Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a 'niche-based approach on stemness' for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a 'fibroblast-like' spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Mesenchymal stem cells (MSCs) were first discovered in bone marrow, which showed the ability to form adhesive colonies in culture and strong osteogenic potential¹. Pittinger et al.² further found their multi-directional differentiation potential towards bone, fat, and cartilage. Although all mesenchymal stem cells from different sources have the potential for multi-directional differentiation, bone marrow mesenchymal stem cells have the strongest chondrogenic differentiation potential compared with mesenchymal stem cells derived from other tissues, making them considered the best candidate cells for bone tissue engineering³. However, many studies have proved that BMMSCs from different origins present site-specific characteristics and properties such as osteogenic differentiation capability and cellular proliferative activity^4,5. This may be due to different germ layers between the jaw and appendicular bones or the iliac crest⁶.

Jaw bone marrow mesenchymal stem cells (JBMMSCs) arise from neural crest cells of the neuroectoderm, while femur-derived BMMSCs originate from the mesoderm⁷. Compared with BMMSCs derived from long bones and the iliac crest, JBMMSCs have a higher proliferation rate, ALP activity, and osteogenic potential⁸. Besides, the application effect of BMMSCs in tissues and organs may vary depending on different cell types and environments⁹. Repair of jaw defects mainly depends on the recruitment of jaw-derived mesenchymal stem cells. Therefore, studying JBMMSCs can provide an experimental basis for its clinical application in jaw bone tissue engineering¹⁰. However, basic research and clinical applications mainly focus on BMMSCs derived from appendicular and axial bones¹¹. Research on JBMMSCs is limited, and this may be due to the low content of cancellous bone in the mandible and the fact that rats' jaws have even less cancellous bone content¹². Therefore, it is difficult to separate jaw bone-derived marrow mesenchymal stem cells using the ordinary bone marrow flushing method, which is commonly used in isolating BMMSCs from appendicular bones or the iliac crest¹³. Based on the methods of Hong et al.¹⁴ and Cheng et al.¹⁵, we hypothesized that the combination of dense bone digestion and the bone marrow flushing method could efficiently isolate rat JBMMSCs.

This study aims to establish an efficient method for isolating rat JBMMSCs and provide sufficient seed cell sources for jaw bone tissue engineering.

The protocol was approved by the Institutional Animal Ethics Committee of the Chinese PLA General Hospital. Thirteen-week-old male Wistar rats were used for the experiment. Details on the animals, reagents, and equipment are listed in the Table of Materials.

1. Experimental preparation

1. Sterilize all surgical instruments, including ophthalmic scissors, tweezers, and bone rongeurs, at high temperature and pressure.
2. Prepare culture media in advance.
   1. Prepare α-MEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.
   2. Prepare 0.1% type II collagenase in phosphate-buffered saline (PBS).
   3. Prepare 50 mL of PBS with 1% penicillin-streptomycin in a disposable sterile 50 mL centrifuge tube.

2. Isolation and cultivation of rat JBMMSCs

1. Isolate the rat mandible following the steps below:
   NOTE: To maintain sample sterility, all metal surgical instruments must be disinfected at high temperature on an alcohol lamp flame for at least three seconds before use.
   1. Anesthetize the rat with 2% pentobarbital sodium (2 mL/kg) by intraperitoneal injection.
   2. Sacrifice the anesthetized rat by cervical vertebrae dislocation (following institutionally approved protocols) after its respiratory rate decreases and the muscles relax completely.
   3. Immerse the rat in 75% ethanol for 30 min to disinfect it and then transfer it to the ultra-clean table.
   4. Place the rat on a disposable sterile cloth or in a sterile container and keep it in the supine position.
   5. Cut and separate the skin and muscle from the angulus oris to the joint along the ramus, and then separate the condyle from the glenoid fossa by cutting the temporomandibular joint disc and bilateral peripheral ligaments horizontally with ophthalmic scissors.
      NOTE: Determine the position of the temporomandibular joint by moving the mandible and observing the position of the condyle externally.
   6. Cut and separate the muscles and connective tissues of the lateral mandible using scissors from the vestibular groove on the buccal side of the lower incisors to the left and right sides.
   7. Incise the lingual muscles of the mandible from the lingual frenum to the posterior region of the mandible.
   8. Remove the mandible from the skull.
   9. Divide the mandible into halves by cutting the symphysis between the lower incisors with ophthalmic scissors.
   10. Remove the remaining attached muscles, fascia, and other soft tissues on the mandible carefully using ophthalmic scissors and forceps.
2. Isolate JBMMSCs.
   1. Place the beak of the rongeur at the junction of the incisors and the mesial part of the first molar, cut off the connection between the lower incisors and the mandible with the rongeur, and extract the lower incisors completely.
   2. Remove all molars with a bone rongeur.
   3. Remove the mandibular ramus with a bone rongeur and expose the marrow cavity by separating the central part of the mandible along the distal edge of the last molar¹⁶.
   4. Suck α-MEM complete medium with a 1 mL disposable sterile syringe. Insert the needle into the bone marrow cavity and flush the bone marrow repeatedly into the culture dish with α-MEM complete medium until the bone turns white.
   5. Collect the bone marrow flushing solution into a 15 mL centrifuge tube and centrifuge it at 800 x g for 3 min at room temperature.
   6. Discard the supernatant with a pipette, resuspend the cells with 10 mL of α-MEM complete medium, and plate them into a new 10 cm culture dish.
   7. Divide the flushed mandible into 1-3 mm³ bone slices with a rongeur and digest them with 3 mL of 0.1% type II collagenase for 90 min in a 200 rpm/min shaker at 37 °C.
   8. Centrifuge the digested mandible at 800 x g for 3 min at room temperature and discard the supernatant.
   9. Culture the harvested cells and digested bone slices with α-MEM complete medium at 37 °C in a 5% CO₂ humidified incubator.
   10. Change half of the culture medium with fresh medium after 72 h and then change it completely every 2 days.
       NOTE: Do not move the culture dish for the first three days.
   11. Passage the adherent cells at a ratio of 1:2 when they reach 80%-90% confluency. Remove bone slices during the second subculture. Use the cells of the P2 or P3 generations for experiments.

3. Flow cytometry for Identification of cell surface markers

1. Resuspend JBMMSCs of the P2 generation in flow cytometry buffer. Count the cells using an automated cell counter and adjust the concentration to 3 × 10⁶ cells/mL.
2. Add 100 µL of cell suspension and 2 µL of monoclonal antibodies (CD11b/c, CD29, CD34, CD44, CD45, and CD90) to each microcentrifuge tube. Incubate for 30 min at 4 °C^17,18.
3. Wash the sample twice with 200 µL of flow cytometry buffer and centrifuge it for 5 min at 250 x g at room temperature. Remove the supernatant.
4. Add 100 µL of flow cytometry buffer and 2 µL of PE-labeled fluorescent secondary antibody to each tube. Incubate at 4 °C for 30 min.
5. Repeat step 3.3.
6. Resuspend JBMMSCs in 300-500 µL of flow cytometry buffer per tube.
7. Conduct the flow cytometric analysis using a flow cytometer.
   NOTE: Use homologous antibodies as negative controls to eliminate background staining caused by the non-specific combination of antibodies.

4. Cell proliferation determination

1. Seed JBMMSCs of the P3 generation into a 96-well plate at a density of 5 × 10³ cells per well.
2. Add 10 µL of CCK-8 solution to each well.
3. Place the 96-well plate in the incubator for 2 h and measure the absorbance (OD value at 450 nm) using a microplate reader.
   NOTE: Perform the test at the same time every day for seven consecutive days.

5. Colony formation capability

1. Seed 1000 JBMMSCs from passage 3 into 10 cm culture dishes and culture at 37 °C with 5% CO₂.
2. Change the culture medium every 3 days.
3. Perform crystal violet staining after continuous cultivation for 15 days.
4. Next, fix the cells with methanol for 5 min and stain with 2% crystal violet for 5 min.

6. Multilineage differentiation of JBMMSCs

NOTE: Use JBMMSCs of P3 generation for multilineage differentiation. The Control group was cultured with α-MEM complete medium.

1. Perform osteogenic differentiation.
   1. Inoculate 1 × 10⁵ JBMMSCs per well in a six-well plate.
   2. When the cell confluence reaches 70%, change the medium to osteogenic induction medium.
      NOTE: Change the osteogenic induction medium every 3 days.
   3. Perform Alkaline Phosphatase (ALP) staining after 7 days of osteogenic induction.
   4. Remove the medium from the six-well plate, fix the cells with 4% paraformaldehyde at room temperature for 30 min, and then wash them with PBS three times.
   5. Stain the cells with ALP staining solution at room temperature for 30 min and protect them from light.
   6. Rinse the cells until no staining solution remains, and observe the samples under a microscope.
   7. Perform Alizarin red staining after 21 days of osteogenic induction.
   8. Stain the cells with Alizarin red staining solution for 5 min after fixing the cells with 4% paraformaldehyde for 30 min.
   9. Wash the cells with PBS until no Alizarin red staining solution remains.
   10. Observe the number of calcium nodules under a microscope.
2. Perform adipogenic differentiation.
   1. Inoculate 1 × 10⁵ JBMMSCs per well in a six-well plate.
   2. When the cell confluence reaches 70%, change the medium to adipogenic induction medium.
      NOTE: Change the adipogenic induction medium every three days.
   3. Perform Oil red O staining after 14 days of adipogenic induction.
   4. Remove the medium from the six-well plate, fix the cells with Oil red O fixing solution for 20-30 min, and then wash them with PBS three times.
   5. Remove the fixing solution and wash the cells with distilled water twice.
   6. Immerse the cells in 60% isopropanol for 20-30 s.
   7. Remove 60% isopropanol and stain the cells with Oil red O staining for 20-30 min.
   8. Remove the staining solution, rinse the cells with 60% isopropanol for 20-30 s, and then wash them with distilled water.
   9. Stain the nucleus with hematoxylin solution for 1-2 min.
   10. Remove the hematoxylin solution and wash the cells 2-3 times with distilled water. Add Oil red O buffer for 1 min and remove it.
   11. Observe the number of lipid droplets under a microscope.
3. Perform chondrogenic differentiation.
   1. Inoculate 1 × 10⁵ JBMMSCs per well in a six-well plate.
   2. When the cell confluence reaches 70%, change the medium to chondrogenic induction medium.
      NOTE: Change the chondrogenic induction medium every 3 days.
   3. Perform Alcian blue staining after 21 days of chondrogenic induction.
   4. Remove the medium from the six-well plate, fix the cells with 4% paraformaldehyde at room temperature for 30 min, and then wash them with PBS three times.
   5. Stain the cells with Alcian blue staining at 37 °C for 1 h.
   6. Rinse the cells until no staining solution remains, and observe them under a microscope.

7. Real-time PCR

1. Extract total RNA using the RNA extraction kit and measure the RNA concentration with an enzyme marker (following the manufacturer's instructions).
2. Synthesize cDNA using 500 ng of RNA¹⁹.
3. Perform qRT-PCR on a Real-time system with 10 µL of qRT-PCR mix containing 1 µL of cDNA, 3.5 µLof double-distilled water, 5 µL of SYBR, and 0.5 µL of primer mix (0.1 µM).
   NOTE: Primer sequences are listed in Table 1. The thermal cycling conditions are as follows: 50 °C for 2 min, 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 2 min, and then increments of 0.5 °C for 5 s from 60 °C to 95 °C for the melting curve.
4. Use GAPDH as an internal control¹⁶.

After 72 h of cell inoculation, most cells were suspended and round in shape, with very few adhered to the wall (Figure 1B). By the fifth day, adherent cell colonies appeared, exhibiting spindles or fibroblast-like shapes (Figure 1C). By the seventh day, adherent cells reached 90% confluency, forming a "fish school" shape with a small number of intermittent suspended cells (Figure 1D). Passaged cells grew rapidly and were passaged every 3 days. The cell morphology was relatively homogeneous, predominantly spindle-shaped, and arranged in a vortex-like pattern after confluency (Figure 1E-G).

Flow cytometry analysis revealed high expression levels of mesenchymal stem cell surface markers CD90, CD29, and CD44, with positive rates of 97.2%, 97.7%, and 95.7%, respectively. Conversely, hematopoietic stem cell surface markers CD45, CD34, and CD11b/c showed low expression, with positive rates of 1.37%, 0.66%, and 1.49%, respectively (Figure 2A). After 2 weeks of culture, JBMMSCs demonstrated colonization, as evidenced by crystal violet staining (Figure 2B). They exhibited stable expansion ability in vitro, with the cell growth curve following a typical 'S-like' pattern (Figure 2C). The cell proliferation process included a latent period, logarithmic growth phase, and plateau phase. Rapid growth commenced after two days, followed by a slowdown after six days. Individual cells exhibited characteristics of self-replication and proliferation.

The cells exhibited blue-purple ALP staining particles on the 7^th day and red mineralized nodules on the 21^st day. Additionally, after 14 days of adipogenic induction, the cells displayed red beaded lipid droplets, while chondrogenic differentiation induction resulted in the formation of blue cartilage-like tissue (Figure 3A). Furthermore, after 7 days of osteogenic induction, the mRNA expression levels of ALP, OCN, and Runx2 in JBMMSCs were elevated¹⁵ (Figure 3B).

Figure 1: Isolation and culture of JBMMSCs. (A) An overview of the protocol. (B-D) Images of cells cultured for 3 days, 5 days, and 7 days in primary culture, respectively. (E-G) Images of the P1, P2, and P3 cells, respectively. Scale bars: 100 µm. Please click here to view a larger version of this figure.

Figure 2: Identification of JBMMSCs. (A) Flow cytometry analysis results of rat JBMMSCs. Flow cytometry analysis revealed that these cells were positive for CD29, CD44, and CD90, consistent with BMMSCs' characteristics. Conversely, they were negative for CD11b/c, CD34, and CD45. (B) Representative images of JBMMSCs colonies stained with crystal violet (scale bars: 100 µm). (C) The cell growth curve showed the cell proliferation rate.The data and the error bars denote mean ± standard deviation, n = 3. Please click here to view a larger version of this figure.

Figure 3: Multilineage (osteogenic, adipogenic, and chondrogenic) differentiation of JBMMSCs. (A) After 7 days of osteogenic induction, increased ALP activity was observed. At 21 days post-osteogenic induction, numerous mineralized nodules were stained red with Alizarin red staining. Oil red O staining revealed a significant number of lipid droplets. Alcian blue staining highlighted the cells induced into chondrocytes following chondrogenic induction. (B) mRNA expression of osteogenic related gene ALP, OCN, and Runx2 of JBMMSCs after osteogenic induction for 7 days. (***P < 0.001, vs. the control group). The data and the error bars denote mean ± standard deviation, n = 3. Scale bars: 100 µm. Please click here to view a larger version of this figure.

Table 1: Primers used in Real-time PCR.

Bone marrow mesenchymal stem cells (BMMSCs) represent a subset of non-hematopoietic stem cells residing in bone marrow, characterized by their self-renewal capabilities, multi-directional differentiation potential, and supportive functions for hematopoiesis. These cells play pivotal roles in various physiological processes such as tissue regeneration, angiogenesis, and the regulation of cellular activities²⁰. Consequently, BMMSCs are frequently utilized in tissue repair and regenerative engineering as an optimal seed cell source²¹.

Jaw bone marrow mesenchymal stem cells (JBMMSCs), initially isolated from human jaw bone marrow aspirates in 2005 by Matsubara et al.²², exhibit distinctive differentiation characteristics attributable to the unique origins and developmental pathways of the jaw compared to other skeletal sites^4,23. Given the distinct developmental and pathological mechanisms governing craniofacial bone, prioritizing JBMMSCs in the repair of craniofacial bone defects appears imperative, owing to their homologous developmental traits²³. Furthermore, JBMMSCs exhibit heightened histocompatibility with the oral microenvironment owing to their shared embryonic tissue origins^24,25. Despite these advantages, a standardized and safe isolation protocol for JBMMSCs remains elusive, and research on JBMMSCs remains relatively limited.

The isolation and cultivation of BMMSCs currently lack standardization, with commonly employed methods including immunomagnetic bead sorting, flow cytometry sorting, density gradient centrifugation, and whole bone marrow adherent culture²⁶. Among these, the immunomagnetic bead method and flow cytometry sorting method isolate cells by recognizing specific antigens on the cell surface. However, these methods involve cumbersome operations, require specialized instruments, and may affect cell activity despite yielding high-purity cells²⁷. The density gradient centrifugation method separates cells through centrifugation and stratification, which can alter the cell microenvironment, leading to slower cell growth and increased cellular aging²⁸. Conversely, the whole bone marrow adherent culture method separates and purifies BMMSCs by intermittently replacing the medium and passaging based on varying adherent abilities among different cell types in the bone marrow. This method inflicts minimal damage to cells and is straightforward to execute¹⁶.

Compared with the femur, the jaw is smaller in size and has a narrower bone marrow cavity, with interference from cells in the tooth or periodontal ligament. The bone marrow flushing and adhesion method requires a relatively larger amount of bone marrow and is suitable for large animals and humans²⁹. JBMMSCs in rats extracted by applying the traditional bone marrow flushing method are rare in number, slow to proliferate, and of poor quality^30,31. The content of JBMMSCs in bone marrow is very low, and they are mainly found in the compact bone and endosteum. Bu-Kyu Lee et al.³² used the mandibular aspirates to isolate JBMMSCs, while the total aspiration time for 10 mL of marrow blood for the mandible was five times longer than that for the iliac crest, and the initial yield of MSCs from the mandible was three times lower than that from the iliac crest. Flushing the bone marrow alone cannot effectively isolate the cells in the compact bone and endosteum.

In recent years, studies have found that compact bone is a new and reliable source of BMMSCs, and a relatively simple method of BMMSC isolation has been derived, the bone slice digesting culture method³³. BMMSCs isolated by this method have high purity³⁴. Guo et al.³⁵have successfully isolated bone mesenchymal stem cells from mouse femurs using the bone slice digesting culture method. Yamazaet al.¹² and Cheng et al.¹⁵ applied the bone slice digesting culture method to the isolation of mouse mandibular bone marrow mesenchymal stem cells and verified MSC properties by cell proliferation, immunophenotype, and multilineage differentiation. This experiment used a combination of flushed whole bone marrow adhesion and bone slice digesting to isolate and culture primary rat JBMMSCs. The bone marrow was thoroughly washed out from the cavity without filtration to maintain the original cellular composition and growth factors in the bone marrow. Then, the bone slices were cut into small pieces and digested using type II collagenase to facilitate cells to climb out of bone fragments. Finally, the bone slices were inoculated onto a culture dish and cultured to allow cells to crawl out of the bone slices. The combination method makes the culture system of the primary cell simultaneously contain components of hematopoietic stem cells, cancellous bone, and cortical bone, which can better simulate the microenvironment of bone marrow mesenchymal stem cells in the body and is more conducive to maintaining the original biological characteristics of cells. This method is therefore called "a niche-based approach on stemness"³⁶. Luet al.³⁷ also emphasized the importance of stem cell niches in the isolation of BMMSCs, but they only involved the microenvironment in the bone marrow without cortical bone. This method has been successfully applied in isolating bone marrow mesenchymal stem cells from mice jaw³⁸.

We also successfully isolated and cultivated rat JBMMSCs with this niche-based approach. First, the isolated cells exhibited a fibroblast-like shape and adhered to plastic culture plates in vitro. Second, the cells positively expressed the CD90, CD29, and CD44 surface markers and negatively expressed CD45, CD34, and CD11b/c. Third, the cells could differentiate into osteocytes, adipocytes, and chondrocytes. The obtained cells had a good morphology, a fast proliferation rate, and differentiation ability, which met the minimum criteria for identifying human-derived mesenchymal stem cells proposed by the International Society for Cellular Therapy¹⁷.

The isolation of JBMMSCs has been developed and used to isolate cells in humans. Matsubara et al.²² were the earliest to isolate JBMMSCs from human alveolar bone marrow samples during oral surgery. Possible complications may occur during the mandible aspiration process, including damage to adjacent tissue, infection due to oral bacterial contamination, etc. Zong et al.³⁹ flushed the bone marrow cavity of the cancellous bone specimen pieces to obtain human JBMMSCs. Park et al.⁴⁰ isolated human JBMMSCs from alveolar bone chips obtained during implant drilling using a sequential digestion method. Mason et al.⁴¹ directly isolated human JBMMSCs from the jaw bone marrow cores and aspirate of patients undergoing routine dental implant placement. There was a high success rate of isolating cells from either aspirates or core and a higher success rate of the combination. However, although the above methods have successfully isolated JBMMSCs from humans, the differences in effectiveness between methods need to be compared. Currently, this presented method has only been used in mice and rats, and there is no research on applying this method to the isolation of human JBMMSCs. There are limitations to this method, especially when it is applied to humans. First, this method of combining bone marrow flushing and bone slice digestion is relatively complicated and requires more steps. Second, complex processes place higher demands on the sterility of operations, which is critical to the protocol. Third, it is impossible to flush the entire bone marrow cavity of the human jawbone, so exploring some equivalent methods, such as flushing cancellous bone fragments, is necessary.

In summary, this study successfully isolated and cultured rat JBMMSCs using a combination of bone marrow flushing and bone slice digestion based on the principle of "stem cell niche." The cell identification results confirmed that this method can isolate sufficient and high-purity JBMMSCs, providing ample cell sources for jawbone tissue engineering, particularly in bone defect repair, which is of great significance.

The authors have nothing to disclose.

This study was supported by the Health Care Projects of the Military Commission Logistics Department (19BJZ22), Beijing Natural Science Foundation (7232154), and Fourth Mil Med Univ. clinical research projects (2021XB025).