Abstract
BackgroundAngiogenesis is of utmost importance for tissue regeneration and repair. Human dental pulp stromal cells (hDPSCs) possess angiogenic potential, as they secrete paracrine factors that may alter the host microenvironment. However, more insight into how hDPSCs guide endothelial cells (ECs) in a paracrine fashion is yet to be obtained. Therefore, the current study aimed to investigate the effect(s) of conditioned medium derived from hDPSCs (hDPSC-CM) on EC behavior in vitro.MethodshDPSCs were harvested from third molars scheduled for surgical removal under informed consent. The angiogenic profile of hDPSC-CM was identified using human angiogenesis antibody array and enzyme-linked immunosorbent assay (ELISA). Using real-time reverse transcription-polymerase chain reaction (RT-PCR) and ELISA, the mRNA and protein expression level of specific angiogenic biomarkers was determined in human umbilical vein endothelial cells (HUVECs) exposed to hDPSC-CM. The effect of hDPSC-CM on HUVEC attachment, proliferation and migration was evaluated by crystal violet staining, MTT, transwell migration along with real-time cell monitoring assays (xCELLigence; ACEA Biosciences, Inc.). A Matrigel assay was included to examine the influence of hDPSC-CM on HUVEC network formation. Endothelial growth medium (EGM-2) and EGM-2 supplemented with hDPSC-CM served as experimental groups, whereas endothelial basal medium (EBM-2) was set as negative control.ResultsA wide range of proangiogenic and antiangiogenic factors, including vascular endothelial growth factor, tissue inhibitor of metalloproteinase protein 1, plasminogen activator inhibitor (serpin E1), urokinase plasminogen activator and stromal cell-derived factor 1, was abundantly detected in hDPSC-CM by protein profiling array and ELISA. hDPSC-CM significantly accelerated the adhesion phases, from sedimentation to attachment and spreading, the proliferation rate and migration of HUVECs as shown in both endpoint assays and real-time cell analysis recordings. Furthermore, Matrigel assay demonstrated that hDPSC-CM stimulated tubulogenesis, affecting angiogenic parameters such as the number of nodes, meshes and total tube length.ConclusionsThe sustained proangiogenic and promaturation effects of hDPSC-CM shown in this in vitro study strongly suggest that the trophic factors released by hDPSCs are able to trigger pronounced angiogenic responses, even beyond EGM-2 considered as an optimal culture condition for ECs.
Highlights
Angiogenesis is of utmost importance for tissue regeneration and repair
A growing number of studies have reported that DPSCs release proangiogenic and antiangiogenic proteins under different culture conditions, affecting different steps in the angiogenic process [12]. Human dental pulp stromal cell (hDPSC) are capable of promoting tube formation in human umbilical vein endothelial cells (HUVECs) both in a paracrine fashion and in a coculture system in vitro [5]. In agreement with these studies, we have shown that local administration of secretome from hDPSCs, grown under hypoxic conditions, promoted bone healing during distraction osteogenesis and reduced healing time through blood vessel formation [13]
Human adult third molars, scheduled for routine extraction, were collected from healthy patients aged 18–25 years under informed consent, in accordance with the protocol approved by the Ethical Research Committee at the University of Bergen, Norway (2009/610REK vest). hDPSCs were isolated from pulp tissue by enzymatic dissociation
Summary
Human dental pulp stromal cells (hDPSCs) possess angiogenic potential, as they secrete paracrine factors that may alter the host microenvironment. The discovery of postnatal dental pulp progenitor cells, exhibiting similar properties to bone marrow mesenchymal stem cells (MSCs) [4], has encouraged attempts to regenerate the damaged pulp tissue and/or maintain its functional integrity [2, 3]. These precursor cells have high regenerative potential when activated in the damaged pulp and exert paracrine/trophic effects that can alter the host microenvironment [5]. A balanced interplay of proangiogenic and antiangiogenic signaling cues, such as matrix metalloproteinases, growth factors, enzymes, cytokines, chemokines, and adhesion molecules, is required during blood vessel formation and development [9]
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