Chitosan-Collagen 3D Matrix Mimics Trabecular Bone and Regulates RANKL-Mediated Paracrine Cues of Differentiated Osteoblast and Mesenchymal Stem Cells for Bone Marrow Macrophage-Derived Osteoclastogenesis

Abstract

Recent studies have identified the regulatory mechanism of collagen in bone ossification and resorption. Due to its excellent bio-mimicry property, collagen is used for the treatment of several bone and joint disease such as arthritis, osteoporosis, and osteopenia. In bone, the biological action of collagen is highly influenced by the interactions of other bone materials such as glycosaminoglycan and minerals. In view of the above perceptions, collagen was crosslinked with chitosan, hydroxyapatite (H), and chondroitin sulfate (Cs), to produce a natural bone-like 3D structure and to evaluate its effect on bone homeostasis using bone marrow mesenchymal stem cells, osteoblast, and bone marrow macrophages. The XRD and micro-CT data confirmed the arrangement of H crystallites in the chitosan-collagen-H-Cs (CCHCs) three-dimensional (3D)-matrix and the three-dimensional structure of the matrix. The stimulatory osteoblastogenic and exploitive osteoclastogenic activity of 3D-matrices were identified using differentiated osteoblasts and osteoclasts, respectively. Besides, osteogenic progenitor's paracrine cues for osteoclastogenesis showed that the differentiated osteoblast secreted higher levels of RANKL to support osteoclastogenesis, and the effect was downregulated by the CCHCs 3D-matrix. From that, it was hypothesized that the morphology of the CCHCs 3D-matrix resembles trabecular bone, which enhances bone growth, limits bone resorption, and could be a novel biomaterial for bone tissue engineering.

Keywords: RANKL; Runx2; biomechanical properties; bone homeostasis; chitosan-composite 3D matrix; mesenchymal stem cells; osteoclast; ovariectomized mice; rheological properties.

Figure 1

Schematic representation of molecular interactions of chitosan, gum Arabic, hydroxyapatite, and chondroitin sulfate with collagen polypeptides.

Figure 2

Rheological properties of chitosan-collagen-based bio-mimic three-dimensional (3D) matrices. Stiffness (a), shrinkage factor (b), water binding capacity (c), porosity (d), XRD spectra (e), TGA spectra (f), and micro-CT images (g) of chitosan-collagen-based bio-mimic 3D matrices. CC: chitosan-collagen 3D matrix, CCH: chitosan-collagen-hydroxyapatite 3D matrix, CCCs: chitosan-collagen-chondroitin sulfate 3D matrix and CCHCs: chitosan-collagen-hydroxyapatite-chondroitin sulfate 3D matrix. The experiments were done three times with similar results. * p < 0.05 vs. CC 3D matrix.

Figure 3

The effect of chitosan-collagen-based bio-mimic 3D matrices on bone cells’ differentiation (a,b), cellular alkaline phosphatase (ALP) (c,d) (Supplementary Figure S1 shows the level of ALP normalized with the cell number), and histological staining of alkaline phosphatase (e) using naphthol AS-MX phosphate and fast blue RR dye; cells were stained with naphthol AS-MX phosphate and fast blue RR after 14 days of culture and arrows show positively-stained cells. Scale bars: 40 μm. BMMSC: bone marrow-derived mesenchymal stem cells. CC: chitosan-collagen 3D matrix, CCH: chitosan-collagen-hydroxyapatite 3D matrix, CCCs: chitosan-collagen-chondroitin sulfate 3D matrix, and CCHCs: chitosan-collagen-hydroxyapatite-chondroitin sulfate 3D matrix. The experiments were done three (a–d) or two (e) times with similar results. * p < 0.05 vs. zero days; different letters indicate statistical significance among 3D matrices for 14 days.

Figure 4

Cellular calcium deposition of bone cells cultured on chitosan-collagen based bio-mimic 3D matrices for 14 days. The cellular mineral level (a), Alizarin red (b), and von Kossa (c) staining of bone cells. Scale bars: 40 μm. The presence of apatite was confirmed using FTIR spectra (d,e); control-cells grown on a regular tissue culture plate. BMMSC: bone marrow-derived mesenchymal stem cells; MC3T3-E1, pre-osteoblast. CC: chitosan-collagen 3D matrix, CCH: chitosan-collagen-hydroxyapatite 3D matrix, CCCs: chitosan-collagen-chondroitin sulfate 3D matrix, and CCHCs: chitosan-collagen-hydroxyapatite-chondroitin sulfate 3D matrix. The experiments were done three (a) or two (b–e) times with similar results. * p < 0.05 vs. zero days; different letters indicate statistical significance among 3D matrices for 14 days.

Figure 5

(a) The effect of chitosan-collagen-based bio-mimic 3D matrices on hydroxyproline and collagen synthesis in bone cells; * p < 0.05 vs. CC. (b) Immunocytochemistry evaluation of collagen I expression of bone cells cultured for 14 days. Scale bars: 100 μm. CC: chitosan-collagen 3D matrix, CCH: chitosan-collagen-hydroxyapatite 3D matrix, CCCs: chitosan-collagen-chondroitin sulfate 3D matrix, and CCHCs: chitosan-collagen-hydroxyapatite-chondroitin sulfate 3D matrix.

Figure 6

Hematoxylin-eosin staining of bone cells cultured on 3D matrices, 100× magnification. Cell nuclei are stained dark purple and 3D matrices pink. Scale bars: 200 µm. Arrows show stained cells. BMMSC: bone marrow-derived mesenchymal stem cells; MC3T3-E1, pre-osteoblast. CC: chitosan-collagen 3D matrix, CCH: chitosan-collagen-hydroxyapatite 3D matrix, CCCs: chitosan-collagen-chondroitin sulfate 3D matrix, and CCHCs: chitosan-collagen-hydroxyapatite-chondroitin sulfate 3D matrix.

Figure 7

Protein (a,b) and mRNA expression (c) of bone cells. (a,b) Western blot analysis of collagen I and osteocalcin proteins expressed in bone cells. GAPDH was a loading control. (c) Osteogenic mRNA expression of differentiated bone cells cultured in 3D matrices. mRNA expression of bone cells was normalized with GAPDH. Blank (B)-cells cultured in a six-well culture plate. Expression of osteogenesis regulatory mRNA was normalized with GAPDH of bone cells cultured at different times (0, 3, 7, and 14 days). BMMSC: bone marrow-derived mesenchymal stem cells; MC3T3-E1, pre-osteoblast. CC: chitosan-collagen 3D matrix, CCH: chitosan-collagen-hydroxyapatite 3D matrix, CCCs: chitosan-collagen-chondroitin sulfate 3D matrix, and CCHCs: chitosan-collagen-hydroxyapatite-chondroitin sulfate 3D matrix. Data are from the experiment repeated thrice with similar results; * p < 0.05 vs. blank (b); * p < 0.05 vs. CC (c); different letters indicate statistical significance among 3D matrices.

Figure 8

Scanning electron microscope (scale bars: i = 1.0 mm, ii and iii = 200 μm) images of bone cells cultured on 3D matrices for 14 days. The small inset in the SEM image is showing the expanded magnification at 50 μm. CC: chitosan-collagen 3D matrix, CCH: chitosan-collagen-hydroxyapatite 3D matrix, CCCs: chitosan-collagen-chondroitin sulfate 3D matrix, and CCHCs: chitosan-collagen-hydroxyapatite-chondroitin sulfate 3D matrix.

Figure 9

(a) Effect of inducers (mCSF, RANKL, and rPTHr11) and CCHCs matrix conditioned medium (MCM) on osteoclast formation from ovariectomized mice. (a) Tartrate-resistant acid phosphatase (TRAP) positive osteoclast cells; scale bars: 200 μm. (b) Total number (mono-, bi-, tri-, and multi-nucleated) of osteoclasts formed from ovariectomized mice bone marrow macrophages (BMM), 1: mCSF-RANKL, 2: rPTHr11, 3: mCSF-RANKL-rPTHr11, 4: mCSF-RANKL-MCM, 5: rPTHr11-MCM, 6: mCSF-RANKL-rPTHr11-MCM, 7: MCM, * p < 0.05 vs. the mCSF-RANKL-treated group. The osteoclast precursor cells were treated with inducers in the presence or absence of MCM. The combination of mCSF-RANKL-rPTHr11 supported mature osteoclast formation. No osteoclasts formed in the MCM alone group. The experiments were done three times with similar results.

Figure 10

Different stages of osteogenic cells in osteoclast formation. Primary osteocytes and undifferentiated- and differentiated-bone cells (mesenchymal stem cells and osteoblasts) were co-cultured for 10 days with osteoclast precursor cells isolated from the femur and tibia of wild mice bone marrow. (a) Images of TRAP-stained osteoclasts; scale bars: 200 μm. (b) Total number (mono-, bi-, tri-, and multi-nucleated) of osteoclasts formed after co-culture; different letters indicate statistical significance between treatments. (c) The level of RANKL expression produced from different bone cells; different letters indicate statistical significance between control and MCM. (d) The level of osteoprotegerin (OPG) expression produced from different bone cells. Undiff OB: undifferentiated osteoblast, Diff OB: differentiated osteoblast, Undiff BMMSC: undifferentiated bone marrow mesenchymal stem cell, Diff BMMSC: differentiated bone marrow mesenchymal stem cell, OC: primary osteocytes, MCM: matrix conditioned medium. * p < 0.05 (n = 3).