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.


| INTRODUC TI ON
Large bone defects resulting from trauma, infection, tumour resection or congenital deformities severely affect the original contours and functions. Autologous bone grafts, routinely regarded as the "golden standard" for bone transplantation, require a second surgical site, inevitably causing new tissue damages and bringing seriously physical, psychological and economic burdens to patients. 1 To attain non-invasive and much safer customized rehabilitation of bone defects, mesenchymal stem cells (MSCs) have been suggested to facilitate the fabrication of customized, bioactive tissue-engineered bone grafts based on the bone scaffold made by three-dimensional printing (3DP), so as to provide promising evidence for clinical settings. 2,3 To date, with the in-depth study, MSCs are extensively used as seed cells for bone tissue regeneration, especially MSCs isolated from bone marrow, characterize better osteogenic differentiation capacity and are frequently used in bone tissue engineering. 4 However, the acquisition number of human bone marrow mesenchymal stem cells (hBMSCs) cannot meet the huge demands for bone repair with invasive process, and the cellular activities are much influenced by the donor's age and health status, greatly restricting the clinical application. 5 In contrast, MSCs from the Wharton's jelly of the neonatal umbilical cord, named human umbilical cord mesenchymal stem cells (hUCMSCs), possess a higher cell yield, rapid proliferation capacity, amounts of secreted cytokines related to cell migration, inflammation, immune regulation, angiogenesis (vascular endothelial growth factor [VEGF], insulin-like growth factor-1, transforming growth factor and platelet-derived growth factor [PDGF]), neurogenic and wound healing processes. [6][7][8] Above all, hUCMSCs do not express the major histocompatibility complex II (MHC II) and immunoregulatory factor B7 costimulatory molecules, which are responsible for the alloimmune response. 9 Therefore, it is believed that hUCMSCs own the characteristics of both seed cells and cytokines in the process of fabrication of tissue-engineered bone, which will be a promising choice to enhance the bioactivity of tissue-engineered bone graft. 10 Numerous researches have also shown that hUCMSCs have superior osteogenic properties attached to the scaffolds and are suitable seed cells for bone tissue engineering. 11 Given that some studies hold the osteogenic ability of hUCMSCs is weaker than that of hBMSCs, it is worthy to improve the osteogenesis ability of hUCMSCs. 12 Besides, due to the lack of nutrition and oxygen supply, the closer MSCs to the centre of the scaffolds, the harder to survive. Studies demonstrated that within 200 μm distance from the blood vessels is critical for the survival and retention of viable cells. 13,14 Considering that VEGF plays a vital role in facilitating angiogenesis as well as stimulating osteogenesis by regulating related growth factors, osteoblasts, BMSCs, hUCMSCs or human adipose mesenchymal stem cells (hADMSCs) derived VEGF is much emphasized. 8,[15][16][17][18] An optimal concentration of VEGF is required for angiogenesis and osteogenesis coupling for intramembranous ossification on account that a higher or lower concentration of VEGF could result in compromised bone formation. 17 In light of these phenomena, a co-culture system of osteogenic cells (stem cells, osteoblasts, etc) and angiogenic cells (endothelial progenitor cells, endothelial cells, etc) has been raised, which has been validated there is a synergistic effect in promoting osteogenesis and angiogenesis, favourable for the survival and prognosis of osteoblasts in the scaffolds, thus ensuring a successful ossification of tissue-engineered bone. 19,20 Nevertheless, the culture of endothelial progenitor cells and endothelial cells in vitro is difficult, and the number of isolated cells is very less with limited proliferation ability. 21 Besides, in the clinical application of co-culturing these two kinds of cells, the risks of immune rejection and disease transmission will increase for the different cell sources. As a result, to lower the aforementioned risks, one single kind of MSCs with abundant sources should be applied to differentiate into osteoblasts and endothelial cells, respectively, before sent for co-culture. To date, studies concerning dual-directional differentiation of one single kind of stem cell are scarce.
Studies show that the mixing ratio of the two kinds of cells and the culture medium used for co-culture directly influence the outcome of osteogenesis. 22 On account of different cell types and treatment methods used by the researchers, no acceptable standard of co-culture has been recognized so far. Most researchers prefer to adopt the method of mixing two types of cells with the ratio of 1:1, cultured in the osteogenic induction medium or the co-culture medium (50% osteogenic induction medium and 50% endothelial growth medium). 20,23,24 By far, there has been no report elaborating on the culture condition concerning the bi-directional differentiation of hUCMSCs.
In this study, hUCMSCs were pre-induced to differentiate into osteoblasts and endothelial cells, respectively, and then co-cultured under different ratios in the screened culture medium. By comparing 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 dualdirectional 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. the rehabilitation effects of critical-sized calvarial bone defect with tissue-engineered bone graft fabricated by incorporating different ratios of induced cells onto 3D printed tricalcium phosphate (TCP) scaffold, the osteogenesis outcome of the co-cultured cells from bi-directional differentiation of pre-induced hUCMSCs was explored and analysed, so as to provide a state-of-the-art method for the application of hUCMSCs in bone tissue engineering.

| Isolation, cultivation and identification of hUCMSCs
The experiment design and examinations of this study were briefly illustrated ( Figure S1). With the informed consents from three volunteers, the umbilical cords were obtained for cell isolation and histological observation. Briefly, after sterilization and thorough washing, the umbilical vein, two arteries and umbilical cord envelope were removed, and the Wharton's jelly was obtained. After cut into pieces, 0.1% type II collagenase (Sigma-Aldrich) was used for digestion overnight at 4°C. Then, the tissue suspension was screened, plated in mesenchymal stem cells medium (SM, Table 1) and incubated in 5% CO 2 at 37°C. After 3 days, the adherent growth of hUCMSCs was observed. Passage 4-8 was chosen for subsequent experiments.

| Co-culture of dual-directional induction of hUCMSCs
For the co-culture of the dual-directional induction of hUCMSCs, a series of culture media were selected (Table 1). When cell confluence reached 70%-80%, the culture medium would be changed from the proliferation medium (PM) to the induction medium. Before sent for co-culture, the os-hUCMSCs (osteogenically induced hUC-MSCs) and en-hUCMSCs (angiogenically induced hUCMSCs) were pre-induced for 3 days. A gradient ratio was selected for co-culture study with different culture medium ( Table 2). For co-culture, preinduced os-hUCMSCs and en-hUCMSCs were mixed with different ratios accordingly and incubated for another 3 days before in vitro and in vivo study.

| Determination of osteogenesis and angiogenesis capacity of co-cultured hUCMSCs
Proliferation study (n = 6): For the observation period of 1 to 7 days, the cultured cells were incubated with the culture medium and Alamar Blue (Invitrogen) (v/v = 10:1) for 3 hours before examined at 570 nm and 600 nm for the optical density (OD). The percentage of proliferation was calculated by the following formula: Matrigel angiogenesis assay (n = 5): To investigate the angiogenesis capacity, the matrigel angiogenesis assay was performed.
Briefly, 10 μL ice-cold matrigel (354230, BD Biosciences) was added into the angiogenesis slides (ibidi) and sent for incubation at 37°C for 30 minutes. After that, 50 μL cell suspension (2 × 10 4 cells) was dropped into each well and imaged after 2.5 hours incubation for microtubule formation.
Acetylated low-density lipoprotein labelled with Dil (Dil-ac-LDL) phagocytosis test (n = 5): Before testing, the culture medium was changed to EIM containing 10 μg/mL Dil-ac-LDL (Invitrogen). After incubation at 37°C for 4 hours followed by several PBS washes, the nuclei staining was carried out with 5 μg/mL Hoechst (Sigma-Aldrich). The confocal laser scanning microscope (CLSM, Leica) was used for observation.
Immunofluorescent staining (n = 3): After endothelial induction, the cells were fixed before penetrated by the frozen methanol at 20°C for 10 minutes. Blocking buffer (Beyotime) was employed to block the endogenous peroxidase at room CST) or rabbit anti-mouse (1:2000; CST) for 1 hour. Finally, chemiluminescence detection reagents (ECL) was dropped onto the membrane and exposed to X-ray film. The gel imaging system (Bio-Rad) was used for photography, and the final results were analysed by Image J.

| In vivo study and analysis
With the permission of Guangzhou Medical University Ethics

| Statistical analysis
All the presented data were expressed as the mean ± standard deviation. Tukey's multiple comparisons test in one-way ANOVA was used to a comparison of two groups, and Sidak's multiple comparisons test was used to analyse the mean of multiple comparisons tests at multiple time points. The significance level was set at P < .05.

| Characteristics of the umbilical cord and hUCMSCs from Wharton's jelly
The umbilical cord tissue structure was demonstrated by HE staining.
Two darkly stained round arteries coupled with one oval-shaped vein  Figure 1C). Flow cytometry showed that more than 99% of the cells expressed the surface markers CD73 and CD90 of MSCs, but did not express the haematopoietic markers CD34 and CD45 ( Figure 1D).

| Osteogenic differentiation of hUCMSCs
To evaluate the osteogenic differentiation capacity of hUCMSCs, ALP activity of quantitative and qualitative analysis, osteogenesisrelated genes expressions and mineralized nodules were studied.
After 4, 7 and 10 days of osteogenic induction, ALP staining of hUCMSCs in OM became purple, darker than that in PM, with the darkest colour observed on the 4th day (Figure 2A), which was in accordance with quantitative examination of ALP activity ( Figure 2B), showing that the ALP activity increased significantly during the observed period in OM compared with that in PM, and reached the highest level on the 4th day, which was twice and three times higher than that of the 7th and 10th day, respectively.
Meanwhile, the mRNA expressions of ALP and OPN increased significantly after osteogenic induction at all detection time points and gradually increased with time, while the expressions of RUNX2 and COL1 genes increased significantly only on the 4th day ( Figure 2C). ARS showed scattered calcium nodules at the 3rd week after induction and patches after 5 weeks. No calcium nodules were found in the PM group ( Figure 2D).

| Endothelial differentiation of hUCMSCs
Three days after EIM induction, the induced hUCMSCs became fluorescent-labelled ac-LDL found around the blue nuclei, while the non-induced MSCs did not possess the same function ( Figure 3C).
Immunofluorescence staining showed that the cell connected in a circular pattern after induction, expressing both arterial endothelial

cells' marker EphrinB2 and vein endothelial cells' marker EphB4 in
the cytoplasm ( Figure 3D). QRT-PCR examination detected that the expressions of angiogenic genes EFNB2, EPHB4, VEGF and bFGF on the 4th, 7th and 10th day after induction, and EFNB2, VEGF and bFGF enhanced on the 4th day. All the tested genes expressions declined on the 7th and 10th day significantly ( Figure 3E). In contrast, except for the expression of anti-angiogenic gene SPROUTY1 declined significantly, the expressions of SERPINF1 and ANGPTL1 increasing over the whole period were contrary to that of EFNB2, VEGF and bFGF ( Figure 3F).

| Screening of the co-culture medium
When pre-induced os-hUCMSCs and en-hUCMSCs were co-cultured for 17 days, ARS showed that cells co-cultured in PM with the ratio  Figure 4B). Therefore, PMM (3:1) was selected for the subsequent co-culture medium.

| Effects of co-culture on the osteogenesis and angiogenesis of hUCMSCs
After co-culture, the cell proliferation rate and ALP activity of 3:1 group were similar to those of 4:0 in OM, which was the positive control ( Figure 5A,B), while the osteogenesis-related genes RUNX2, ALP and protein expression in group 3:1 were higher than that of 4:0 ( Figure 5C,D). The proliferation rate of 2:2 and 1:3 group maintained at a higher standard in the overall observation ( Figure 5A).
Considering the osteogenesis-related genes and protein expression, ALP, RUNX2 and its protein of 2:2 group were the highest ( Figure 5C,D). In contrast, ALP activity of 1:3 group on the 1st and 2nd day showed the highest compared with the others (Figure 5B), while ALP gene expression decreased after 3 days' co-culture, with RUNX2 gene and its protein still higher than that of 4:0 group ( Figure 5C,D).
For angiogenesis analysis, compared with 0:4 group, ANG mRNA expression of 3:1 group was the highest on the 3rd day, as well as PDGFB mRNA expression of 2:2 group, VEGF mRNA expression of 1:3 ( Figure 5C). All these differences were statistically significant.
CD146, the marker of pericyte, related to vascular stability, its gene and protein expression declined from 3:1 group to 0:4 group gradually ( Figure 5C,E).
To further illustrate the angiogenetic capacity of the co-culture system, the matrigel tube formation assay was adopted. After 3 days pre-induction (0 day), the more percentage of en-hUCMSCs mixed, the more vascular tubules formed on the matrigel, with a more complete and continuous circular shape ( Figure 6). However, after 3 days co-culture, the 0:4 group still possessed good angiogenesis ability, but the tube formation in the other groups predominantly weakened, with only a small amount of branching structures ( Figure 6).

| Characterization of the tissue-engineered construct with co-cultured dual-directional differentiated hUCMSCs
SEM showed that the struts of the 3D printed TCP scaffolds were dedicated and uniform with pore size ranging from 350 to 400 μm ( Figure 7A). At 3.5 k magnification, microporous and nanoporous structures could be observed ( Figure 7B). Live/dead staining results showed that green-stained viable cells attached to the scaffold with hardly visible red-stained dead cells ( Figure 7C). Similarly, the hierarchical porous structure was beneficial to the growth and migration of MSCs, suggesting the 3D scaffolds possessed excellent cytocompatibility, and this combination was feasible for bone graft fabrication ( Figure 7D).

| Rehabilitation of the critical-sized bone defect
After the operation, all rats recovered soundly. From micro CT,

| D ISCUSS I ON
With increasing demands of bioactive bone grafts in clinical settings, MSCs have been regarded as an ideal approach compared with cytokines in addition which often bring about unexpected side effects for patients, to enhance the osteogenic, angiogenic, immunoregulatory capacity of bio-scaffolds. Considering that larger the bone scaffolds, harder the seeded cells to live inside for the limited perfusion and migration of capillaries, in this study, dual-directional differentiations of hUCMSCs were conducted, coupled with enhanced osteogenic capacity with co-culture strategy, so as to obtain osteoblasts and endothelial cells' functions in fabricating bioactive tissueengineered bone graft for critical-sized bone defects rehabilitation.
In terms of osteogenic differentiation, the results of the two-dimensional induction culture showed that hUCMSCs were much weaker than that of hBMSCs. 12 After culture in OM, the ALP ac- is, hBMSCs are more likely to express the same genes as osteoblasts, while hUCMSCs tend to express the same genes as embryonic stem cells. 25 Batsali et al found that most of the genes positively related to WNT signalling pathway were significantly reduced in hUCM-SCs during osteogenic differentiation compared with hBMSCs for WNT-1 inducible-signalling pathway protein-1 (WISP1) could be used to induce the increased expressions of osteogenesis-related genes significantly. 27 Thus, it is speculated that the inadequate activation of WNT signalling pathway in hUCMSCs may lead to relatively lower osteogenesis ability.
For angiogenic induction, reported methods included cytokine-induction, 28 hypoxia-induction, 29,30 co-culture induction 31,32 and gene transfection-induction. 33 Angiogenesis-related cytokines-induced differentiation of MSCs into endothelial cells are the most commonly used and currently a more successful approach.
Herein, EIM was prepared by adding VEGF and bFGF into EGM.
After 4 days induction, tube formation and phagocytosis characterization of endothelial cells were found in pre-induced hUCMSCs, which was in accordance with other reports. 34,35 Meanwhile, the expressions of EphrinB2, a specific marker of the artery, and EphB4 protein specific for the vein increased. For in vivo study, biocompatible 3D printed β-TCP scaffolds 49,50 were used as carriers, and the bi-directional pre-induced differentiated hUCMSCs of different ratios as seed cells were combined to fabricate bioactive tissue-engineered bone graft to repair the critical-sized cranial bone defects of rats, which further validated that 3:1 group showed the optimal rehabilitation outcome compared with 4:0 group, in accordance with other researches. Zhou et al

F I G U R E 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 inoculated endothelial cells derived from rabbit BMSCs and BMSCs at the ratio of 1:1 onto β-TCP scaffolds for repairing 1.5 cm ulnar segmental defect of rabbit with a satisfying outcome. 51 Though the co-culture system of bi-directional pre-differentiated hUCMSCs could effectively promote osteogenesis, better than that of osteogenic induction alone, the optimal pre-induction time and co-culture conditions need to be further studied for clinical instructions.
At present, many in vitro studies have shown that there are three communication modes between osteoblasts and endothelial cells.
One is soluble cytokines secreted by endothelial cells, such as bone morphogenetic protein-2 (BMP-2), VEGF, PDGF acting on osteoblasts in the form of paracrine to promote their differentiation. 52 Secondly, the extracellular matrix plays a pivotal role in the communication between two kinds of cells. [53][54][55] Finally, multiple scholars believe that the direct contact between cells is the premise of endothelial cells to promote the functions of osteoblasts, and gap junctions are decisive. 56 Besides, in vivo studies of long bone have found that H-type blood vessels are important structures for coupling osteogenesis and angiogenesis, 57 which rely on the Notch signalling pathway of vascular endothelial cells. 58 Therefore, in our study, whether there are the same or similar communication modes in vitro and in vivo of co-cultured bi-directional pre-induced hUCMSCs is worth further studies. Nevertheless, there are still many shortcomings in this study. First and foremost, bi-directional pre-induced hUCMSCs should be labelled for identifying the phenotypic changes after pre-induction and co-culture by flow cytometry, and the cell transformation after implantation. Secondly, for instructing the clinical application of hUCMSCs, human originating cells were directly used in non-immunodeficient SD rats, and no immunosuppressive agents were injected after the operation.
Although literature says that the immunogenicity of hUCMSCs is much lower, the immune response will happen in this process inevitably. Besides, the genome and proteome of the dual-directional differentiated hUCMSCs should be conducted and analysed for clarification of induction efficiency and further knowledge of the cells' crosstalk. All these problems will be gradually solved in the follow-up experiments.

| CON CLUS ION
Our study validated that hUCMSCs possess a slightly weaker osteogenic differentiation ability and can be induced and differentiated into endothelial-like cells with endothelial function in a short time. The osteogenesis capacity of bi-directional differentiated hUCMSCs into osteoblasts and endothelial cells cocultured in the PM was dramatically enhanced. Besides, the 3:1 group showed the optimal osteogenesis capacity both in vitro and in vivo when cultured in the same proportion of mixed medium.
This co-culture strategy of bi-directional pre-induction of hUCM-SCs provides a novel approach for the construction of bioactive tissue-engineered bone graft for clinical transplantation with enhanced osteogenesis.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.