Safety and Homing of Human Dental Pulp Stromal Cells in Head and Neck Cancer.

BACKGROUND: Head and neck cancer (HNC) is one of the most common cancers, associated with a huge mortality and morbidity. In order to improve patient outcomes, more efficient and targeted therapies are essential. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) express tumour homing capacity, which could be exploited to target anti-cancer drug delivery to the tumour region and reduce adverse side-effects. Nevertheless, dental pulp stromal cells (DPSCs), an MSC-like population present in teeth, could offer important clinical benefits because of their easy isolation and superior proliferation compared to BM-MSCs. Therefore, we aimed to elucidate the tumour homing and safe usage of DPSCs to treat HNC. METHODS: The in vivo survival as well as the effect of intratumourally administered DPSCs on tumour aggressiveness was tested in a HNC xenograft mouse model by using bioluminescence imaging (BLI), (immuno)histology and qRT-PCR. Furthermore, the in vitro and in vivo tumour homing capacity of DPSCs towards a HNC cell line were evaluated by a transwell migration assay and BLI, respectively. RESULTS: Intratumourally injected DPSCs survived for at least two weeks in the tumour micro-environment and had no significant influence on tumour morphology, growth, angiogenesis and epithelial-to-mesenchymal transition. In addition, DPSCs migrated towards tumour cells in vitro, which could not be confirmed after their in vivo intravenous, intraperitoneal or peritumoural injection under the tested experimental conditions. CONCLUSIONS: Our research suggests that intratumourally delivered DPSCs might be used as safe factories for the continuous delivery of anti-cancer drugs in HNC. Nevertheless, further optimization as well as efficacy studies are necessary to understand and improve in vivo tumour homing and determine the optimal experimental set-up of stem cell-based cancer therapies, including dosing and timing.

Chorioallantoic Membrane Assay as Model for Angiogenesis in Tissue Engineering: Focus on Stem Cells.

Tissue engineering aims to structurally and functionally regenerate damaged tissues, which requires the formation of new blood vessels that supply oxygen and nutrients by the process of angiogenesis. Stem cells are a promising tool in regenerative medicine due to their combined differentiation and paracrine angiogenic capacities. The study of their proangiogenic properties and associated potential for tissue regeneration requires complex in vivo models comprising all steps of the angiogenic process. The highly vascularized extraembryonic chorioallantoic membrane (CAM) of fertilized chicken eggs offers a simple, easy accessible, and cheap angiogenic screening tool compared to other animal models. Although the CAM assay was initially primarily performed for evaluation of tumor growth and metastasis, stem cell studies using this model are increasing. In this review, a detailed summary of angiogenic observations of different mesenchymal, cardiac, and endothelial stem cell types and derivatives in the CAM model is presented. Moreover, we focus on the variation in experimental setup, including the benefits and limitations of in ovo and ex ovo protocols, diverse biological and synthetic scaffolds, imaging techniques, and outcome measures of neovascularization. Finally, advantages and disadvantages of the CAM assay as a model for angiogenesis in tissue engineering in comparison with alternative in vivo animal models are described. Impact statement The chorioallantoic membrane (CAM) assay is an easy and cheap screening tool for the angiogenic properties of stem cells and their associated potential in the tissue engineering field. This review offers an overview of all published angiogenic studies of stem cells using this model, with emphasis on the variation in used experimental timeline, culture protocol (in ovo vs. ex ovo), stem cell type (derivatives), scaffolds, and outcome measures of vascularization. The purpose of this overview is to aid tissue engineering researchers to determine the ideal CAM experimental setup based on their specific study goals.

Angiogenic Effects of Human Dental Pulp and Bone Marrow-Derived Mesenchymal Stromal Cells and their Extracellular Vesicles.

Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) possess paracrine proangiogenic properties, which are, at least partially, induced by their extracellular vesicles (EVs). However, the isolation of BM-MSCs is associated with several drawbacks, which could be overcome by MSC-like cells of the teeth, called dental pulp stromal cells (DPSCs). This study aims to compare the angiogenic content and functions of DPSC and BM-MSC EVs and conditioned medium (CM). The angiogenic protein profile of DPSC- and BM-MSC-derived EVs, CM and EV-depleted CM was screened by an antibody array and confirmed by ELISA. Functional angiogenic effects were tested in transwell migration and chicken chorioallantoic membrane assays. All secretion fractions contained several pro- and anti-angiogenic proteins and induced in vitro endothelial cell motility. This chemotactic potential was higher for (EV-depleted) CM, compared to EVs with a stronger effect for BM-MSCs. Finally, BM-MSC CM, but not DPSC CM, nor EVs, increased in ovo angiogenesis. In conclusion, we showed that DPSCs are less potent in relation to endothelial cell chemotaxis and in ovo neovascularization, compared to BM-MSCs, which emphasizes the importance of choice of cell type and secretion fraction for stem cell-based regenerative therapies in inducing angiogenesis.

Preconditioning of Human Dental Pulp Stem Cells with Leukocyte- and Platelet-Rich Fibrin-Derived Factors Does Not Enhance Their Neuroregenerative Effect.

Pathologies of the central nervous system are characterized by loss of brain tissue and neuronal function which cannot be adequately restored by endogenous repair processes. This stresses the need for novel treatment options such as cell-based therapies that are able to restore damaged tissue or stimulate repair. This study investigated the neuroregenerative potential of the conditioned medium of human dental pulp stem cells (CM-hDPSCs) on neural stem cell (NSC) proliferation and migration as well as on neurite outgrowth of primary cortical neurons (pCNs). Additionally, the effect of leukocyte- and platelet-rich fibrin (L-PRF) priming on the neuroregenerative potential of the hDPSC secretome on NSCs and pCNs was evaluated. L-PRF contains factors that enhance stem cell-induced regeneration, but its effect on hDPSC-mediated neuroregeneration is unknown. This study demonstrated that CM-hDPSCs enhanced neuritogenesis. Moreover, CM-hDPSCs had a chemoattractant effect on NSCs. Although priming hDPSCs with L-PRF increased brain-derived neurotrophic factor secretion, no additional effects on the paracrine-mediated repair mechanisms were observed. These data support the neuroregenerative potential of hDPSCs, and although priming had no additional effect, the potential of L-PRF-primed hDPSCs on distinct regenerative mechanisms remains to be clarified.

Angiogenic Properties of 'Leukocyte- and Platelet-Rich Fibrin'.

Leukocyte- and Platelet-Rich Fibrin (L-PRF) is an autologous platelet concentrate, consisting of a fibrin matrix enriched with platelets, leukocytes and a plethora of cytokines and growth factors. Since L-PRF is produced bedside from whole blood without the use of an anti-coagulant, it is becoming a popular adjuvant in regenerative medicine. While other types of platelet concentrates have been described to stimulate blood vessel formation, little is known about the angiogenic capacities of L-PRF. Therefore, this study aimed to fully characterize the angiogenic potential of L-PRF. With an antibody array, the growth factors released by L-PRF were determined and high levels of CXC chemokine receptor 2 (CXCR-2) ligands and epidermal growth factor (EGF) were found. L-PRF induced in vitro key steps of the angiogenic process: endothelial proliferation, migration and tube formation. In addition, we could clearly demonstrate that L-PRF is able to induce blood vessel formation in vivo, the chorioallantoic membrane assay. In conclusion, we could demonstrate the angiogenic capacity of L-PRF both in vitro and in vivo, underlying the clinical potential of this easy-to-use platelet concentrate.

Stem Cells for Cartilage Repair: Preclinical Studies and Insights in Translational Animal Models and Outcome Measures.

Due to the restricted intrinsic capacity of resident chondrocytes to regenerate the lost cartilage postinjury, stem cell-based therapies have been proposed as a novel therapeutic approach for cartilage repair. Moreover, stem cell-based therapies using mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs) have been used successfully in preclinical and clinical settings. Despite these promising reports, the exact mechanisms underlying stem cell-mediated cartilage repair remain uncertain. Stem cells can contribute to cartilage repair via chondrogenic differentiation, via immunomodulation, or by the production of paracrine factors and extracellular vesicles. But before novel cell-based therapies for cartilage repair can be introduced into the clinic, rigorous testing in preclinical animal models is required. Preclinical models used in regenerative cartilage studies include murine, lapine, caprine, ovine, porcine, canine, and equine models, each associated with its specific advantages and limitations. This review presents a summary of recent in vitro data and from in vivo preclinical studies justifying the use of MSCs and iPSCs in cartilage tissue engineering. Moreover, the advantages and disadvantages of utilizing small and large animals will be discussed, while also describing suitable outcome measures for evaluating cartilage repair.