Small extracellular vesicles with nanomorphology memory promote osteogenesis

Abstract

Nanotopographical cues endow biomaterials the ability to guide cell adhesion, proliferation, and differentiation. Cellular mechanical memory can maintain the cell status by retaining cellular information obtained from past mechanical microenvironments. Here, we propose a new concept "morphology memory of small extracellular vesicles (sEV)" for bone regeneration. We performed nanotopography on titanium plates through alkali and heat (Ti8) treatment to promote human mesenchymal stem cell (hMSC) differentiation. Next, we extracted the sEVs from the hMSC, which were cultured on the nanotopographical Ti plates for 21 days (Ti8-21-sEV). We demonstrated that Ti8-21-sEV had superior pro-osteogenesis ability in vitro and in vivo. RNA sequencing further confirmed that Ti8-21-sEV promote bone regeneration through osteogenic-related pathways, including the PI3K-AKT signaling pathway, MAPK signaling pathway, focal adhesion, and extracellular matrix-receptor interaction. Finally, we decorated the Ti8-21-sEV on a 3D printed porous polyetheretherketone scaffold. The femoral condyle defect model of rabbits was used to demonstrate that Ti8-21-sEV had the best bone ingrowth. In summary, our study demonstrated that the Ti8-21-sEV have memory function by copying the pro-osteogenesis information from the nanotopography. We expect that our study will encourage the discovery of other sEV with morphology memory for tissue regeneration.

Keywords: Nanotopographic; Osteogenesis; PEEK; Small extracellular vesicles; hMSCs.

Graphical abstract

Fig. 1

Scheme illustration of small extracellular vesicles with nanomorphology memory promote osteogenesis.

Fig. 2

Characterization of nanotopographic on titanium (Ti) and morphological changes of hMSCs on corresponding surfaces. (A) Surface morphologies characterization of different samples under SEM, Scale bars = 1 μm (first panel) and 200 nm (second panel). (B) Contact angles of different samples. (C) AFM images of the different samples. (D, E, F, G) Ra, Rq, Maximum peak depth and Minimum peak depth of the different Ti samples. (H) SEM of morphologies of hMSCs on different Ti samples, Scale bars = 20 μm (first panel) and 10 μm (second panel). (I) Fluorescent images of cell morphologies on different Ti samples, Scale bars = 100 μm. (J) Quantitative cell spreading area based on SEM images. (K) Quantitative cell aspect ratio based on SEM images. (L) Cell viability based on CCK 8. (M) Live-Dead staining of hMSCs cultured on different Ti samples, Scale bars = 500 μm. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001 versus the Ti group, n.s. not significant.

Fig. 3

Nanotopographic promote hMSC differentiation and characterization of sEV. (A) Scheme illustration of nanotopographic promote hMSC differentiation and obtained sEV through different centrifugation. (B) ALP and ARS staining of hMSC cultured on nanotopographic after 21 days. (C, D) Quantitative results of ALP and ARS staining of hMSC cultured on nanotopographic after 21 days. (E) TEM image of Ti-21-sEV and Ti8-21-sEV, Scale bars = 100 nm. (F) Expression of CD9 and CD63 protein was assessed by a Western blot assay. (G) Nanoparticle tracking analysis (NTA) of sEV. (H) Internalization of PKH26-labeled sEV by hMSC, Scale bars = 200 μm **p < 0.01, ***p < 0.001.

Fig. 4

Pro-osteogenesis ability of Ti8-21-sEV in vitro. (A) Scheme illustration of sEV secreted from hMSC cultured on Ti nanotopographic after 21 days (Ti8-21-sEV) and Ti8-21-sEV promote osteogenesis. (B) ALP and ARS staining of hMSC after inculbated with Ti8-21-sEV and Ti-21-sEV for 7 days, Scale bars = 200 μm. (C) Immunofluorescence staining (IF) of osteogenic makers including RUNX2, OCN and ALP of hMSC after inculbated with Ti8-21-sEV and Ti-21-sEV, Scale bars = 200 μm. (D, E, F) Osteogenesis-related makers OPN, RUNX2 and ALP were detected through qRT-PCR. *p < 0.05, **p < 0.01.

Fig. 5

Pro-osteogenesis ability of Ti8-21-sEV in vivo. (A) Scheme illustration of mouse fracture treatment through Ti-21-sEV and Ti8-21-sEV. N = 6 for each group. (B) Micro-CT images of mouse fracture treated by Ti-21-sEV and Ti8-21-sEV after 14 and 21 days. (C) Three-point bending test for measuring the biomechanical strength of the healing bone after 14 and 21 days. *P < 0.05, ***P < 0.001. ns, not significant. (D, E, F, G) TV, BV/TV, Tb. Th and Tb. Sp of the new bone tissue were quantitated based on Micro-CT. (H) Histological staining and immunohistochemical staining of OCN of new bone tissue after 14 and 21 days, Scale bars = 200 μm and 50 μm, respectively.

Fig. 6

Mechanistic analysis of Ti8-21-sEV promote osteogenesis. (A, C) GO term associated with cell differentiation and we displayed the top 10 GO terms associated with cell differentiation using bubble plot. (B, D) Top 20 KEGG pathways of differentially expressed genes. (E) Potential molecular signaling pathways by sEV which secreted from hMSC cultured on nanotopography on Ti surface influence osteogenic differentiation.

Fig. 7

3D printed PEEK scaffolds loaded with sEV for bone regeneration. (A) Scheme illustration of implantation of 3D printed PEEK scaffolds loaded with sEV into femoral condyle defect of rabbit. (B) SEM images of PEEK plates, PEEK/PDA plates and PEEK/PDA/Ti8-21-sEV plates. Black arrow: Ti8-21-sEV, Scale bars = 1 μm. (C) 2D (transverse, coronal, and sagittal) and 3D reconstruction images based on Micro CT after 12 weeks. (D, E, F, G, H, I) Quantitative results of BV, BV/TV, Tb. Th, Tb. N, Tb. Sp and BMD among the different groups. (J) Van-Gieson (VG) and Toluidine blue staining of histological sections after implanted for 12 weeks. **p < 0.01, ***p < 0.001, ns, not significant.