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. 2020 Jun 25;5(4):844-858.
doi: 10.1016/j.bioactmat.2020.06.005. eCollection 2020 Dec.

Doping bioactive elements into a collagen scaffold based on synchronous self-assembly/mineralization for bone tissue engineering

Huanhuan Liu  1 , Mingli Lin  2 , Xue Liu  1 , Ye Zhang  1 , Yuyu Luo  3 , Yanyun Pang  1 , Haitao Chen  1 , Dongwang Zhu  1 , Xue Zhong  1 , Shiqing Ma  1 , Yanhong Zhao  1 , Qiang Yang  4 , Xu Zhang  1   5
Affiliations

Doping bioactive elements into a collagen scaffold based on synchronous self-assembly/mineralization for bone tissue engineering

Huanhuan Liu et al. Bioact Mater. .

Abstract

Pure collagen is biocompatible but lacks inherent osteoinductive, osteoimmunomodulatory and antibacterial activities. To obtain collagen with these characteristics, we developed a novel methodology of doping bioactive elements into collagen through the synchronous self-assembly/mineralization (SSM) of collagen. In the SSM model, amorphous mineral nanoparticles (AMN) (amorphous SrCO3, amorphous Ag3PO4, etc.) stabilized by the polyampholyte, carboxymethyl chitosan (CMC), and collagen molecules were the primary components under acidic conditions. As the pH gradually increased, intrafibrillar mineralization occurred via the self-adaptive interaction between the AMNs and the collagen microfibrils, which were self-assembling; the AMNs wrapped around the microfibrils became situated in the gap zones of collagen and finally transformed into crystals. Sr-doped collagen scaffolds (Sr-CS) promoted in vitro cell proliferation and osteogenic differentiation of rat bone marrow mesenchymal stromal cells (rBMSCs) and synergistically improved osteogenesis of rBMSCs by altering the macrophage response. Ag-doped collagen scaffolds (Ag-CS) exhibited in vitro antibacterial effects on S. aureus, as well as cell/tissue compatibility. Moreover, Sr-CS implanted into the calvarial defect of a rat resulted in improved bone regeneration. Therefore, the SSM model is a de novo synthetic strategy for doping bioactive elements into collagen, and can be used to fabricate multifunctional collagen scaffolds to meet the clinical challenges of encouraging osteogenesis, boosting the immune response and fighting severe infection in bone defects.

Keywords: Bioactive elements; Bone tissue engineering; Collagen scaffold; Synchronous self-assembly/mineralization.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image 1
Graphical abstract
Scheme 1
Scheme 1
Flow diagram of the experimental procedure.
Fig. 1
Fig. 1
(a) Low-magnification image of nanocomplexes of CMC/ASC at pH 7. Inset: High magnification of CMC/ASC and the corresponding SAED pattern indicating the amorphous phase. (b, c) Image of aggregates of ASC nanoparticles (open arrows) irregularly covering self-assembled collagen fibrils after 3 days in the MFS model. (d) Low-magnification image of CMC/ASC at pH 2. (e) Image of collagen fibrils with pre-assembly and post-assembly scenes (indicated by the red dashed line) after 24 h of mineralization in the SSM model. (f) Image of the spindle -like ASC aggregated in gap zones of the fibril and lined up along the long axis regularly after 3 days of mineralization in the SSM model. (g) Low-magnification image of CMC/ASP at pH 2. (h) Image of collagen fibrils with pre-assembly and post-assembly scenes after 24 h of mineralization in the SSM model. (i) Image of the spindle -like ASP arranged along the long axis after 3 days of mineralization in the SSM model.
Fig. 2
Fig. 2
(a) Micro-CT of CS and Sr-CS. (b) Images of lyophilized collagen scaffold. (c) Low-magnification SEM images of the area indicated by a rectangle with a red dashed line in (b). (d) High-magnification image of the area indicated by a rectangle with a red dashed line in (c).
Fig. 3
Fig. 3
(a) ATR-FTIR spectra of various scaffolds showed the typical peaks of pure collagen scaffold and decrease in the intensity of the amide I, II, and III bands in Sr-CS and Ag-CS. (b) Weight percent of various scaffolds. (n = 3, mean ± SD, **P < 0.01 vs CS group). (c) Absolute and (d) cumulative concentration of Sr2+ released from collagen scaffolds by SSM and MFS after immersion in pH 7.4 phosphate-buffered saline (PBS) for 21 days. (e) Absolute and (f) cumulative concentration of Ag + released in pH 7.4 phosphate-buffered saline (PBS) (n = 3; mean ± SD).
Fig. 4
Fig. 4
(a) The antibacterial rate of pure collagen scaffolds and Ag-doped collagen scaffolds after 24 h, 48 h and 72 h (n = 3, mean ± SD, **P < 0.01 vs CS group). (b) Fluorescence microscopic inspection of CS and Ag-CS showing the viability of the bacteria on samples after 24 h, 48 h and 72 h. (c) Image showing the adherence of S. aureus (red arrow) on CS and Ag-CS samples.
Fig. 5
Fig. 5
(a) CCK-8 assay results of rBMSCs distributed on different collagen scaffolds after culturing for 1, 3, 5 and 7 days (n = 3, mean ± SD, *P < 0.05 vs CS group; **P < 0.01 vs CS group). (b) Fluorescence microscopic inspection of cells cultured with the CS group and Sr-CS group for 1, 3, 5 and 7 days (AO/EB staining). The scale bars are 100 μm. The fluorescence microscopic images were taken after staining the nuclei (blue) and actin filaments (red) in both groups of samples for 3 days to observe the precise morphology of cells. The scale bars are 10 μm. (c) ALP activity of rBMSCs distributed on different collagen scaffolds after culturing for 7 and 14 days. (n = 3, mean ± SD, *P < 0.05 vs CS group; **P < 0.01 vs CS group). (d,e) The relative mRNA levels of osteogenesis-related genes (RUNX2, OCN, ALP) in rBMSCs cultured in different groups for 7 days and 14 days (n = 3, mean ± SD, *P < 0.05 vs CS group; **P < 0.01 vs CS group).
Fig. 6
Fig. 6
(a) Fluorescence microscopic images of RAW264.7 cells obtained after staining nuclei (blue) and actin filaments (red). RAW264.7 cells cultured with the CS group and Sr-CS group for 2 days. The scale bars are 20 μm (b) The relative mRNA levels of cytokines and osteogenic-related genes of RAW 264.7 cells stimulated by macrophage-conditioned CS and Sr−CS for 3 days. (c) ALP activity of rBMSCs distributed in different macrophage-conditioned media after culturing for 7 and 14 days (n = 3, mean ± SD, *P < 0.05 vs CS group; **P < 0.01 vs CS group). (d,e) The relative mRNA levels of osteogenesis-related genes (RUNX2, OCN, ALP) in rBMSCs cultured in different macrophage-conditioned medium groups for 7 days and 14 days (n = 3, mean ± SD, *P < 0.05 vs CS group; **P < 0.01 vs CS group).
Fig. 7
Fig. 7
(a) Reconstructed 3D micro-CT images of new bone regeneration at 1, 2, 3 and 6 months post-operation. The scale bars are 5 mm. (b) Quantitative analysis of regenerated bone in values of BV/TV for the control group and groups filled with CS or Sr-CS (n = 3, mean ± SD, **P < 0.01 vs Sr-CS group).
Fig. 8
Fig. 8
(a,b) Histological analysis of bone formation at 1, 2, 3, and 6 months by H&E and Masson's trichrome staining of rat cranial defects and surrounding tissue at different test times: blank control, filled with CS, and filled with Sr-CS. (black arrows, host bone; red arrows, new bone; black stars, osteoid; blue arrows, fibrous tissue). The scale bars are 500 μm. (c) Immunohistochemical staining for CD68+ cells in the three groups at 1, 2, 3, and 6 months. The scale bars are 100 μm.
Fig. 9
Fig. 9
(a) Schematic diagram showing the interactions between CMC, metal cations and acid radicals. At pH 7, with protonation of HCO3-, ASC can be stabilized by the acidic polymer CMC. In contrast, at pH 2, CMC competes with mineral anions for protonation, thereby causing deprotonation of the mineral anions to a certain degree to form ASC. (b) Schematic diagram showing that various bioactive elements were incorporated into collagen fibrils via the SSM model. In this model, the amorphous mineral nanoparticles are first stabilized by CMC and then mixed and interacted with the collagen microfibrils. With further collagen self-assembly, nanoparticles are trapped and localized in the gap zones. (c) Schematic diagram showing various bioactive elements aggregated on the surfaces of collagen fibrils via the MFS model.
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