Platelet-Rich Plasma as an Autologous and Proangiogenic Cell Delivery System

Abstract

Angiogenesis is a key factor in early stages of wound healing and is crucial for the repair of vascularized tissues such as the bone. However, supporting timely revascularization of the defect site still presents a clinical challenge. Tissue engineering approaches delivering endothelial cells or prevascularized constructs may overcome this problem. In the current study, we investigated platelet-rich plasma (PRP) gels as autologous, injectable cell delivery systems for prevascularized constructs. PRP was produced from human thrombocyte concentrates. GFP-expressing human umbilical vein endothelial cells (HUVECs) and human bone marrow-derived mesenchymal stem cells (MSCs) were encapsulated in PRP gels in different proportions. The formation of cellular networks was assessed over 14 days by time-lapse microscopy, gene expression analysis, and immunohistology. PRP gels presented a favorable environment for the formation of a three-dimensional (3D) cellular network. The formation of these networks was apparent as early as 3 days after seeding. Networks increased in complexity and branching over time but were only stable in HUVEC-MSC cocultures. The high cell viability together with the 3D capillary-like networks observed at early time points suggests that PRP can be used as an autologous and proangiogenic cell delivery system for the repair of vascularized tissues such as the bone.

Figures

Figure 1

Network analysis. Image analysis method of PRP gels (seeding density: 2.5 × 103 cells/μl gel) with encapsulated PKH26 Red-labeled MSCs and GFP-HUVECs (green). Shown are representative pictures of a 50% HUVEC–50% MSC coculture after 14 days of culture. (a) Red-labeled MSCs, (b) GFP-labeled HUVECs, and (c) combined image. (d) Tubular structures are marked with the polygons (grey). Scale bar = 200 μm.

Figure 2

MSC mediated shrinkage of PRP gels. PRP gels (120 μl) were seeded either with or without cells in a 96-well plate. Different types of cells were encapsulated (seeding density: 2.5 × 103 cells/μl gel): 100% MSCs (100 M), 100% HUVECs (100 H), 75% HUVECs–25% MSCs (75 HM), 50% HUVECs–50% MSCs (50 HM). Shown are representative pictures from day 0 (d0), day 7 (d7), and day 14 (d14). White dashed lines indicate the outline of gels. Shrinkage was only observed when MSCs were present in gels.

Figure 3

Time-lapse pictures of HUVEC and MSC mono- and cocultures. GFP-positive HUVECs (green) and PKH26 Red prestained MSCs were encapsulated in PRP (seeding density: 2.5 × 103 cells/μl gel) and time-lapse microscopy ran for two weeks. Four different cell proportions were seeded: 100% MSCs (100 M, first column), 100% HUVECs (100 H, second column), 75% HUVECs–25% MSCs (75 HM, third column), and 50% HUVECs–50% MSCs (50 HM, fourth column). Representative pictures of five time points are shown: day 0 (d0), day 3 (d3), day 7 (d7), day 10 (d10), and day 14 (d14). Cellular organization towards formation of tube-like networks starting from day 3 was observed in mono- and cocultures in PRP over time but not in the condition of 100% MSCs. After one week, a complex cellular network could be detected in both cocultures which was still apparent after two weeks. Cellular networks in HUVEC monocultures disintegrated after 10 days. Scale bar = 200 μm.

Figure 4

High-resolution images of HUVEC and MSC mono- and cocultures after 2 weeks. Representative images are shown from mono- and cocultures at day 14 after seeding (seeding density: 2.5 × 103 cells/μl gel) with (a) 100% MSCs (100 M); (b) 100% HUVECs (100 H); (c) 75% HUVECs–25% MSCs (75 HM); and (d–f) 50% HUVECs–50% MSCs (50 HM). Network formation occurred in 100 H and both cocultures but was only stable in the presence of MSCs which integrate in cellular networks as demonstrated in e and f (white arrow heads). Scale bar = 100 μm (a–d), 25 μm (e, f).

Figure 5

Analysis of cellular networks. Image analysis was performed of mono- and cocultures (100% HUVECs (100 H), 75% HUVECs–25% MSCs (75 HM), and 50% HUVECs–50% MSCs (50 HM)) in PRP (seeding density: 2.5 × 103 cells/μl gel) (n = 3). Networks were measured by manually marking cellular networks. The relative percentage of prestained green (HUVECs) and red (MSCs) cells was investigated for day 0, day 3, day 7, day 10, and day 14 in the following regions: relative percentage of green or red signal in the entire image (a, b), relative percentage of marked network area (c), relative percentage of green or red signal in marked network area (d, e), and the relative percentage of green or red signal beside marked network area (f, g). For 100 H monocultures, networks decreased after one week whereas in both cocultures, networks were present and stable after two weeks (c). Cellular networks were mainly made out of HUVECs (d), with a minor contribution of MSCs (e). Two-way ANOVA with Tukey's post hoc test for multiple comparison was applied to test for significant differences over time (compared to day 0: (A) 50 HM p < 0.01 (all time points); (B) 75 HM p < 0.01 (day 3) and p < 0.001 (day 7–14); and (C) 100 H p < 0.001 (day 3) and p < 0.01 (day 7)) and between different culture conditions (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to 50 HM, ###p < 0.001 compared to 75 HM). n = 3.

Figure 6

Gene expression of HUVEC and MSC mono- and cocultures in PRP at days 7 and 14. Gene expression (normalized to 18s expression) at day 7 and day 14 is displayed as fold change to day 0. Dashed lines indicate a fold change of 1, that is, unchanged gene expression. MSC monocultures (100 M), HUVEC monocultures (100 H), and cocultures 75% HUVECs–25% MSCs (75 HM) and 50% HUVECs–50% MSCs (50 HM) were investigated (seeding density: 2.5 × 103 cells/μl gel). (a) Gene expression of VEGF, showing downregulation in 100H cultures. (b) An upregulation of angiopoetin-1 (Ang 1), a crucial growth factor of angiogenic processes, was detected in all conditions at day 14 as well as in the cocultures on day 7. (c) Gene expression of Tie 2, one of the receptors binding Ang 1. (d–f) Depicted is the expression of pericyte markers CD146, NG 2, and PDGFRβ1, respectively. CD146 was upregulated at day 7 in MSC monocultures (d). At day 7, both cocultures showed a higher NG 2 expression compared to monocultures (e). PDGFRβ1 upregulations were detectable in all cultures at day 14 (f). MSCs upregulated connexin 43 (g), indicating the formation of gap junctions, whereas no differential regulation was observed for collagen IV (h). Two-way ANOVA with Tukey's post hoc test for multiple comparison was applied to test for differences between experimental groups; ∗p < 0.05. n = 3.

Figure 7

Immunohistology for CD146 and connexin 43 (Cx 43) of HUVEC and MSC mono- and cocultures in PRP at day 14. Immunohistology was performed on cryosections of mono- and cocultures with 100% MSCs (100 M), 100% HUVECs (100 H), 75% HUVECs–25% MSCs (75 HM), and 50% HUVECs–50% MSCs (50 HM) (seeding density: 2.5 × 103 cells/μl gel). Negative controls (NC) are shown in the first row. In comparison to the NC, positive signals of CD146 were detectable in the presence of HUVECs, whereas connexin 43 protein levels were only apparent in a mono- or coculture with MSCs. Scale bars = 100 μm.