The present protocol describes an efficient procedure for isolating and culturing of human mandibular bone marrow-derived mesenchymal stem cells using the whole bone marrow adherence method. The cultured cells were identified by cell proliferation assays, flow cytometry, and multilineage differentiation induction.
Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various origins have been found to obtain hMSCs recently, while bone marrow was still considered the main source. Bone marrow-derived MSCs (BMSCs) from different donor bone sites have distinct characteristics due to microenvironmental factors. Studies have shown that BMSCs from maxillofacial bone may have greater proliferative and osteogenic capacities than BMSCs from long bones or the iliac crest. And maxillofacial BMSCs were considered more suitable for stem cell therapy in the maxillofacial tissues. The mandible, especially the ascending ramal area with sufficient marrow, was a feasible donor site for harvesting BMSCs. This study described a protocol for harvesting, isolating, and culturing human mandibular bone marrow-derived MSCs (hmBMSCs). Furthermore, immunophenotyping of hmBMSCs, proliferation assays, and in vitro induction of osteogenic, adipogenic, and chondrogenic differentiation was performed to identify the cultured cells. Applying this protocol can help the researchers successfully obtain enough high-quality hmBMSCs, which is necessary for further studies of the biological function, microenvironmental effects, and clinical applications.
Human mesenchymal stem cells (hMSCs) are multipotent cells that can be differentiated into various cell types, such as osteocytes, adipocytes, and chondrocytes from mesodermal lineage, hepatocytes, and pancreocytes from endodermal lineage, and neurocytes from ectodermal lineage1. Thus, hMSCs have shown great potential in tissue regeneration. Furthermore, hMSCs are powerful immunomodulators that can regulate the microenvironment in the host tissues and effectively treat chronic refractory diseases2. Therefore, hMSCs have been widely used in cell therapy of clinical studies. Consequently, it is important to obtain sufficient hMSCs with high-quality in a convenient way successfully.
Since hMSCs were first reported in the bone marrow, many alternative MSCs sources have been found, such as adipose tissue, synovium fluid, skeletal muscle, amniotic fluid, endometrium, dental tissues, and umbilical cord1,3. However, bone marrow remains the principal source of hMSCs for most preclinical and clinical studies, and bone marrow-derived MSCs (BMSCs) are taken as a standard for comparing MSCs from other sources4. For years, the iliac crest or long bones (the tibia and femur) have been the most popular anatomic locations for obtaining bone marrow1,5. However, the iliac crest or long bones have different embryonic origins and development patterns compared with the maxillofacial bones5,6. Many clinical, laboratory and developmental studies proved that BMSCs from different origins showed site-specific properties, and the grafted BMSCs retain the properties of the donor site after implantation at the recipient site5,6,7,8,9. From the perspective of developmental origin, the maxillofacial tissues, such as the maxilla, mandible, dentin, alveolar bone, pulp, and periodontal ligament, arise exclusively from neural crest cells. In contrast, the iliac crest and long bones are formed by mesoderm. In addition, mandibles are created by intramembranous ossification, while axial and appendicular skeletons undergo endochondral ossification. Moreover, compared to the iliac crest and long bones, some clinical studies have shown that maxillofacial bone marrow-derived hMSCs had better cellular proliferative activity and differentiation capability6,8. Therefore, BMSCs from the maxillofacial areas are expected to be a better choice for maxillofacial tissue regeneration and maxillofacial chronic refractory diseases therapy.
The mandible is composed of two-layer thick cortical bones with cancellous bone marrow in between so that it can load the power of chewing. Therefore, the mandible, especially the ascending ramal area, is usually used as a donor site to obtain autologous bone grafts in craniomaxillofacial surgeries10. And in surgeries such as mandibular sagittal split ramus osteotomy and mandibular angle reduction plasty, parts of mandibular cortical and cancellous bone have to be removed to achieve a pleasing facial contour. Those discarded cancellous bones could be a potential resource for hMSCs. However, few published studies described the protocol to isolate rapidly and culture high-quality human mandibular bone marrow-derived MSCs (hmBMSCs).
The present study uses a modified whole bone marrow adherence method to introduce a reliable and reproducible protocol for the isolation and culture of hmBMSCs. And the stem cells were identified by flow cytometric immunophenotyping of MSCs, proliferation assays, and multilineage differentiation induction. Applying this standard procedure may help the researchers obtain high-quality human mandibular bone marrow-derived MSCs, which is important in further studies of the biological function, microenvironmental effects, and clinical applications.
The procedure for harvesting human mandibular bone specimens was approved by the Ethics Committee of the School of Stomatology, the Fourth Military Medical University. The study followed the ethical guidelines of the 1975 Declaration of Helsinki. All donors for the present study were informed of the possible risks and the study objectives. The age of the donors ranged between 18-40 years, and there was no gender bias. Written consent was obtained from all the human participants.
1. Surgery preparation
2. Harvesting human mandibular bone specimen
3. Isolation and cultivation of human mBMSCs
4. Cell passage
5. Flow cytometric analysis
6. Cell proliferation assay
7. Multilineage differentiation
A mandibular bone specimen was successfully collected from the patient. And the time from cutting with the ultrasonic osteotome blade to placing the bone fragment into the centrifuge tube is about 5 min. None of the potential complications happened in and after the resection procedure, including damage of the inferior alveolar nerve or dental root, infection, vascular rupture and bleeding, mucosal injury, accidental bone fracture, etc. The hmBMSCs were successfully cultured, passaged, and differentiated without contamination.
In the present study, many adhered cells were seen under the microscope on the third day after the initial culture. On the seventh day, most of the adherent cells were already attached to the culture dish. Normally, the cultured cells reached 70%-80% confluence after 7-9 days of initial culture. After passage, the P3 cells were usually thought to be purified MSCs, which showed spindle-shaped, plastic-adherent, and fibroblast-like morphology (Figure 2). And the identification experiments were conducted in P3 to P5 cells. Cell proliferation assay, the cells growth rate increased rapidly from the third day of culture, and the increased rate slowed down on day 6 (Figure 2).
According to the definition given by the International Society for Cellular Therapy, MSCs have a positive and negative expression of particular surface molecules. In this study, the flow cytometric analysis of culture cells showed positive expression of CD44, CD90, and negative expression of CD45, CD34, which is consistent with the definition4,11 (Figure 3). In the Multilineage differentiation ability experiments, the cultured cells showed strong osteogenic, adipogenic, and chondrogenic differentiation capability. After 7 days of osteogenic induction, the extracellular matrix calcium deposition emerged under the microscope. After 21 days of osteogenic induction, the cultured cells showed obvious mineralization nodes, and the nodes were stained red with alizarin red staining (Figure 4). For adipogenesis, lots of accumulated round lipid droplets stained red by Oil-red-O staining were seen in the cytoplasm after 21 days of adipogenic induction (Figure 4). After 21 days of chondrogenic differentiation induction, the cell pellet slides showing cartilage-like tissue with cartilage lacuna can be stained blue (Figure 4).
Figure 1: Donor site selection on 3D CT image. (A) 3D CT image of the donor mandible from a 21-year-old female patient. (B) Surgical simulation of the bilateral sagittal split ramus osteotomy of the mandible. (C) Surgical planning shows the setback of a split mandible. (D) 3D CT image showing the cortical bone (the translucent part), the cancellous bone rich in bone marrow, and the inferior alveolar nerve (red) based on different CT values, which can guide the surgeons to select the donor site and avoid nerve injury. Cancellous bone chips were harvested from the region in the black rectangle. Please click here to view a larger version of this figure.
Figure 2: Microscopic morphology and growth curve of P3 human mandible-derived bone marrow stem cells (hmBMSCs). (A–C) Spindle-shaped, plastic-adherent, and fibroblast-like morphology of P3 hmBMSCs (Scale bar = 200 µm, 100 µm, 50 µm for A, B, C, respectively). (D) The cell growth curve showed that the growth rate of hmBMSCs increased rapidly from the third day of culture and the increase rate slowed down on the sixth day of culture. Please click here to view a larger version of this figure.
Figure 3: Cell surface antigen expression on hmBMSCs was detected by flow cytometry. Flow cytometry analysis showed that hmBMSCs were negative for CD45 (A) and CD44 (C), positive for CD44 (B), and CD90 (D). Please click here to view a larger version of this figure.
Figure 4: Osteogenic, adipogenic, and chondrogenic differentiation of hmBMSCs. (A) ALP staining of hmBMSCs after 7 days of culture without osteogenic differentiation induction (Scale bar = 100 µm). (B) ALP staining of hmBMSCs after 7 days of osteogenic differentiation induction (Scale bar = 100 µm). (C) Quantitative results of ALP staining positive area (***P < 0.001). (D) Alizarin red staining of hmBMSCs without osteogenic differentiation (Scale bar = 100 µm). (E) The formation of obvious mineralization nodes can be seen after 21 days of osteogenic differentiation of hmBMSCs, and it can be stained red with alizarin red staining (Scale bar = 100 µm). (F) Quantitative results of alizarin red staining (***P < 0.001). (G) Oil red O staining of hmBMSCs without adipogenic differentiation (Scale bar = 50 µm); (H) Round lipid droplets were seen after 21 days of adipogenic differentiation of hmBMSCs, and the lipid droplets were stained red with oil red O staining (Scale bar = 50 µm). (I) Alcian blue staining was positive after 21 days of chondrogenic differentiation induction (Scale bar = 50 µm). Please click here to view a larger version of this figure.
Recently, the hMSCs therapy has shown great promise in tissue regeneration and the treatment of many refractory diseases, such as immune dysfunction diseases, systemic hematological diseases, cancers, or trauma, in numerous clinical trials1,14,15,16,17. Among various sources of MSCs, bone marrow remains the most widely used and easily accessed source. We used human mandibular cancellous bone chips to successfully culture BMSCs using the whole bone marrow adherence method described in the present protocol. To date, there are four main approaches to isolate stem cells from bone marrow, including the whole bone marrow adherence method, density gradient centrifugation method, fluorescent cell sorting method, and magnetic-activated cell sorting method10. The whole bone marrow adherence method is simple, easy to operate, cheap, and can get large amounts of adherent cells. However, the limitation of this method was the low purity of primary cultured BMSCs, which were mixed with hematopoietic cells and fibroblasts. After refreshing the culture medium of primary cells regularly, the non-adherent hematopoietic cells were discarded along with the discarded medium. Also, the fibroblasts can be cleared through cell passage, and P3 cells were highly purified BMSCs. So P0 to P2 cells cannot be used for cell therapy, which means extra time was needed to purify the stem cells. Using fluorescent cell sorting and magnetic-activated cell sorting methods, one can get more purified BMSCs, while the two methods are expensive, and a long selection time can impair cell viability11. To prove that the cultured cells were MSCs, we referred to the definition of human MSCs proposed by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy, which included plastic adherent character, positive and negative expression of certain phenotypes, such as CD45, CD90 and so on, and multilineage differentiation ability18.
In most studies, femurs and iliac crest were the main sources of BMSCs, compared to maxillofacial bones, such as the mandible and maxilla9,16. However, the site-specific characteristic theory of hBMSCs in recent studies showed that hBMSCs from different bones had different characters in differentiation ability, proliferative activity, osteogenesis, and immunity6,8. The site-specific difference may be related to different embryological origins, adaptation to functional demands at each skeletal site, microenvironment, local vascular supply, hormonal effects, etc. Furthermore, studies showed that grafted iliac bone exhibited more rapid vertical loss than the jawbone within 6 months after the bone graft8. Otherwise, studies have found that the proliferative activity of MSCs from mandibular marrow was superior to those from iliac marrow5,8,19,20. And this proliferative activity difference was attributed to the characters that the mandible had more blood supply and a faster bone turnover rate than the ilium5,6,8. Studies also revealed that BMSCs from the mandible expressed a higher level of Runx-2 and OCN than those from femurs, and the osteogenic ability of BMSCs from the mandible was equal to or higher than those from femurs and ilium5,19,21. The adherence to titanium ability of hmBMSCs was also found stronger than BMSCs from femurs, which suggested hmBMSCs were more appropriate to be used in oral implantology5. In addition, a 3-year clinical study to reconstruct the alveolar defect found that the regenerated bone of MSCs from the dental pulp was composed of a fully compact bone with a higher matrix density, while the hm BMSCs regenerated spongy bone similar to normal human alveolar bone struct22. In conclusion, hmBMSCs were ideal therapeutic stem cells for maxillofacial regeneration and other diseases due to the same embryological origin and their superior characteristics.
However, the mandible has less bone marrow than femurs and ilium, so it is important to obtain enough bone marrow and BMSCs from the mandible for clinical use. Human iliac aspirates can obtain large amounts of bone marrow to isolate MSCs. Researchers also used the mandibular aspirates to obtain MSCs, while the initial yield of the MSCs from mandibular aspirates was three times lower than that of iliac aspirates21. Additional incisions were needed to collect enough mandible marrow aspirates, adding additional surgical trauma. Furthermore, studies have shown that the proliferative potential of the MSCs from mandible bone chips may be superior to those from mandible marrow aspirates8,21. Therefore, in this study, the discarded mandible cancellous bone chips were used to isolate MSCs. Because both sides of the mandible were included in sagittal split ramus osteotomy or mandibular angle reduction plasty, we can get enough mandible marrow from the patients without any extra harm. Recently, computer-assisted technology has been widely used in oral and maxillofacial surgery to improve the surgical effect and reduce surgical complications23. To avoid injury of the mandible and nerve during mandible bone marrow harvesting, the 3D CT image of the donors' mandible was obtained, and surgical planning of the donors was analyzed to decide the donor sites and to implement surgical simulation in the study; thus, none of the surgical complications happened. The ultrasonic osteotome blade, a tissue-specific device allowing surgeons to make precise osteotomies while protecting adjacent soft tissue24, was also used to avoid soft tissue injury and preserve the obtained bone marrow activity.
In summary, this study described a reliable, simple, safe, and cheap protocol to isolate and culture adequate human mandibular MSCs, which can be used in cell therapies of dental and maxillofacial tissues.
The authors have nothing to disclose.
The study was supported by the National Natural Sciences Foundation of China (No.81903249) and the Natural Science Basic Research Program of Shaanxi province (No.2019JQ-701, No.2022JZ-50).
4% Paraformaldehyde | PlantChemMed | PC-00005 | |
Adipogenic differentiation medium | OriCell, Cyagen Biosciences | HUXMX-90031 | |
Alcian blue solution | OriCell, Cyagen Biosciences | ALCB-10001 | |
Alcohol | Macklin | e809056 | |
Alizarin red staining solution | Solarbio | G1452 | |
ALP staining solution | Beijing ComWin Biotech Co.,Ltd. | CW0051S | |
Autoclave | ALP Co., Ltd., Japan | CL-40L | |
CCK-8 solution | Yeasen Biotech Co., Ltd. | 40203ES80 | |
Cell filter (70 μm pore size) | BD Biosciences | 352350 | |
Cell incubator | Thermo Fisher Scientific | 41334177 | |
Centrifuge | Eppendorf | 5805ZP761456 | |
Centrifuge tube (50 mL, 15 mL) | Sangon Biotech | F600888-9001 | |
Cetylpyridinium chloride | Aladdin | H108696 | |
Chondrogenesis differentiation medium | OriCell, Cyagen Biosciences | HUXMX-90041 | |
Clean bench/Laminar flow cabinet | BIOBASE | BBS-DDS00030 | |
Culture dish (6 cm) | Thermo | 150462 | |
Culture flasks (25 cm) | Thermo | 156367 | |
Culture plates (96-well, 6-well) | Corning-Costar | 352350 | |
Disposable sterile gloves and masks | Sangon Biotech | F516018-9001;F516038-9001 | |
Disposable sterile syringe (1 mL) | Shaanxi longkangxin Medical Instrument Co., Ltd | 1.00009E+11 | |
Dulbecco's modified Eagle's medium with high glucose (HG-DMEM) | Hyclone | SH30022.01B | |
EDTA | Solarbio | E8040-500g | |
Fetal bovine serum (FBS) | PlantChemMed | PC-00001 | |
Flow cytometer | Beckman Coulter | EPICS XL | |
Fluorescein-isothiocyanate (FITC)-conjugated mouse monoclonal anti-human antibody against CD34 | Biolegend | 343503 | |
Fluorescein-isothiocyanate (FITC)-conjugated mouse monoclonal anti-human antibody against CD44 | Biolegend | 338804 | |
Fluorescein-isothiocyanate (FITC)-conjugated mouse monoclonal anti-human antibody against CD90 | Biolegend | 328108 | |
Hemocytometer | Koraba | 30119480698 | |
Icebox | Sangon Biotech | F615002-0001 | |
Image J software | National Institute of Mental Health | ||
Light microscope | OLYMPUS | IX71-2L20944 | |
Microcentrifuge tubes | Sangon Biotech | F601620-0010 | |
Microplate spectrophotometer | BioTek-EPOCH | 259091 | |
Microtome | Feica | 1003001 | |
Mimics software | Materialise | ||
Minimum essential medium alpha (α-MEM) | Hyclone | SH30265.01 | |
Oil red O staining solution | Solarbio | G1261 | |
Osteogenic differentiation medium | OriCell, Cyagen Biosciences | HUXMA-90021 | |
Penicillin and streptomycin | PlantChemMed | PC-86115 | |
Phosphate buffer saline (PBS) | PlantChemMed | PC-00003 | |
Phycoerythrin (PE)-conjugated mouse monoclonal anti-human antibody against CD45 | Biolegend | 304008 | |
Pipette | SORFA | 320511 | |
ProPlan CMF 3.0 | Materialise | ||
Scissors, tweezers and knives | Shanghai Jinzhong Surgical instrument Co., Ltd | ZJA030,YAA110,J11010 | |
Sterile wet gauze | HENAN PIAOAN GROUP Co., Ltd | ||
Trypsin | Gibco | 17075029 | |
Ultrasonic osteotome blade | Stryker Instruments | 5450-815-107 |
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