Mesenchymal Stromal Cell Differentiation for Generating Cartilage and Bone-Like Tissues In Vitro

Abstract

In the field of tissue engineering, progress has been made towards the development of new treatments for cartilage and bone defects. However, in vitro culture conditions for human bone marrow mesenchymal stromal cells (hBMSCs) have not yet been fully defined. To improve our understanding of cartilage and bone in vitro differentiation, we investigated the effect of culture conditions on hBMSC differentiation. We hypothesized that the use of two different culture media including specific growth factors, TGFβ1 or BMP2, as well as low (2% O₂) or high (20% O₂) oxygen tension, would improve the chondrogenic and osteogenic potential, respectively. Chondrogenic and osteogenic differentiation of hBMSCs isolated from multiple donors and expanded under the same conditions were directly compared. Chondrogenic groups showed a notable upregulation of chondrogenic markers compared with osteogenic groups. Greater sGAG production and deposition, and collagen type II and I accumulation occurred for chondrogenic groups. Chondrogenesis at 2% O₂ significantly reduced ALP gene expression and reduced type I collagen deposition, producing a more stable and less hypertrophic chondrogenic phenotype. An O₂ tension of 2% did not inhibit osteogenic differentiation at the protein level but reduced ALP and OC gene expression. An upregulation of ALP and OC occurred during osteogenesis in BMP2 containing media under 20% O₂; BMP2 free osteogenic media downregulated ALP and also led to higher sGAG release. A higher mineralization was observed in the presence of BMP2 during osteogenesis. This study demonstrates how the modulation of O₂ tension, combined with tissue-specific growth factors and media composition can be tailored in vitro to promote chondral or endochondral differentiation while using the same donor cell population.

Keywords: chondrogenesis; donor comparison; osteochondral constructs; osteogenesis.

Figure 1

Schematic overview of the experimental design. Created with BioRender.com.

Figure 2

Gene expression measured at day 14 by real-time polymerase chain reaction (qPCR) of chondrogenic and osteogenic scaffolds. A significant upregulation of the chondrogenic markers Collagen type II, Aggrecan and SOX9, and the hypertrophic marker Collagen type X, was observed in the chondrogenically differentiated constructs compared with osteogenic samples. The Sox9/Runx2 and Col2A1/Coll10A1 ratios were also greater in chondrogenic samples. Furthermore, ALP gene expression was consistently downregulated in chondrogenically differentiated constructs under 2% oxygen tension. An upregulation of ALP was observed in the osteogenically differentiated constructs, with the exception of BMP2 combined with 20% O₂. Statistical significance was defined as * p < 0.05 and ** p < 0.01.

Figure 3

Gene expression measured at day 14 of differentiation by real-time polymerase chain reaction (qPCR). VEGF gene expression was significantly upregulated in the osteogenic groups supplemented with BMP2. Relative quantification of target mRNA was performed according to the comparative Ct method. Values represent the mean and standard deviation of four independent hBMSC donors in experimental triplicate. Statistical significance was defined as * p < 0.05 and ** p < 0.01.

Figure 4

Biochemical analysis of chondrogenically and osteogenically differentiated constructs after 2 weeks in culture shows a significantly higher sulphated glycosaminoglycan deposition in chondrogenically differentiated constructs independent of oxygen tension. (A) Bisbenzimide Höchst 33528 dye was used to quantify the DNA in proteinase K digests of scaffolds. (B–F) Dimethylmethylene blue (DMMB) at pH 3 was used to determine the sulphated glycosaminoglycan (GAG) produced by mesenchymal stem cells (MSCs): (B) sulphated glycosaminoglycan produced by MSCs and deposited inside the scaffold: (C) GAG/DNA per scaffold: (D) culture media GAG normalized to DNA: (E) cumulative GAG deposited into the scaffold and released in culture media; (F) Total GAG/DNA. Values represent the mean ± SD of four independent hBMSC donors in experimental triplicate or quadruplicate. Statistical significance was defined as * p < 0.05, and ** p < 0.01.

Figure 5

GAG produced and released into culture media from scaffolds over 2 weeks in culture. In the first 7 days, the osteogenically differentiated groups released significantly more GAG into the culture media compared with the chondrogenically differentiated constructs. The absence of BMP2 in the osteogenic media kept the media GAG higher until day 14. sGAG content in culture medium was determined spectrophotometrically following reaction with 1.9-dimethylmethylene blue (DMMB) pH 3. Values represent the mean ± SD of three independent hBMSC donors in experimental quadruplicate or quintuplicate. Statistical significance was defined as * p < 0.05 and ** p < 0.01.

Figure 6

Chondrogenic and osteogenic hBMSC-constructs after 2 weeks of culture. Images showing a higher magnification of donor 1. Scale bar: 200 µm. Inset: Images showing the full section of the constructs 1. Scale bar: 1 mm. Scaffolds were stained with safranin O/Fast Green, Von Kossa and immunohistochemically labelled for collagen type I and collagen type II. Chondrogenically differentiated groups showed higher Safranin O, Collagen Type I and Collagen type II deposition. The osteogenically differentiated groups with BMP2 are positive for Von Kossa staining.

Figure 7

Chondrogenic and osteogenic hBMSC-constructs after 2 weeks of culture. Images showing a higher magnification of donor 2. Scale bar: 200 µm. Inset: Images showing the full section of the constructs 1. Scale bar: 1 mm. Scaffolds were stained with safranin O/Fast Green, Von Kossa and immunohistochemically labelled for collagen type I and collagen type II. Chondrogenically differentiated groups showed higher Safranin O, Collagen Type I and Collagen type II deposition. The osteogenically differentiated groups with BMP2 are positive for Von Kossa staining.

Figure 8

Chondrogenic and osteogenic hBMSC-constructs after 2 weeks of culture. Images showing a higher magnification of donor 3. Scale bar: 200 µm. Inset: Images showing the full section of the constructs 1. Scale bar: 1 mm. Scaffolds were stained with safranin O/Fast Green, Von Kossa and immunohistochemically labelled for collagen type I and collagen type II. Chondrogenically differentiated groups showed higher Safranin O, Collagen Type I and Collagen type II deposition. The osteogenically differentiated groups with BMP2 are positive for Von Kossa staining.

Figure 9

Chondrogenic and osteogenic hBMSC-constructs after 2 weeks of culture. Images showing a higher magnification of donor 4. Scale bar: 200 µm. Inset: Images showing the full section of the constructs 1. Scale bar: 1 mm. Scaffolds were stained with safranin O/Fast Green, Von Kossa and immunohistochemically labelled for collagen type I and collagen type II. Chondrogenically differentiated groups showed higher Safranin O, Collagen Type I and Collagen type II deposition. The osteogenically differentiated groups with BMP2 are positive for Von Kossa staining.