3D printed HUVECs/MSCs cocultures impact cellular interactions and angiogenesis depending on cell-cell distance

Abstract

Vascularization is a crucial process during the growth and development of bone ¹, yet it remains one of the main challenges in the reconstruction of large bone defects. The use of in vitro coculture of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) has been one of the most explored options. Both cell types secrete specific growth factors that are mutually beneficial, and studies suggested that cell-cell communication and paracrine secretion could be affected by a number of factors. However, little is known about the effect of cell patterning and the distance between cell populations on their crosstalk. In the present study, we showed that the separation and distance between ECs and MSCs populations affects angiogenesis by modulating cell-cell communication. HUVECs grown farther apart from MSCs (˃400 μm) presented characteristics of an early stage of angiogenesis (migration/proliferation). Results showed an increase in the up-regulation of VEGF, FGF-2, and ITGA3 (integrins) but a smaller fold change in the expression of VE-Cadherin and Ang-1. HUVECs were also still highly proliferative. On the contrary, HUVECs incubated closer (≤200 μm) to MSCs, showed signs of stabilization, mainly an increase in Ang-1 and VE-cadherin expression, as well as tighter monolayers. Conditioned media collected from HUVECs and MSCs grown ≤200 μm apart preferentially promoted tube formation, a later stage of angiogenesis, due in part to a significant increase in Ang-1 paracrine secretion. In addition, in groups in which fibers were printed farther apart (400 μm), cells produced EVs with a significantly increase cargo. Finally, in vivo experiment results showed an increase in blood vessels density and new bone thickness after 12 weeks of implantation in rat cranial defect, further suggesting the higher efficiency of indirect ECs/MSCs contact in prompting the release of paracrine signals that stimulate the angiogenesis of local tissues, and enhanced subsequent bone regeneration.

Keywords: 3D printing; Angiogenesis; Bone tissue engineering; EVs; Paracrine signaling.

Figure I:. Methods.

(a) Chart showing the different experimental groups: Endothelial Cells (ECs), Mesenchymal Stem Cells (MSCs), Mixed, and Separated with a distance of 0μm (D0), 200μm (D200), 400μm (D400) between fibers. ECs/MSCs groups were used as controls, and are made of adjacent fibers containing only ECs/MSCs, respectively. To investigate the effect of separation of cell population, a mixed group was used. The bioink for the mixed group contains both ECs/MSCs, and the fibers are also printed adjacent to each other. To investigate the effect of distance between cell population, 3 groups were used. For the D0, D200, D400 groups, ECs and MSCs are encapsulated in different bioinks, and the fibers are printed alternatively and with a distance of 0, 200, 400μm between them. The Mix and Do groups were eventually chosen for in vivo experiments. (b) After printing, 3D printed samples were incubated for 48h in serum free media. After 48h, the media was collected and the ECs from the samples were magnetically sorted. (c) Micrographs of 3D printed samples (D0, D200, D400). All samples are 8mm diameter and 1mm thick discs. All samples were stained directly after printing, using calcein, showing live cells in green. Using ImageJ, the distance between fibers was calculated: d(D200)= 195.1±25.4μm and d(D400)=381.7±25.9μm.

Figure II:. ECs grown in close (≤200μm) coculture condition show characteristics of a later stage angiogenesis.

For all figures, groups not sharing a letter are statistically different. (a) Schematic of the experiment. Cells were collected from the 3D printed samples after 48h of incubation. HUVECs were sorted using CD31 MACS, and used in subsequent experiments. (b) FACS analysis, pre and post-separation. The bottom row confirms the positive selection of HUVECs (c) Rt-PCR (n=9) showed an increase in gene expression of VEGF and FGF-2 for cells isolated from D400 samples and an increase in the fold change in mRNA of Ang-1 for cells isolated from D0/D200 samples. (d) Cell confluency (n=3) after 24h of incubation. D400 cells exhibit a significantly higher proliferation rate (p<0.05) than the other groups. (e) Permeabilization Assay (n=3). Diffusion of Dextran-FITC was observed through a confluent monolayer of HUVECs. Cells isolated from D400 samples showed the highest permeability. Rt PCR showed an increase in gene expression of ITGA5 for the same cells.

Figure III:. The crosstalk between hMSC and HUVECs is regulated by the separation and distance of cell populations.

Groups not sharing a letter are significantly different. (a) Schematic of the experiments. After 48h of incubation, CdM was collected and concentrated. Different growth factors concentration was measured using ELISAs. Finally the CdM was used to supplement media in functional Angiogenic assays. (b) Concentration of VEGFA, FGF-2 and Ang-1 in CdM from all experimental groups (n=9). For each growth factor, groups not sharing a letter are statistically different (p<0.05). Paracrine secretions were significantly increased in coculture groups where HUVECs were separated from MSCs (D0, D200). (c) CdM was used to supplement media in a proliferation assay (n=3). HUVECs confluency after 24h was measured. No statistical difference was observed between coculture groups. (d) CdM was used to supplement media for migration assay (n=3). Picture of the wound were taken 12h after “scratching” a confluent monolayer of HUVECs, and the wound closure was calculated. Groups incubated with CdM from coculture in which HUVECs and MSCs were grown the farther apart (D400) show a significant increase (p<0.05) in migratory activity. (e) CdM was used to supplement media for a tube formation assay (n=3). HUVECs incubated with CdM from coculture in which HUVECs and MSCs were grown separated but close (<200 μm) (D0, D200) formed a significantly longer network (p<0.05).

Figure IV:. EVs cargo increases with distance between cell populations and promotes early angiogenesis.

For each figure, groups not sharing a letter are significantly different (p<0.05). (a) EVs and protein per EVs quantification (n=3). Proteins concentration per EVs increases with the distance between cell populations in co-culture. (b) Schematics of experiment. After 48h of incubation, CdM was collected from all experimental groups and EVs were collected by centrifugation. EVs were then quantified and their Angiogenic potential was evaluated in functional Angiogenic assays. (c) Tube Formation Assay (n=3). EVs supplemented media was used to incubate HUVECs on Matrigel. Total length of network formed by tube like structures was measured. No statistical difference was observed between coculture groups. (d) Proliferation Assay (n=3). EVs supplemented media was used to grown HUVECs. Confluency was measured after 24 and 48h of incubation. HUVECs grown with D400 EVs showed a higher proliferation rate. (e) Migration Assay (n=3). HUVECs grown in D400 EVs supplemented media showed a faster migration.

Figure V:. Healing of critical-size bone defect was improved using optimized 3DP scaffolds (D0 scaffolds)

(a) Representative images of H&E staining after 4 weeks. The black arrows are pointing to blood vessels. (b) Histomorphological quantification of blood vessels/mm², new bone formation and new bone thickness. (c) Masson’s trichrome staining of full cross section of the defect. (d) OCT B-Scan images of scaffolds after 4 weeks implantation.