Chitosan enhances mineralization during osteoblast differentiation of human bone marrow-derived mesenchymal stem cells, by upregulating the associated genes

Abstract

Objectives: Chitosan is widely used as a scaffold for bone tissue engineering. However, up-to-date, no previous detailed study has been conducted to elucidate any mechanism of osteogenesis by chitosan itself. Here, we have evaluated effects of chitosan-coated tissue culture plates on adhesion and osteoblast differentiation processes of human mesenchymal stem cells (hMSCs), isolated from adult bone marrow.

Materials and methods: Tissue culture plates coated with chitosan at different coating densities were used to evaluate the effects on hMSC adhesion and osteoblast differentiation. hMSCs were induced to differentiate into osteoblasts on the chitosan-coated plates and were evaluated using established techniques: alkaline phosphatase assay, demonstration of presence of calcium and real time PCR.

Results: The cells adhered to plates of lower coating density of chitosan, but formed viable cell aggregates at higher coating density (100 μg/sq.cm). Coating density of 25 μg/sq.cm, supporting cell adhesion was chosen for osteoblast differentiation experiments. Differentiating hMSCs showed higher mineral deposition and calcium content on chitosan-coated plates. Chitosan upregulated genes associated with calcium binding and mineralization such as collagen type 1 alpha 1, integrin-binding sialoprotein, osteopontin, osteonectin and osteocalcin, significantly.

Conclusions: We demonstrate for the first time that chitosan enhanced mineralization by upregulating the associated genes. Thus, the study may help clinical situations promoting use of chitosan in bone mineralization, necessary for healing non-union fractures and more.

Figure 1

Characterization of adult bone marrow derived human mesenchymal stem cells. (a) Morphology of the cells under phase contrast microscope shows spindle shaped fibroblast like cells. (b) The immunofluorescent micrograph shows the cells stained positive for vimentin. (c) Phase contrast and (d) Oil Red O staining confirmed the differentiation into the adipogenic lineage. (e–h) Differentiation into osteogenic lineage was confirmed by phase contrast and histochemical staining for calcium. (e) Phase contrast micrograph and (f) Alizarin Red S staining on day 14 of differentiation. (g) Phase contrast micrograph and (h) von Kossa staining on day 21 of differentiation. (i) The Immunophenotyping by flow cytometry showed the isolated cells positive for mesenchymal stem cell markers like CD90, CD73, CD105, CD44, CD166 and negative for haematopoetic markers like CD34 and CD45. The total magnification of the micrographs is 100× except (c, d) which is 200×.

Figure 2

hMSCs culture on tissue culture plates coated with different concentrations of chitosan. (a) Cells on untreated plates. (b–f) Cells on chitosan coated plates. (b–d) hMSCs attached and showed fibroblast like morphology on chitosan plates of coating density 5, 10 and 25 μg/sq. cm. There was uniform cell distribution on these plates. (e) Chitosan plate with a coating density of 50 μg/sq. cm also showed adherent hMSCs but there was non‐uniform cell distribution. (f) Chitosan plate with a coating density of 100 μg/sq. cm did not show any adherent hMSCs but there was formation of non‐adherent cell aggregates on this plate. A coating density of 25 μg/sq. cm which supported hMSCs adhesion was selected for further osteoblast differentiation experiments. Phase contrast micrographs with a total magnification of 40×.

Figure 3

hMSC aggregates on chitosan coated plates. (a) 6 days old cell aggregates on chitosan coated plates (100 μg/sq. cm). (b) The aggregates re‐plated into normal tissue culture plates showed attached spindle shaped cells spreading from the aggregates. (c, d) Osteogenic differentiation experiments with the cell aggregates on chitosan coated plates. (c) Alizarin Red S staining for the aggregates in non osteogenic medium for 21 days showed no calcium deposition. (d) Cell aggregates grown in osteogenic media for 21 days gave a positive staining with Alizarin Red S, indicating osteoblast differentiation and mineralization. (a, b) Phase contrast micrographs with a total magnification of 100×. (c, d) Phase contrast micrographs with a total magnification of 400×.

Figure 4

Phase contrast micrographs of cells undergoing osteoblast differentiation on chitosan coated plates. (a–c) Osteoblast differentiation on the untreated plates. (d–f) Osteoblast differentiation on the chitosan coated plates. There were more amounts of mineral deposits by the cells differentiated on chitosan coated plates. (e, f) There was formation of bone nodules by day 14 of differentiation on chitosan coated plates which increased in size by day 21. Total magnification is 100×.

Figure 5

Alkaline phosphatase (ALP) as a marker of osteoblast differentiation. (a–d) Cells undergoing osteoblast differentiation stained for ALP by BCIP–NBT method after 7 days of induction. (a, b) Staining for ALP on untreated plates. (c, d) Staining for ALP on chitosan coated plates. Chitosan coated plates showed more intensely stained cells. (a, c) ALP positive cells were mainly present in the periphery of both the plates (Gross view). (b, d) Phase contrast micrographs with a total magnification of 100×. (e) Alkaline phosphatase assay. The ALP expression pattern was similar on both the untreated (UT) and chitosan coated plates (chitosan). There was significant increase in the ALP expression by the hMSCs in osteogenic induction medium (OST) compared to the hMSCs in non osteogenic medium (C, control). The second week of differentiation showed the peak in ALP expression which is believed to coincide with the initiation of mineralization in both the plates. (f) ALPL mRNA expression levels. mRNA expression levels for ALPL was quantified at different stages of osteoblast differentiation by real time PCR. hMSCs on the UT and chitosan coated plates (chitosan) showed different pattern of expression levels. The peak in ALPL expression was significantly higher on chitosan coated plates (*P < 0.05).

Figure 6

Histochemical staining for calcium. (a–d) Alizarin Red S staining on day 14 of osteogenic differentiation. (a, b) The staining on untreated plate. (c, d) The staining on chitosan coated plates showing more intense staining indicating more calcium deposits or mineralization. (e–h) von Kossa Staining on day 21 of osteogenic differentiation. (e, f) The staining on untreated plate. (g, h) The staining on chitosan coated plate. The Von Kossa staining confirmed the enhancement of mineral matrix deposition by the chitosan coated plates which were evident from the more black deposits on these plates. (b, d, f, h) Phase contrast micrographs with a total magnification of 100×.

Figure 7

Calcium assay by O‐Creolphthalein Complexone method. The amount of calcium in the deposited mineral matrix was quantified on day 21 of osteoblast differentiation and was expressed per mg of total protein. Cells on chitosan coated plates (chitosan) showed significantly high (*P < 0.05) calcium deposits compared to the untreated plates (UT).

Figure 8

Real time PCR for osteoblast differentiation associated gene expression analysis. The graphs represent the relative fold difference in the gene expression on chitosan coated (chitosan) plates with respect to the untreated (UT) plates. The fold difference was calculated by the 2^−ΔΔCT method. Cells on chitosan coated plates showed significantly high fold difference in the expression of OPN, IBSP and COL1A1, the three genes regulating respective proteins associated with calcium binding and bone matrix formation. ALPL showed a marked increase and a significantly high expression on day 14 and 21 of differentiation respectively. ON, a later stage and OCN, a final stage differentiation marker also showed significant fold difference on chitosan coated plates. Another key transcription factor OSX, regulating osteoblast differentiation, also showed a marked increase in expression on chitosan coated plates (*P < 0.05, **P < 0.01 and ***P < 0.001).