TGFβ3 recruits endogenous mesenchymal stem cells to initiate bone regeneration

Abstract

Background: The recruitment of a sufficient number of endogenous mesenchymal stem cells (MSCs) is the first stage of in-situ tissue regeneration. Transforming growth factor beta-3 (TGFβ3) could recruit stem or progenitor cells and endothelial cells to participate in tissue regeneration. However, the mechanism of TGFβ3 recruiting MSCs toward bone regeneration has remained obscure.

Methods: We estimated the promigratory property of TGFβ3 on human bone marrow MSCs (hBMSCs) cocultured with the vascular cells (human umbilical artery smooth muscle cells or human umbilical vein endothelial cells) or not by Transwell assay. After the addition of the inhibitor (SB431542) or Smad3 siRNA, the levels of MCP1 and SDF1 in coculture medium were tested by ELISA kit, and then the migratory signaling pathway of hBMSCs induced by TGFβ3 was investigated by western blot analysis. In vivo, a 2-mm FVB/N mouse femur defect model was used to evaluate chemokine secretion, endogenous cell homing, and bone regeneration induced by scaffolds loading 1 μg TGFβ3 through qPCR, immunofluorescent staining, immunohistochemical analysis, and Micro-CT, compared to the vehicle group.

Results: TGFβ3 (25 ng/ml) directly showed a nearly 40% increase in migrated hBMSCs via the TGFβ signaling pathway, compared to the vehicle treatment. Then, in the coculture system of hBMSCs and vascular cells, TGFβ3 further upregulated nearly 3-fold MCP1 secretion from vascular cells in a Smad3-dependent manner, to indirectly enhance nearly more than 50% of migrated hBMSCs. In vivo, TGFβ3 delivery improved MCP1 expression by nearly 7.9-fold, recruited approximately 2.0-fold CD31⁺ vascular cells and 2.0-fold Sca-1⁺ PDGFR-α⁺ MSCs, and achieved 2.5-fold bone volume fraction (BV/TV) and 2.0-fold bone mineral density, relative to TGFβ3-free delivery.

Conclusions: TGFβ3, as a MSC homing molecule, recruited MSCs to initiate bone formation in the direct-dependent and indirect-dependent mechanisms. This may shed light on the improvement of MSC homing in bone regeneration.

Keywords: MCP1; Mesenchymal stem cell; Recruitment; TGFβ3; Vascular cells.

Fig. 1

Transwell assay for TGFβ3-induced hBMSC migration. a Analysis for migrated hBMSCs induced by 0 and 25 ng/ml TGFβ3 after 24-hour incubation. b Analysis for migrated hBMSCs induced by 0 and 25 ng/ml TGFβ3 with the pretreatment of inhibitor for TβRI/II. c Expression of TβRII in hBMSCs induced by TGFβ3 assessed by western blot analysis. Migrated cells were stained purple with crystal violet. Scale bar: 100 μm. *P < 0.05, **P < 0.01. TGFβ3 transforming growth factor beta-3, SB SB431542

Fig. 2

Transwell assay for hBMSC migration. a Analysis for the migration of hBMSCs with or without hUVECs and hUASMCs. b In the coculture system of hBMSCs and hUASMCs, representative light photomicrographs of migrated hBMSC induced by 0-100 ng/ml TGFβ3 after 24-hour incubation. c In the coculture system of hBMSCs and hUVECs, representative light photomicrographs of migrated hBMSCs induced by 0-100 ng/ml TGFβ3 after 24-hour incubation. d Quantitative analysis of migrated cell density for (c) and (d). Migrated cells were stained purple with crystal violet. Scale bar: 100 μm. *P < 0.05, **P < 0.01, ***P < 0.005, ***P < 0.001. hUASMC human umbilical artery smooth muscle cell, hUVEC human umbilical vein endothelial cell, MSC mesenchymal stem cell, TGFβ3 transforming growth factor beta-3

Fig. 3

TGFβ3 upregulated MCP1 secretion from vascular cells by TβRII/Smad3 signaling. a Secretion of MCP1 and SDF1 from vascular cells in the coculture system hBMSCs and vascular cells with TGFβ3. b Secretion of MCP1 from hBMSCs, hUVECs, and hUASMCs in different systems. c Expression of TβRII in hUVECs and hUASMCs induced by TGFβ3 assessed by western blot analysis. d Expression of p-Smad3 and Smad3 in hUVECs and hUASMCs induced by TGFβ3 assessed by western blot analysis. **P < 0.01, ***P < 0.005, ****P < 0.001. hUASMC human umbilical artery smooth muscle cell, hUVEC human umbilical vein endothelial cell, MSC mesenchymal stem cell, TGFβ3 transforming growth factor beta-3

Fig. 4

Knockdown of Smad3 in vascular cells inhibited TGFβ3-induced hBMSC migration. a Expression of Smad3 in hUVECs and hUASMCs were transfected with siRNA Smad3 as assessed by western blot analysis. b Relative density of Smad3 for (a). c Secretion of MCP1 in different cells. d Transwell assay for hBMSC migration in the coculture system of hBMSC and vascular cells with or without knockdown of Smad3. Migrated cells were stained purple with crystal violet. Scale bar: 100 μm. **P < 0.01, ****P < 0.001. hUASMC human umbilical artery smooth muscle cell, hUVEC human umbilical vein endothelial cell, MSC mesenchymal stem cell, siRNA small interfering RNA, TGFβ3 transforming growth factor beta-3

Fig. 5

TGFβ3 recruited endogenous MSCs to initiate bone formation. a Expression of MCP1 in regenerated tissue in the TGFβ3 and vehicle groups at 3 days post implantation. b Immunohistochemical analysis for CD31⁺. Scale bar: 100 μm. c Number of CD31⁺ cells. d Immunofluorescent images of Sca-1 and PDGFR-α in scaffolds; green, Sca-1; red, PDGFR-α; blue, DAPI. Scale bar: 20,000 nm. White arrows, Sca-1⁺ PDGFR-α⁺ MSCs. e Recruited MSC%. f 3D and 2D center-sagittal view images of regenerated bone mass in the TGFβ3 and vehicle groups at 8 weeks post implantation. Scale bar: 10 mm. g BV/TV and BMD of the regenerated bone in (f). *P < 0.05, **P < 0.01, ****P < 0.001. BMD bone mineral density, BV/TV bone volume fraction, MCP1 monocyte chemotactic protein 1, MSC mesenchymal stem cell, TGFβ3 transforming growth factor beta-3

Fig. 6

Schematic representation of the direct and indirect mechanism of TGFβ3-induced MSC migration based on this study. Black arrow, direct mechanism; red arrow, indirect mechanism. MCP1 monocyte chemotactic protein 1, MSC mesenchymal stem cell, Tgfbr1 TGFβ type I, Tgfbr2 TGFβ type II, TGFβ3tTransforming growth factor beta-3