Objective: In vitro comparison of chondrogenic differentiation ability of bone marrow extracted mesenchymal stem cells (MSCs) and buffy coat. Methods: MSCs of New Zealand white rabbits were cultured in vitro and adherent cells were passaged. The cells were inoculated on polyglycolic acid (PGA) scaffold (3 mm in diameter and 2 mm in height) liquid for 21 days. Under the same conditions, without subculture, buffy coat was directly inoculated into the cell scaffold. The degree of chondrogenic differentiation was compared by Safranin-Ostaining, histological scoring and biochemical functional detection. Results: The chondrogenic differentiation ability of the buffy coat group was better than that of the MSC group. Safranin-Ostain was stronger in buffy coat group than in MSC group. The Bern Score was also higher in the buffy coat group than in the MSC group and the total amount of sulfated glycosaminoglycans (GAGs) in buffy coat group was higher than that in MSC group. Conclusion: The chondrogenic differentiation ability of buffy coat is higher than that of MSC. Through this result, it was found buffy coat can also differentiate into sub-cartilage without stimulating growth factor. Thus, buffy coat can make a great source in artificial cartilage engineering.
According to World Health Organization statistics, there are currently more than 50 million patients with osteoarthritis in China, a figure that will continue to increase as the population ages. Cartilage injury is an important cause of knee osteoarthritis. However, there is currently no effective method of articular cartilage regeneration. Therefore, the exploration and development of a new cartilage regeneration method have important value.
With the development of tissue engineering, repairing damaged cartilage tissue has become a hot topic. The main factors of tissue engineered cartilage are seed cells, scaffold materials, and cell growth factors. Currently used cells include chondrocytes, mesenchymal stem cells (MSCs), and induced pluripotent stem cells [
Numerous studies have demonstrated the therapeutic potential of mononuclear cells (MNCs) extracted from bone marrow for tissue regeneration after ischemic femoral head necrosis, myocardial infarction, and cartilage injury [
Healthy 16-week-old New Zealand white rabbits were purchased from the experimental animal center of Yanbian University. All of the rabbits were housed during the experimental study under 12:12 hour light/dark conditions at approximately 27˚C. The experimental procedure was approved by the Institutional Animal Care and Use Committee of Yanbian University Hospital (2015036).
Rabbit bone marrow-derived buffy coat and MSCs were obtained from 16-week-old male (n = 12) New Zealand white rabbits. Two milliliters of bone marrow blood was harvested from the iliac crest. The samples were diluted in phosphate buffered saline (PBS) and the buffy coat was isolated by centrifugation using a Ficoll density gradient (17-1440-02; Amersham Biosciences, Sweden) at 1000 × g for 30 min [
The bone marrow (2 mL) aspirates were obtained aseptically from the iliac crest, while the buffy coat was obtained by Ficoll gradient centrifugation at 1000 × g for 30 min. The buffy coat (0.5 mL) was dispensed with a pipette and placed in a 1.5-mL tube. The concentrated buffy coat was centrifuged at 1000 × g for 5 min. After supernatant removal, 400 μL was collected and 100 μL of concentrated buffy coat seeding was collected onto a PGA scaffold (5 × 106 MNCs/scaffold) and incubated in a CO2 incubator for 90 minutes and then placed in chondrogenic medium.
The PGA scaffold in sheet form was purchased from Albany International Inc. (Mansfield, MA, USA). The PGA scaffold was cut by a biopsy punch to 2 mm thick and 3 mm in diameter. The PGA scaffolds were submerged in ethylene oxide for sterilization.
Chondrogenic medium (Dulbecco’s modified Eagle’s medium supplemented with ITS mixture, 50 mg/mL ascorbate 2-phosphate, 100 nM dexamethasone, 40 mg/mL proline, and 1.25 mg/mL bovine serum albumin), without TGF-β, a typical chondrogenic inducer. The cell-seeded scaffolds were placed in 24-well plates and incubated until 4 weeks at 37˚C under 5% CO2 to induce chondrogenic differentiation (n = 6/scaffold).
The percentage of viable and dead cells was measured using a live/dead viability/ cytotoxicity assay (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions. One-day samples were incubated in a solution containing 2 mM ethidium homodimer-1 and 4 mM calcein AM for 30 min. After being washed in PBS, the construct was placed on a glass slide and carefully pressed flat using a cover glass. The fluorescence image was visualized using a laser canning confocal microscope (Zeiss LSM510 Meta; Carl Zeiss, Jena, Germany). The number of viable (green fluorescence) or dead (red fluorescence) cells was quantified by Image-Proplus 4.0 image analysis software (Media Cybernetics, Silver Spring, MD, USA). Cell viability was determined by dividing the number of green cells (viable cells) by the total cells (green cells + red cells) [
The PGA scaffolds were fixed with 4% paraformaldehyde for 4 days, embedded in paraffin, cut into ultrathin sections, and stained with Safranin O/solid green staining in accordance with the literature. The prepared paraffin sections were dewaxed to water; stained with hematoxylin for 3 minutes; washed with 1% hydrochloric acid for 15 seconds and water for 3 minutes, a 0.02% solid green water solution for 10 seconds, and 1% acetic acid to remove residual solid green; and stained with 0.1% Safranin O for 6 minutes. They were then dried in air, dehydrated, xylene transparent, and finally sealed with neutral gum for microscopic observation.
To minimize the effects of subjective bias, three researchers independently evaluated the quality of the engineered cartilage and graded the Safranin O-fast green-stained slides using the Bern Score, which consists of seven categories and assigns a score of 0 - 9 [
The specimens were dried and digested with papain. The glycosaminoglycan (GAG) content was detected using the 1,9-dimethyl methylene blue (DMB) color method as follows: 0, 0.00625, 0.0125, 0.025, 0.05, 0.1 mg/mL chondroitin sulfate standard solution (50 µL) added to 200 µL of DMB reagent mixture, left to react in the dark for 30 minutes and then examined at a 530-nm wavelength colorimetric absorbance to create the standard GAG curve. Aliquots of the supernatant of the experimental and control groups (1 mL and 50 µL, respectively) were collected after the centrifugation, and 200 µL of DMB was added for 30 minutes. The supernatant absorbance was determined by the same method, and the supernatant GAG was determined by the standard curve. DNA content was detected by Qubit Fluorometer in papain-digested specimens as described previously [
Student’s t test in Microsoft Excel 2007 was used to analyze the data. The data are expressed as mean ± standard deviation. Group comparisons were made using single factor analysis of variance, and values of p < 0.05 were considered statistically significant (*p < 0.05, **p < 0.01, and ***p < 0.001).
The MSCs and buffy coat were inoculated into PGA cell stents for 1 day and a live/dead cytotoxicity assay (
Cells in the experimental and control groups were cultured on the PGA scaffolds for 21 days and were stained with Safranin O stain (
Analysis of these sections using the Bern Score supports this observation. The cells in the constructs with high cell densities showed increased Safranin-O staining in the buffy coat group, which demonstrated a rounded cell shape and higher extracellular matrix (ECM) production (
DMB colorimetry was performed in the experimental and control groups after 21 days of culture on the PGA scaffold for the quantitative detection of GAG content. The results showed that the GAG content of the buffy coat group was greater than that of the MSC group (n = 7; p < 0.001) (
higher in the MSC group than in the buffy coat group (n = 7; p < 0.001;
The results showed that the cartilage differentiation of the experimental group and the buffy coat group was much more than that of the single MSCs in vitro. The bone marrow cells and cytokines were cultured in vitro with different growth factors.
The extracted concentration of bone marrow MSC and MNC from the iliac crest of the rabbit was 607.8/mL and 2.3 × 106/mL, respectively [
The same number of cells was inoculated into the same cell scaffold, and we found no difference in the initial cell coverage or viability (
The MSC concentration in the buffy coat extracted from the bone marrow of the rabbit from Ficoll was 2.34 × 104 MNCs [
In this experiment, buffy coat extraction was related to cartilage formation when the red blood cell (RBC) content was high as previously described by Horn et al. [
The ultimate goal of tissue engineering is its clinical application, and the ability to resolve MSC differentiation without the application of interventional conditions is a key factor in achieving such clinical goals. The buffy coat addresses this problem and can become the best source of cells for tissue engineering.
This study was supported by the grant of the National Natural Science Foundation of China (81560353); China Postdoctoral Science Foundation (2016M601394); Jilin Postdoctoral Science Foundation, China; The Science and Technology Research Project of the Department of Education, Jilin, China.
The authors who have contributed certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.
Healthy 16-week-old New Zealand white rabbits were purchased from the experimental animal center of Yanbian University. All of the rabbits were housed during the experimental study under 12:12 hour light/dark conditions at approximately 27˚C. The experimental procedure was approved by the Institutional Animal Care and Use Committee of Yanbian University Hospital (2015036).
Jin, L.H., Zhang, Z.Y., Liu, Y.Q., Gurung, K., Jin, Y.J., He, Y.M., Jin, Y. and Jin, Z.H. (2018) Comparison of Chondrogenic Ability between Mesenchymal Stem Cells and Buffy Coat in Vitro. Journal of Biosciences and Medicines, 6, 86-94. https://doi.org/10.4236/jbm.2018.63006