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

Therapeutic Potential of Dental Pulp Stem Cells and Leukocyte- and Platelet-Rich Fibrin for Osteoarthritis.

Osteoarthritis (OA) is a degenerative and inflammatory joint disorder with cartilage loss. Dental pulp stem cells (DPSCs) can undergo chondrogenic differentiation and secrete growth factors associated with tissue repair and immunomodulation. Leukocyte- and platelet-rich fibrin (L-PRF) emerges in regenerative medicine because of its growth factor content and fibrin matrix. This study evaluates the therapeutic application of DPSCs and L-PRF in OA via immunomodulation and cartilage regeneration. Chondrogenic differentiation of DPSCs, with or without L-PRF exudate (ex) and conditioned medium (CM), and of bone marrow-mesenchymal stem cells was compared. These cells showed differential chondrogenesis. L-PRF was unable to increase cartilage-associated components. Immature murine articular chondrocytes (iMACs) were cultured with L-PRF ex, L-PRF CM, or DPSC CM. L-PRF CM had pro-survival and proliferative effects on unstimulated and cytokine-stimulated iMACs. L-PRF CM stimulated the release of IL-6 and PGE2, and increased MMP-13, TIMP-1 and IL-6 mRNA levels in cytokine-stimulated iMACs. DPSC CM increased the survival and proliferation of unstimulated iMACs. In cytokine-stimulated iMACs, DPSC CM increased TIMP-1 gene expression, whereas it inhibited nitrite release in 3D culture. We showed promising effects of DPSCs in an in vitro OA model, as they undergo chondrogenesis in vitro, stimulate the survival of chondrocytes and have immunomodulatory effects

Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct <i>in vitro</i>

In the present study, we evaluated the differentiation potential of human dental pulp stem cells (hDPSCs) toward Schwann cells, together with their functional capacity with regard to myelination and support of neurite outgrowth in vitro. Successful Schwann cell differentiation was confirmed at the morphological and ultrastructural level by transmission electron microscopy. Furthermore, compared to undifferentiated hDPSCs, immunocytochemistry and ELISA tests revealed increased glial marker expression and neurotrophic factor secretion of differentiated hDPSCs (d-hDPSCs), which promoted survival and neurite outgrowth in 2-dimensional dorsal root ganglia cultures. In addition, neurites were myelinated by d-hDPSCs in a 3-dimensional collagen type I hydrogel neural tissue construct. This engineered construct contained aligned columns of d-hDPSCs that supported and guided neurite outgrowth. Taken together, these findings provide the first evidence that hDPSCs are able to undergo Schwann cell differentiation and support neural outgrowth in vitro, proposing them to be good candidates for cell-based therapies as treatment for peripheral nerve injury

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

The insulin-like growth factor system and adenocarcinoma of the colon

The insulin-like growth factor (IGF) system is important in normal growth and development. However, it is also known to be involved with malignant transformation and cellular proliferation. IGF binding proteins modulate the biological activity of IGF-I, either potentiating or inhibiting its activity, as well as determining how much enters the circulation at any one time. IGF binding protein-4 (IGFBP-4), for example is believed to be inhibitory to the effects of IGF-I. This thesis shows that the colon cancer cell lines Colo 205, HT29 and WiDR proliferate in response to IGF-I, and that IGFBP-4 at high concentrations inhibits their growth. However, it was found that with lower concentrationsof IGFBP-4, proliferation in HT29 and WiDR cells increased. Nevertheless in two cell lines, IGFBP-4 partially negated the proliferative effects of IGF-I. An antibody against IGFBP-4 was used to show that endogenous IGFBP-4 plays an important role in modifying cell growth. In order to start in vivo experiments which required considerable quantities of IGFBP-4, this protein was produced in an expression system and purified using an immunoaffinity column method. The rhIGFBP-4 thus produced was shown to be functional and to inhibit colorectal cancer cell growth in vitro. A nude mouse model of colon cancer was produced and the expression of components of the IGF system in this model determined using PCR. Experiments were performed using conditioned medium from Colo 205 cells to investigate IGFBP-4 protease activity. This thesis shows that manipulation of the IGF system is a potential target for further research into treatment for adenocarcinoma of the colon

Improvement of purine and pyrimidine antimetabolite-based anticancer treatment by selective suppression of mycoplasma-encoded catabolic enzymes

Most mycoplasmas are present as commensals, colonising the mucosa of our respiratory and gastrointestinal tract. Experimental data suggest that the long-term association of certain mycoplasma species with mammalian cells might favour host-cell transformation and malignancy. Moreover, increased mycoplasma infection has been noted in several cancers. Despite efforts to develop target-specific anticancer drugs, current cancer treatment still relies on the use of nucleobase or nucleoside-based analogues. Here, we provide experimental evidence that nucleoside-metabolising catabolic enzymes expressed by mycoplasmas substantially compromise the efficacy of nucleoside antimetabolites used in the treatment of cancer. We also suggest potential methods for improving future chemotherapy by suppressing mycoplasma-mediated catabolism of the anticancer nucleoside analogues.status: publishe

The Effect of Leukocyte- and Platelet-Rich Fibrin on Central and Peripheral Nervous System Neurons—Implications for Biomaterial Applicability

Leukocyte- and Platelet-Rich Fibrin (L-PRF) is a second-generation platelet concentrate that is prepared directly from the patient’s own blood. It is widely used in the field of regenerative medicine, and to better understand its clinical applicability we aimed to further explore the biological properties and effects of L-PRF on cells from the central and peripheral nervous system. To this end, L-PRF was prepared from healthy human donors, and confocal, transmission, and scanning electron microscopy as well as secretome analysis were performed on these clots. In addition, functional assays were completed to determine the effect of L-PRF on neural stem cells (NSCs), primary cortical neurons (pCNs), and peripheral dorsal root ganglion (DRG) neurons. We observed that L-PRF consists of a dense but porous fibrin network, containing leukocytes and aggregates of activated platelets that are distributed throughout the clot. Antibody array and ELISA confirmed that it is a reservoir for a plethora of growth factors. Key molecules that are known to have an effect on neuronal cell functions such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) were slowly released over time from the clots. Next, we found that the L-PRF secretome had no significant effect on the proliferative and metabolic activity of NSCs, but it did act as a chemoattractant and improved the migration of these CNS-derived stem cells. More importantly, L-PRF growth factors had a detrimental effect on the survival of pCNs, and consequently, also interfered with their neurite outgrowth. In contrast, we found a positive effect on peripheral DRG neurons, and L-PRF growth factors improved their survival and significantly stimulated the outgrowth and branching of their neurites. Taken together, our study demonstrates the positive effects of the L-PRF secretome on peripheral neurons and supports its use in regenerative medicine but care should be taken when using it for CNS applications

Nanomaterials for Periodontal Tissue Regeneration: Progress, Challenges and Future Perspectives

Bioactive nanomaterials are increasingly being applied in oral health research. Specifically, they have shown great potential for periodontal tissue regeneration and have substantially improved oral health in translational and clinical applications. However, their limitations and side effects still need to be explored and elucidated. This article aims to review the recent advancements in nanomaterials applied for periodontal tissue regeneration and to discuss future research directions in this field, especially focusing on research using nanomaterials to improve oral health. The biomimetic and physiochemical properties of nanomaterials such as metals and polymer composites are described in detail, including their effects on the regeneration of alveolar bone, periodontal ligament, cementum and gingiva. Finally, the biomedical safety issues of their application as regenerative materials are updated, with a discussion about their complications and future perspectives. Although the applications of bioactive nanomaterials in the oral cavity are still at an initial stage, and pose numerous challenges, recent research suggests that they are a promising alternative in periodontal tissue regeneration

The Impact of Advanced Glycation End-Products (AGEs) on Proliferation and Apoptosis of Primary Stem Cells: A Systematic Review

Stem cell-based regenerative therapies hold great promises to treat a wide spectrum of diseases. However, stem cell engraftment and survival are still challenging due to an unfavorable transplantation environment. Advanced glycation end-products (AGEs) can contribute to the generation of these harmful conditions. AGEs are a heterogeneous group of glycated products, nonenzymatically formed when proteins and/or lipids become glycated and oxidized. Our typical Western diet as well as cigarettes contain high AGEs content. AGEs are also endogenously formed in our body and accumulate with senescence and in pathological situations. Whether AGEs have an impact on stem cell viability in regenerative medicine remains unclear, and research on the effect of AGEs on stem cell proliferation and apoptosis is still ongoing. Therefore, this systematic review provides a clear overview of the effects of glycated proteins on cell viability in various types of primary isolated stem cells used in regenerative medicine