Effect of cyclic mechanical loading on immunoinflammatory microenvironment in biofabricating hydroxyapatite scaffold for bone regeneration

Abstract

It has been proven that the mechanical microenvironment can impact the differentiation of mesenchymal stem cells (MSCs). However, the effect of mechanical stimuli in biofabricating hydroxyapatite scaffolds on the inflammatory response of MSCs remains unclear. This study aimed to investigate the effect of mechanical loading on the inflammatory response of MSCs seeded on scaffolds. Cyclic mechanical loading was applied to biofabricate the cell-scaffold composite for 15 min/day over 7, 14, or 21 days. At the predetermined time points, culture supernatant was collected for inflammatory mediator detection, and gene expression was analyzed by qRT-PCR. The results showed that the expression of inflammatory mediators (IL1B and IL8) was downregulated (p < 0.05) and the expression of ALP (p < 0.01) and COL1A1 (p < 0.05) was upregulated under mechanical loading. The cell-scaffold composites biofabricated with or without mechanical loading were freeze-dried to prepare extracellular matrix-based scaffolds (ECM-based scaffolds). Murine macrophages were seeded on the ECM-based scaffolds to evaluate their polarization. The ECM-based scaffolds that were biofabricated with mechanical loading before freeze-drying enhanced the expression of M2 polarization-related biomarkers (Arginase 1 and Mrc1, p < 0.05) of macrophages in vitro and increased bone volume/total volume ratio in vivo. Overall, these findings demonstrated that mechanical loading could dually modulate the inflammatory responses and osteogenic differentiation of MSCs. Besides, the ECM-based scaffolds that were biofabricated with mechanical loading before freeze-drying facilitated the M2 polarization of macrophages in vitro and bone regeneration in vivo. Mechanical loading may be a promising biofabrication strategy for bone biomaterials.

Keywords: Biofabrication; Bioreactor; Bone biomaterials; Inflammatory microenvironment; Macrophage polarization.

Graphical abstract

Fig. 1

Scheme of experimental setup. A) The cancellous bone of bovine vertebra was sintered and cut into a cylindrical shape; hUCMSCs were seeded on natural 3D scaffold. B) Cell-scaffold composites were cultured in a compressive loading bioreactor (0.06–0.94 MPa; 1 Hz; 15 min/day), and static culture was acted as control. C) Cell-scaffold composites were freeze-dried to obtain ECM-based scaffolds, and the effect of ECM-based scaffolds on macrophage polarization was assessed. D) A rabbit femoral condyle bone defect model was established to assess the efficacy of these ECM-based scaffolds in bone defect repair. E) Experimental design for harvesting samples and assays.

Fig. 2

Characteristics of hUCMSCs. A-D) Osteogenic (A-B, Alizarin red staining) and adipogenic (C-D, Oil red O staining) differentiation identification after 3 weeks of induction culture. Scale bars = 100 μm. E-F) Identification of MSCs immunophenotype by flow cytometry (positive biomarkers including CD73, CD90, and CD105; negative biomarkers including CD19, CD34, CD45, and HLA-DR).

Fig. 3

Gross view under the stereomicroscope. A-B) 3D porous structure of calcined bovine bone scaffold. C) Dynamic change of cellular layers on the scaffold surface after being cultured statically for 7, 14, and 21 days. D) Dynamic change of cellular layers on the scaffold surface after being cultured by mechanical loading for 7, 14, and 21 days. E) Illustration of cell growth on the scaffold surface in the static groups. F) Illustration of cell growth on the scaffold surface in the mechanical loading groups. Scale bars = 2000 μm.

Fig. 4

Calcein AM/PI/Hoechst cell staining and the DNA quantification of cell-scaffold composite. A) Cell-scaffold composites were stained with Calcein AM, PI and Hoechst after being cultured statically or dynamically for 7, 14, and 21 days; MSCs seeded on scaffold did not achieve complete cellular confluence in the static groups (red circles). (Scale bars = 500 μm, n = 3). B) Cell viability assessment by comparing the live cell ratio (n = 3, Mean ± SD, **p < 0.01). C) DNA quantification per scaffold in different groups (n = 6, Mean ± SD, **p < 0.01, ****p < 0.0001).

Fig. 5

Screening of proinflammatory proteins by Proteome Profiler Array (n = 1). A) Five proinflammatory proteins presented the most significantly increased expression at the 7th, 14th, and 21st day in the static groups. B) Alterations of other proinflammatory proteins at the 7th, 14th, and 21st day in the mechanical loading or the static groups. Data were normalized to the DNA content of corresponding group. The five proteins with the highest expression were further analyzed by qRT-PCR.

Fig. 6

The expression of osteogenic (A1-A4) and proinflammatory (A5, B1–B5) genes of MSCs seeded on scaffolds in the mechanical loading or the static groups. n = 3, Mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001; ALP: Alkaline phosphatase, COL1A1: Collagen 1A1, RUNX2: Runt-related transcription factor 2, OCN: Osteocalcin, IL: Interleukin, TNFA: Tumor necrosis factor alpha, GROa: Growth-regulated oncogene alpha, NAP2: Neutrophile-activating protein 2, PF4: Platelet factor 4, RANTES: regulated upon activation normal T cell expressed and secreted.

Fig. 7

The expression levels of macrophage polarization-related genes induced by ECM-based scaffolds. A1-A2) M1 marker genes. A3-A5) proinflammatory genes. B1–B2) M2 marker genes. B3) anti-inflammatory gene. B4–B5) bone regeneration-related genes. n = 3, Mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001; Arg 1: Arginase 1, iNOS: Inducible nitric oxide synthase. E−7d: ECM-based scaffolds from E-Static- or E-Loading-7d group; E−14d: ECM-based scaffolds from E-Static- or E-Loading-14d group; E−21d: ECM-based scaffolds from E-Static- or E-Loading-21d group. Static-derived ECM-based scaffolds: the ECM-based scaffolds that were prepared with static culture before freeze-drying; Loading-derived ECM-based scaffolds: the ECM-based scaffolds that were biofabricated with mechanical loading before freeze-drying.

Fig. 8

Western blot and semi-quantitative analysis of macrophage polarization-related proteins induced by ECM-based scaffolds. The expression of Arg 1 (M2 marker protein) and iNOS (M1 marker protein) after co-culture of ECM-based scaffolds and macrophages. n = 3, Mean ± SD; *p < 0.05, **p < 0.01.

Fig. 9

Repairment of rabbit bone defects in vivo. A-G) Micro-CT 3D reconstruction of in vivo new bone formation (green area) ability of the blank scaffold (white area) group and ECM-based scaffolds (white area) after 8 weeks of implanatation. H) Quantitative analysis of bone volume/total volume (BV/TV) of ECM-based scaffolds after implanted in vivo for 8 weeks based on the Micro-CT evaluation. n = 3, Mean ± SD; **p < 0.01.