 was considered statistically significant.

Results

Isolation and identification of SCAPs were successfully achieved

In this study, SCAPs were extracted from the mandibular third molar embryo of a patient (male, 12 years old) by tissue block method and cultured in cell culture bottles. It was observed that the cells crawled out and grew against the wall, showing a long spindle shape and arranged in a whirlpool shape (Fig. 2A), indicating that they had a good growth state.

Fig. 2
figure 2

Extension and evaluation of SCAPs. A Image of primary SCAPs cells extracted and cultured to P3, observed under a 4 × microscope, showing the typical spindle shaped MSCs morphology for all types of cells. Scale bars = 100 μm. B Flow cytometry was used to determine the expressions of hematopoietic stem cell markers CD34, CD45, and HLA-DR on the surface of SCAPs, as well as the expressions of mesenchymal markers CD73, CD90 and CD105. C Images showing in vitro trilineage differentiation of cultured SCAPs. The trilineage differentiation capacities of the SCAPs were confirmed by alizarin red for extracellular calcium. Oil red O for intracellular lipid accumulation, and Alcian blue for extracellular cartilage ( Scale bars = the left and the right is 100 μm; the middle is 50 μm)

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To identify if the cells were SCAPs, we determined the expressions of typical markers of MSCs using flow cytometry. The result showed the high expression of mesenchymal stem cell markers (CD73, CD90, CD105) and low expression of hematopoietic stem cell markers (CD34, CD45, and HLA-DR), indicating that the extracted cells had high MSCs characteristics and good purity (Fig. 2B). Furthermore, trilineage differentiation capacities of SCAPs were measured, and alizarin red staining demonstrated the formation of calcified nodules, which is a specific index of bone tissue formation. After differentiation induced by lipid formation, the formation of red lipid droplets was observed in oil red O staining, while Alcian blue staining displayed the blue-green acid mucopolysaccharide structure after chondroblast induced differentiation (Fig. 2C).

Lengthening the duration of the MA model and appropriately increasing the frequency of intraarticular injection of SCAPs promoted the lengthening of the mandible

We established a mandibular advancement (MA) model by grinding 3 mm of the lower anterior teeth on both sides of the rat to induce mandibular protrusion, successfully creating a stable protrusive occlusion relationship at this location. After determining the injection sites, SCAPs suspension was injected into the bilateral joint cavities of the rats to regenerate the mandibular condyle and promote the growth and development of the mandible (Supplementary Fig. 1). Rats experienced a slight weight loss in the first three days after surgery, followed by stable vital signs and a steady increase in weight; no accidents occurred in any of the experimental rats throughout the study (Fig. 3A).

Fig. 3
figure 3

Assessing the effect of rat mandibular advancement combined with SCAPs administration. A Changes in body weight of experimental rats. B Schematic diagram of anatomical measurements. Anatomical measurement graph, length of the mandibular body (Go-Id), length of the mandibular ramus (Co-Go). C-D Analysis of mandibular length index in rats after different durations of mandibular advancement combined with SCAPs injection at 4 and 8 weeks (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ns: not statistically significant). E–F Analysis of changes in mandibular length index in rats after altering the frequency of SCAPs injection (***: compared with Control group, p < 0.001; ◇◇◇: Compared with MA group, p < 0.001; ns: not statistically significant)

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The rat mandible samples were collected and subjected to anatomical measurement analysis, with measurement standards as shown in the figure (Fig. 3B). Comparing the lengths of the mandibular body (Go-Id) and the mandibular ramus (Co-Go) under 4 weeks of MA and 8 weeks of MA, it was found that both the mandibular body (Go-Id) and the mandibular ramus (Co-Go) lengths were significantly increased in the 8-week MA compared to the 8-week Control (***p < 0.001), while the mandibular body (Go-Id) length showed no significant increase and the mandibular ramus (Co-Go) length increased in the 4-week MA compared to the 4-week Control (*p < 0.05). After the combined application of SCAPs injection, both the 4-week MA + SCAPs and 8-week MA + SCAPs groups showed an increase in the length of the mandibular body and ramus, with the increase in the ramus length at 8 weeks being non-significant (Fig. 3C-D). Subsequent animal models maintained the mandibular advancement for 8 weeks, and different injection frequencies of SCAPs were found to be most effective in increasing the lengths of the mandibular body (Go-Id) and the mandibular ramus (Co-Go) when injected once a week (MA + SCAPs 4) and once every two weeks (MA + SCAPs 3), with no statistical difference between the two groups (Fig. 3E-F).

To further confirm the above observations, we measured the thickness of HE is staining and safranin-fast green staining of the rat condyles, selecting the posterior-superior region of the condyle as the region of interest (Supplementary Fig. 2). The results showed that the proportion of the total area of the condylar cartilage in the 4-week MA group was greater than that in the 4-week Control group; the proportion of the total area of the condylar cartilage in the 8-week MA group was also higher than that in the 8-week Control group, and the rate of increase in the proportion at 8 weeks was significantly higher than that in the 4-week group, indicating that extending the time of mandibular advancement can increase the thickness of the cartilage layer. Following the co-injection of SCAPs, the total area proportion of the condylar cartilage in the injection group was significantly higher than that in the non-injection group, indicating that SCAPs played a role in thickening the condylar cartilage. (Fig. 4A-A1, B-B1).Upon changing the frequency of SCAPs injection, the thickening was most pronounced with an injection every 2 weeks (MA + SCAPs 3) and every 1 week (MA + SCAPs 4). This suggests that an appropriate injection frequency of SCAPs contributes to the thickening of the condylar cartilage. (Fig. 4C-C1, D-D1) (***p < 0.001; ◇◇: Compared with MA group, p < 0.01; ◇◇◇: Compared with MA group, p < 0.001).

Fig. 4
figure 4

Histological analysis of mandibular advancement combined with SCAP injection effects in the rat model. A- A1 HE stains (7 × Visual 100x) and area ratio analysis of rat condylar cartilage after 4 weeks of MA, 8 weeks of MA, and SCAPs injection. B- B1 Safranin-Fast Green staining (7 × Visual 100x) and area ratio analysis of rat condylar cartilage after 4 weeks of MA, 8 weeks of MA, and SCAPs injection. (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ns: not statistically significant). C- C1 HE stains (7 × Visual 100x) and area ratio analysis of rat condylar cartilage under different SCAPs injection frequencies. D- D1 Safranin-Fast Green staining (7 × Visual 100x) and area ratio analysis of rat condylar cartilage under different SCAPs injection frequencies. (***: compared with Control group, p < 0.001; ◇◇: Compared with MA group, p < 0.01; ◇◇◇: Compared with MA group, p < 0.001; ns: not statistically significant). Scale bar = A, B, C, D is 100 μm

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Prolongating the time of mandibular advancement and higher SCAPs injection frequency up-regulated the expressions of cartilage-related genes

The results of immunohistochemical staining and semi-quantitative analysis showed that compared to the Control group, both the 4-week MA and 8-week MA groups had higher expressions of SOX9 and COL2A1 (***p < 0.001). Compared to the MA group, there was a significant increase in the expressions of SOX9 and COL2A1 in 4-week MA + SCAPs group (***p < 0.001); no significant increase in the expression of SOX9 and COL2A1 in 8-week MA + SCAPs group compared to the MA group, indicating that extending the advancement time could promote the expression of SOX9 and COL2A1, and the injected SCAPs could have the effects within a certain period. (Fig. 5A-A1, B-B1) Maintaining the mandibular advancement model for 8 weeks and changing the injection frequency of SCAPs, it was found that the positive expression of SOX9 was most pronounced with an injection every two weeks (MA + SCAPs 3) and every week (MA + SCAPs 4) (◇◇◇: Compared with MA group, p < 0.001), with no statistical difference between the two groups (ns: not statistically significant), suggesting that increasing the frequency and amount of SCAPs injections may up-regulate the expression of SOX9 and COL2A1. (Fig. 5C-C1, D-D1).

Fig. 5
figure 5

Expression of cartilage-related genes in rats after mandibular advancement and SCAP administration. A-A1 Immunohistochemical staining (7 × Visual 100x) and semi-quantitative analysis of SOX9 in rat condylar cartilage after 4 weeks of MA, 8 weeks of MA, and combined SCAPs treatment. B-B1 Immunohistochemical staining (7 × Visual 100x) and semi-quantitative analysis of COL2A1 in rat condylar cartilage after 4 weeks of MA, 8 weeks of MA, and combined SCAPs treatment. (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ns: not statistically significant). C-C1 Altering the frequency of SCAPs injection, immunohistochemical staining (7 × Visual 100x) and semi-quantitative analysis of SOX9 in condylar cartilage. D-D1 Altering the frequency of SCAPs injection, immunohistochemical staining (7 × Visual 100x) and semi-quantitative analysis of COL2A1 in condylar cartilage. (***: compared with Control group, p < 0.001; ◇◇◇: Compared with MA group, p < 0.001; ns: not statistically significant). Scale bars = A, B, C, D is 100 μm

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Discussion

Studies have indicated that functional orthopedic appliances can cause the condyle and glenoid fossa to shift anteroinferior and undergo remodeling [20,21,22]. It is currently widely believed that the optimal timing for functional mandibular advancement (MA) treatment to improve mandibular growth is during the peak growth period [23,24,25,26]. Patients who miss the peak growth period will see their potential significantly decrease with increasing age. In our experiment, intra-articular injection of SCAPs showed good efficacy in promoting the growth and development of the mandible.

A Literature review revealed that young animal models have good mandibular regeneration potential, but few studies on adult animal models exist

PubMed and other databases were used to review the literature on exogenous stimulation of mandibular condyle or mandible growth and development (Fig. 6).

Fig. 6
figure 6

A systematic review for the current treatments of promoting mandibular extension. PRISMA flow diagram of the literature selection process. PubMed search string ((mandibular condylar chondrocytes) AND (mandibular growth)). Search for "Promoting mandibular growth" through CNKI. After screening, a total of 13 literature reports met the requirements

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By searching the literature review, we found that most of the reports on promoting mandibular regeneration used adolescent or young animals to build animal models, and few reports on adult rats. To understand from a macro perspective whether adult animals that have missed the peak of growth and development can still promote the effect of condylar cartilage regeneration and mandibular lengthening under exogenous stimulation, we conducted literature search and screening using PubMed and other databases. According to the standard procedures and inclusion and exclusion criteria (Fig. 6), almost all studies focused on animal models at young age. And a few studies employed the animal models that had entered adult age, but no effect was deliberately investigated. The omission and insufficiency in this issue prompted us to further explore the therapeutic effect of mandibular remodeling in adult rats.

The biomechanics generated during mandibular advancement can cause changes at the cellular and molecular levels of the condyle

By treating patients with skeletal Class II malocclusion through functional MA, the mandible is caused to shift anteriorly [27], and it has been shown that a fundamental factor regulating cellular activity during morphogenesis is mechanical stress [28]. Numerous experiments on rats and monkeys have also indicated that when the mandible shifts anteriorly through mechanical stress, new bone formation occurs in the condyle and glenoid fossa [29], suggesting that the biomechanics resulting from mandibular advancement can cause changes at the cellular and molecular levels of the condyle and mandible [30]. This provides an important direction for the study of mandibular growth and development. In the design of animal experiments, the duration of functional MA is a core element of the overall experiment. It is known that in the life cycle conversion between humans and experimental rats, one day of rat survival is equivalent to 8.6 days of human survival [31,32,33]. In rat animal experiments, it has been proven that a continuous 30-day functional MA treatment can achieve adaptive morphological changes in the condyle and mandible [34]. These changes are highly related to the duration of functional MA treatment, the direction, magnitude, and type of force [35, 36]. This result is consistent with the duration of orthodontic functional orthopedic MA treatment in current clinical practice, with the conventional MA treatment duration being 6–12 months. Studies have pointed out that by extending the MA duration to 8 weeks in adult rats, new bone can be seen in the condyle, with stable and sustained growth levels, compared to the conventional 4-week MA treatment, which can bring a more stable long-term bone effect [37].

Endochondral ossification of the mandibular condyle is regulated by multiple biomolecules

As the growth center of the mandible, condyle plays a crucial role in increasing the length and height of the mandible. A wealth of research indicates that the growth and endochondral ossification of the mandibular condyle in adolescent rats are influenced not only by mechanical stress [38] but also by a variety of biomolecules, such as Insulin-like Growth Factor-1 (IGF-1) 32, Parathyroid Hormone (PTH) [40], Parathyroid Hormone-related Protein (PTHrP) [41], Indian Hedgehog (Ihh) [42], and so on. In terms of cartilage, both PTH and PTHrP have regulatory effects on the chondrocytes of the mandibular condyle. PTH can enhance the proliferation of chondrocytes and increase the expression of COL II, a collagen that is the main component of the cartilage matrix produced by chondrocytes [43]. Concurrently, it reduces the expression of COL X and runt-related transcription factor 2 (RUNX2), where type X collagen (COL X) is produced by hypertrophic chondrocytes37,38, and its synthesis marks the termination of the cartilage formation process and signals the onset of endochondral ossification. This indicates that PTH promotes cartilage formation and inhibits chondrocyte hypertrophy [40, 44]. PTHrP plays a very important role in the development of the condyle [45, 46]. Research data have confirmed that PTHrP-deficient mice exhibit deformities in the mandibular ramus and body, suggesting that the disruption of PTHrP may impair the intramembranous and endochondral ossification processes of the mandible [45]. PTHrP may also regulate the proliferation, chondrogenic, and osteogenic differentiation processes of chondrocytes by upregulating the expression of SOX9 and COL II while suppressing the expression of RUNX2, COL X, and alkaline phosphatase (ALP) [47,48,49]. In the endochondral ossification process of long bones in limbs, PTHrP and Ihh have also been proven to regulate the elongation of the growth plate. A substantial amount of evidence indicates that PTH and PTHrP can enhance the proliferation of chondrocytes [40, 41, 44, 50].

Regulation of local signaling pathway can promote endochondral osteogenesis and inhibit chondrocyte hypertrophy

As a type of dental-derived stem cells, SCAPs share similar characteristics with Bone Marrow Mesenchymal Stem Cells (BMSCs), possessing the potential for multilineage differentiation. When injected, they can differentiate into osteoblasts and chondrocytes [51, 52], promoting the formation of bone and cartilage in the mandible [53]. Studies have indicated that hypoxia-inducible factor-1α (HIF-1α) within cells can enhance the expression of Sox9 induced by bone morphogenetic protein 2 (BMP2) and cartilage formation [54, 55]. During the endochondral ossification process of the mandibular condyle, SOX9 can regulate the differentiation of chondrogenic progenitor cells into chondrocytes [56], and SOX9 directly triggers the expression of Type II Collagen (COL II) [57]. This process is beneficial for cartilage formation. In animal experiments, HIF-1α has shown to promote cartilage formation and inhibit endochondral ossification [58, 59]. It has also been found that HIF-1α and BMP2 together promote the expansion of the proliferative chondrocyte zone and inhibit chondrocyte hypertrophy and endochondral ossification in vitro [58].

The presence of SCAPs may modulate local signaling pathways, such as the Wnt/β-catenin pathway. Studies have shown that the activation of the Wnt/β-catenin signaling pathway is associated with the hypertrophic phenotype of chondrocytes [60,61,62]. The activation of the Wnt signaling pathway, such as Wnt5a and Wisp-1, can initiate excessive bone remodeling [63,64,65,66]. At the same time, Wnt3a and 5a can also lead to the loss of proteoglycans in chondrocytes and a shift in phenotype, accompanied by a decrease in the expression of COL2A, aggrecan, and Sox-9 [63, 67]. During the process of mesenchymal stem cells differentiating into chondrocytes, the activation of this pathway promotes the expression of the hypertrophic phenotype. However, the dynamic regulation of multiple local signaling pathways can improve the quality of cartilage formation. Moderate activation of the Wnt signaling can promote the proliferation of chondroprogenitor cells, but excessive activation accelerates the hypertrophic phenotype and matrix degradation. Furthermore, the Hedgehog signaling (such as IHH) maintains the proliferative state of chondrocytes by regulating parathyroid hormone-related protein (PTHrP), preventing their premature maturation [52, 68, 69]. The TGF-β/Smad pathway supports the stable production of cartilage matrix by enhancing the expression of Sox9, promoting the synthesis of type II collagen and proteoglycans [70, 71]. In clinical applications, adjusting the treatment duration of TGF-β or inhibiting the excessive activation of Wnt signaling helps to enhance the quality of MSC-derived cartilage tissue [72, 73]. The regulation of this pathway can affect the quality of MSC-derived cartilage tissue, which is significant for cartilage tissue engineering and the repair of cartilage injuries.

Limitations of this study

This study has achieved some results in adult rat models but considering the differences in physiological and anatomical structure between rats and humans, its application in human bodies needs to be further explored. It can be challenging to determine the optimal dose and frequency of injection of SCAPs, and too high or too low a dose can affect the therapeutic effect. In the future, a sustained release system can be added on a cellular basis to avoid repeated injections causing trauma, which is required to be further studied.

Conclusion

Adult rats that have missed their peak growth period still have the potential for continued growth and development of their condylar cartilage. By extending the duration of mandibular advancement and injecting dental-derived stem cells (SCAPs), the growth and development of the mandibular condyle can be stimulated. Furthermore, experimental results indicate that the optimal injection frequency for SCAPs is once a week and once every two weeks. Considering the therapeutic trauma and the cost and effectiveness of the treatment, it is believed that when the injection frequency is set at once every two weeks, the trend of mandibular condylar remodeling is relatively good.

Availability of data and materials

All data supporting the conclusions of this article are included in the paper or supplementary information. Other relevant data are available from the corresponding author upon reasonable request.

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Acknowledgements

We would like to express our gratitude for the National Natural Science Foundation of China (No. 82370995). Thanks to Biorender.com for their assistance in creating the images. The authors declare that they have not use AI-generated work in this manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No.82370995).

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  1. Xin Cheng and Liangching Huang have contributed equally to this work.

Authors and Affiliations

  1. School of Stomatology, Jinan University, Guangzhou, 510632, China

    Xin Cheng, Liangching Huang, Huijuan Wang, SiLong Lei, Chichong Chan & Yue Huang

  2. Division of Histology and Embryology, Medical College, Jinan University, Guangzhou, 510632, China

    Xuesong Yang

  3. International School, Guangzhou Huali College, Zengcheng, Guangzhou, 511325, China

    Xuesong Yang

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Contributions

All authors have read and agreed to the published version of the manuscript. X.C.&L.H.: conceptualization, design, and execution of the experiments, methodology, data collection and analysis, manuscript preparation, and writing. H.W.: SCAPs isolation and identification, helped with in vivo experiments, and collected data. S.L.&C.C.: helped with animals’ experiments and collection of data. X.Y.: administrative support, project supervision, and expert revision. Y.H.: financial support, conceptualization, design of the experiments, data analysis and interpretation, manuscript preparation, and final approval of the manuscript.

Corresponding authors

Correspondence to Xuesong Yang or Yue Huang.

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Ethics approval and consent to participate

All human cell cultures and animal experiments conducted in this study were approved by the Ethics Committees of the Jinan University (No. JNUKY-2023–0110). The approved project was titled “Construction and orthodontic function evaluation of 3D Printed Autologous Tooth-Bone transformation prosthesis based on CEMP1 regulating periodontal regeneration mechanism.” The date of approvals is December 29, 2023. Written informed consents were obtained from patients and their families who provided teeth for further research.

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13287_2024_4055_MOESM1_ESM.pdf

Additional file 1 Establishing a rat model of mandibular advancement with SCAPs administration.Schematic diagram of the occlusion relationship of the anterior teeth in mice. To promote the advancement of the mandible, 3 millimeters of the mandibular incisors of the rat were ground down, and the sagittal view of the incisors was depicted. Concurrently, SCAPs were injected into the temporomandibular joint cavity. The directional arrow indicates the migration trajectory of the anterior teeth after the intervention.The construction process of the rat mandibular advancement model. After deep anesthesia of the rat, a homemade mouth opener was used to expose the lower incisors, and 3mm were ground down on both sides of the lower incisors. The eyes and ears were disinfected, and the fur was shaved, and the injection site was marked.Left and right mandibles of rats

13287_2024_4055_MOESM2_ESM.pdf

Additional file 2 Fields of interest under the microscope. The black square indicates the posterior-superior region of the rat condyle's sagittal 22 section, which serves as the region of interest for experimental observation and analysis. The areas observed in this experiment for HE stains, safranin-fast green staining, and immunohistochemical staining all originate from this region

Additional file 3 The main research contents of selected literatures derived from above systematic review

Additional file 4 The main research contents of selected literatures derived from above systematic review

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Cheng, X., Huang, L., Wang, H. et al. The combination of odontogenic stem cells and mandibular advancement promotes the length of the mandible in adult rats by facilitating the development of condylar cartilage. Stem Cell Res Ther 15, 441 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-04055-x

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Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512