A novel method to improve the osteogenesis capacity of hUCMSCs with dual-directional pre-induction under screened co-culture conditions

Abstract

Objectives: Mesenchymal stem cells (MSCs) based therapy for bone regeneration has been regarded as a promising method in the clinic. However, hBMSCs with invasive harvesting process and undesirable proliferation rate hinder the extensive usage. HUCMSCs of easier access and excellent performances provide an alternative for the fabrication of tissue-engineered bone construct. Evidence suggested the osteogenesis ability of hUCMSCs was weaker than that of hBMSCs. To address this issue, a co-culture strategy of osteogenically and angiogenically induced hUCMSCs has been proposed since thorough vascularization facilitates the blood-borne nutrition and oxygen to transport in the scaffold, synergistically expediting the process of ossification.

Materials and methods: Herein, we used osteogenic- and angiogenic-differentiated hUCMSCs for co-culture in screened culture medium to elevate the osteogenic capacity with in vitro studies and finally coupled with 3D TCP scaffold to repair rat's critical-sized calvarial bone defect. By dual-directional induction, hUCMSCs could differentiate into osteoblasts and endothelial cells, respectively. To optimize the co-culture condition, gradient ratios of dual-directional differentiated hUCMSCs co-cultured under different medium were studied to determine the appropriate condition.

Results: It revealed that the osteogenic- and angiogenic-induced hUCMSCs mixed with the ratio of 3:1 co-cultured in the mixed medium of osteogenic induction medium to endothelial cell induction medium of 3:1 possessed more mineralization nodules. Similarly, ALP and osteogenesis/angiogenesis-related genes expressions were relatively higher. Further evidence of bone defect repair with 3D printed TCP of 3:1 group exhibited better restoration outcomes.

Conclusions: Our work demonstrated a favourable and convenient approach of dual-directional differentiated hUCMSCs co-culture to improve the osteogenesis, establishing a novel way to fabricate tissue-engineered bone graft with 3D TCP for large bone defect augmentation.

Keywords: bone defect; co-culture; dual-directional differentiation; human umbilical cord mesenchymal stem cells; osteogenesis.

Figure 1

Characteristics of the umbilical cord and hUCMSCs from Wharton's jelly. A, HE staining of the umbilical cord tissue. Light pink stood for the Wharton's jelly and dark pink for the arteries and the vein. Higher magnification revealed loose and irregular jelly‐like tissue with nuclei stained dark blue; B, Immunohistochemical staining of the Wharton's jelly, arteries and vein with CD31 and CD34. Positive brown stained cells could only be detected in the arteries and vein; C, Wharton's jelly‐derived P1 cells with homogeneous bipolar spindle‐like shape, lining as a whirlpool; D, Surface markers of P5 cells

Figure 2

Osteogenic differentiation of hUCMSCs. A, ALP staining of non‐ and osteogenically induced hUCMSCs for 4, 7 and 10 d and (B) ALP quantitative analysis; C, The expressions of osteogenesis‐related genes ALP, RUNX2, COL1 and OPN after 4, 7 and 10 d induction; D, ARS of non‐ and osteogenically induced hUCMSCs for 3 and 5 wk. Much more red‐stained depositions observed in OM in the 5th wk. *P < .05; **P < .01; ***P < .001; ****P < .0001

Figure 3

Endothelial differentiation of hUCMSCs. The morphology of induced hUCMSCs was shown as (A) vascular‐like structure indicated by a red arrow under phase contrast microscopy, with (B) angiogenesis in the matrigel and (C) Dil‐ac‐LDL phagocytosis, with blue stained nuclei and red‐stained Dil labelled ac‐LDL; D, Immunofluorescence assay validated the expressions of EphrinB2 and EphB4 proteins; E, The expressions of pro‐angiogenesis‐related genes EFNB2, EPHB4, VEGF and bFGF, coupled with (F) anti‐angiogenesis‐related genes SERPINF1, ANGPTL1 and SPROUTY1 after 4, 7 and 10 d induction. *P < .05; **P < .01; ***P < .001; ****P < .0001

Figure 4

Screening the co‐culture medium for bi‐directional induction of hUCMSCs. A, ARS results of pre‐induced os‐hUCMSCs and en‐hUCMSCs co‐cultured under gradient ratios and different medium for 17 d, and osteogenically induced hUCMSCs in OM was set as the positive control; B, Calcium nodules quantification assay. *P < .05; **P < .01; ***P < .001; ****P < .0001

Figure 5

Co‐culture of hUCMSCs of bi‐directional pre‐induction. A, Cell proliferation curve; B, ALP quantitative study; C, Expressions of osteogenesis‐ and angiogenesis‐related genes after co‐culture for 3 d; D, RUNX2 protein expression after co‐culture for 3 d and (E) CD146 protein expression. *P < .05; **P < .01; ***P < .001; ****P < .0001

Figure 6

Tube formation capacity of the co‐cultured system of 3 d. The more percentage of en‐hUCMSCs mixed, the more vascular tubules formed on the matrigel, while no obvious branches or reticular structure observed in 3:1 and 2:2 groups

Figure 7

Characterization of the tissue‐engineered construct with co‐cultured dual‐directional differentiated hUCMSCs. A, Surface architecture and (B) microstructure of scaffold under SEM; C, Live/dead staining results showed green stained living cells attached to the scaffold; D, Dual‐directional differentiated hUCMSCs spread well onto the scaffold by SEM. Red arrow referred to dual‐directional differentiated hUCMSCs

Figure 8

In vivo rehabilitation outcome of rat's calvarial bone defect. A, Micro CT and histology outcome validating the reconstruction outcome of different approaches. For micro CT, green‐stained tissue referred to the newly formed bone, while the grey stood for the 3D scaffold. For histology analysis, pink‐stained homogeneous osteoid and new bone structure from HE staining and dark red‐stained mature bone and light blue‐stained collagen tissue from Masson trichrome staining could be detected in the 3:1 group. CT: connective tissue; SF: scaffold; OD: osteoid; NB: new bone; HB: host bone; B, Quantitative comparison of new bone volume, trabecular number and thickness, respectively; C, Percentage of new bone by histological study. *P < .05; **P < .01; ***P < .001; ****P < .0001