Schwann cells promote prevascularization and osteogenesis of tissue-engineered bone via bone marrow mesenchymal stem cell-derived endothelial cells

Abstract

Background: Tissue-engineered bone grafts (TEBGs) that undergo vascularization and neurotization evolve into functioning bone tissue. Previously, we verified that implanting sensory nerve tracts into TEBGs promoted osteogenesis. However, the precise mechanisms and interaction between seed cells were not explored. In this study, we hypothesized that neurotization may influence the osteogenesis of TEBGs through vascularization.

Methods: We cultured rat Schwann cells (SCs), aortic endothelial cells (AECs), and bone marrow-derived mesenchymal stem cells (BM-MSCs) and then obtained BM-MSC-derived induced endothelial cells (IECs) and induced osteoblasts (IOBs). IECs and AECs were cultured in an SC-conditioned medium (SC-CM) to assess proliferation, migration, capillary-like tube formation, and angiogenesis, and the vascular endothelial growth factor (VEGF) levels in the supernatants were detected. We established an indirect coculture model to detect the expression of nestin and VEGF receptors in IECs and tissue inhibitor of metalloproteinase (TIMP)-2 in SCs. Then, SCs, IECs, and IOBs were labeled and loaded into a β-tricalcium phosphate scaffold to induce prevascularization, and the scaffold was implanted into a 6-mm-long defect of rat femurs. Three groups were set up according to the loaded cells: I, SCs, and IECs (coculture for 3 days) plus IOBs; II, IECs (culture for 3 days) plus IOBs; III, IOBs. Nestin and TIMP-2 expression and osteogenesis of TEBGs were evaluated at 12 weeks post-implantation through histological and radiological assessments.

Results: We found that SC-CM promoted IEC proliferation, migration, capillary-like tube formation, and angiogenesis, but no similar effects were observed for AECs. IECs expressed nestin extensively, while AECs barely expressed nestin, and SC-CM promoted the VEGF secretion of IECs. In the coculture model, SCs promoted nestin and VEGF receptor expression in IECs, and IECs inhibited TIMP-2 expression in SCs. The promotion of prevascularized TEBGs by SCs and IECs in group I augmented new bone formation at 6 and 12 weeks. Nestin expression was higher in group I than in the other groups, while TIMP-2 expression was lower at 12 weeks.

Conclusions: This study demonstrated that SCs can promote TEBG osteogenesis via IECs and further revealed the related specific characteristics of IECs, providing preliminary cytological evidence for neurotization of TEBGs.

Keywords: Bone marrow-derived mesenchymal stem cells; Bone tissue engineering; Endothelial cells; Nestin; Prevascularization; Schwann cells; TIMP-2.

Fig. 1

Cell culture and identification. a SCs at passage 2. b–f S100 (b), GFAP (c), SOX10 (d), MPZ (e), and GAP43 (f) immunocytochemical or immunofluorescence staining of SCs. g AECs at passage 2. h Factor VIII immunocytochemical staining of AECs. i Pecam-1 immunofluorescence staining of AECs. j vWF immunofluorescence staining of AECs. k BM-MSCs at passage 3. l IECs after induction for 21 days. m–o Flow cytometric detection of CD90 (m), CD29 (n), and CD73 (o) on IEC membrane (blue: isotype negative control, red: IECs). p TEM image of BM-MSCs (× 6610). q TEM image of IECs after induction for 21 days (× 5200). r CD34, Kdr, Nos2, and Nos3 mRNA expression levels in BM-MSCs and IECs, as assessed via real-time qPCR. s Pecam-1 immunofluorescence staining of IECs. t vWF immunofluorescence staining of IECs. u, v ALP (u) and alizarin red S (v) staining of IOBs after induction for 21 days

Fig. 2

The effects of SC-CM on endothelial cells. a Proliferation curves of IECs (red) and AECs (blue) in SC-CM and regular medium. b Migrated IECs or AECs in the SC-CM wells and regular medium wells (scale bar = 50 μm). c OD values of the eluted fluid of the cell-migrated membranes in each group. d Capillary-like tube structures formed by IECs or AECs in Matrigel of SC-CM wells and regular medium wells (scale bar = 100). e Capillary-like mesh structure counting of each group. f New vessels formed by IECs perfused with ink in the rat corneas of each group (scale bar = 100). g Vascular segment counting of the new vessels formed by IECs in rat corneas of each group

Fig. 3

The interaction of SCs and endothelial cells. a VEGF secretion levels in the supernatants in each group as determined by ELISA. b Nestin expression in IECs and AECs by immunofluorescence staining. c Nestin and Flt1 expression in IECs in SC-CM-DMEM or DMEM by double immunofluorescence staining. d Nestin, Flt1, and Kdr expression levels in IECs with or without coculture at days 3 and 7 by WB. e TIMP-2 and MMP-14 expression levels in SCs cocultured with IECs and without coculture by qPCR. f Secreted TIMP-2 expression in the supernatants of the coculture system and the control wells

Fig. 4

Prevascularized scaffolds loaded with labeled cells were applied in rat models. a BM-MSCs, IOBs expressing EGFP, and IECs expressing RFP (scale bar = 100). b SCs labeled with Hoechst 33342 (scale bar = 100). c SCs, IECs, and IOBs adhered and grew on β-TCP scaffolds observed by SEM at day 6 post-seeding. d Labeled SCs, IOBs-EGFP, and IECs-RFP grew on the β-TCP scaffolds to make prevascularized TEBGs (scale bar = 100) at day 6 post-seeding. e The internal fixation with steel plate applied in rat operations. f Femur exposed and drilled for internal fixation. g Six-millimeter-long defect of the middle femur and the steel plate implanted for internal fixation. h Prevascularized TEBG implanted into the femur defect. i The design of the time schedule and group setting during the experiment

Fig. 5

Radiological assessment of TEBGs in vivo. a X-ray images after operation 6 weeks and 12 weeks of the three groups. b, c The micro-CT images of the three groups (b) and the comparison of BV/TV % values (c) after removing the internal fixations at 12 weeks postoperatively

Fig. 6

Histological assessment of TEBGs in vivo. a–f IHC staining of nestin at 12 weeks in groups I (a, b), II (c, d), and III (e, f). g–l IHC staining of TIMP-2 at 12 weeks in groups I (g, h), II (I, J), and III (k, l). m–r H&E and Masson staining at 12 weeks in groups I (m, p), II (n, q), and III (o, r)

Fig. 7

A schematic diagram of the interactions between SCs and endothelial cells in TEBG. SCs promoted nestin and VEGF receptor expression of BM-MSC-derived IECs, and IECs inhibited TIMP-2 secretion of SCs by autologous nestin expression to promote activity of MMP-14 and VEGF, eventually efficiently induced prevascularization and osteogenesis of TEBG