Vascular-like Network Research Articles

Direct Measurement of Contraction Force in Cardiac Tissue Construct in 2D-Plane Using Dual Axis Cantilever Sensor

In this work, we present a technique for a dual axis contraction force measurement of human cell based cardiac tissue constructs. The cardiac tissue constructs consist of a vascular-like network and induced pluripotent stem cell derived cardiomyocytes. Before the force measurements, the cardiac tissue constructs were detached from the culture substrate to allow less restricted contraction. The in-house prepared force sensors are composed of piezoelectric sensing elements and a metallic cantilever for contacting the cardiac tissue constructs. A dedicated measurement platform with embedded signal processing software is used for data acquisition from the sensors. Dual axis force sensor results are compared with our previously developed single axis force sensor technique. Additionally, the proposed dual axis force measurement system can measure two-dimensional displacement trajectories of the cantilever probe tip. We propose a pattern matching method for classification of the captured cardiac contraction cycle patterns and for extracting anomalies in the measured cycles. We demonstrate both single and dual axis peak cardiac construct contraction force measurement results in the ranges of 3.4 - $6.7~\mu \text{N}$ and 9.4 - $10.6~\mu \text{N}$ , respectively. The relative standard deviation of the peak contraction force results varied between 1.0 and 4.1% in eight captured 60 second measurement sequences.

Cancer-associated fibroblasts mediate cancer progression and remodel the tumouroid stroma

BackgroundCancer-associated fibroblasts (CAFs) are highly differentiated and heterogeneous cancer-stromal cells that promote tumour growth, angiogenesis and matrix remodelling.MethodsWe utilised an adapted version of a previously developed 3D in vitro model of colorectal cancer, composed of a cancer mass and the surrounding stromal compartment. We compared cancer invasion with an acellular stromal surround, a “healthy” or normal cellular stroma and a cancerous stroma. For the cancerous stroma, we incorporated six patient-derived CAF samples to study their differential effects on cancer growth, vascular network formation and remodelling.ResultsCAFs enhanced the distance and surface area of the invasive cancer mass whilst inhibiting vascular-like network formation. These processes correlated with the upregulation of hepatocyte growth factor (HGF), metallopeptidase inhibitor 1 (TIMP1) and fibulin-5 (FBLN5). Vascular remodelling of previously formed endothelial structures occurred through the disruption of complex networks, and was associated with the upregulation of vascular endothelial growth factor (VEGFA) and downregulation in vascular endothelial cadherin (VE-Cadherin).ConclusionsThese results support, within a biomimetic 3D, in vitro framework, the direct role of CAFs in promoting cancer invasion, and their key function in driving vasculogenesis and angiogenesis.

Topography-driven alterations in endothelial cell phenotype and contact guidance

Understanding how endothelial cell phenotype is affected by topography could improve the design of new tools for tissue engineering as many tissue engineering approaches make use of topography-mediated cell stimulation. Therefore, we cultured human pulmonary microvascular endothelial cells (ECs) on a directional topographical gradient to screen the EC vascular-like network formation and alignment response to nano to microsized topographies. The cell response was evaluated by microscopy. We found that ECs formed unstable vascular-like networks that aggregated in the smaller topographies and flat parts whereas ECs themselves aligned on the larger topographies. Subsequently, we designed a mixed topography where we could explore the network formation and proliferative properties of these ECs by live imaging for three days. Vascular-like network formation continued to be unstable on the topography and were only produced on the flat areas and a fibronectin coating did not improve the network stability. However, an instructive adipose tissue-derived stromal cell (ASC) coating provided the correct environment to sustain the vascular-like networks, which were still affected by the topography underneath. It was concluded that large microsized topographies inhibit vascular endothelial network formation but not proliferation and flat and nano/microsized topographies allow formation of early networks that can be stabilized by using an ASC instructive layer.

Quantifying Oxygen Levels in 3D Bioprinted Cell-Laden Thick Constructs with Perfusable Microchannel Networks.

The survival and function of thick tissue engineered implanted constructs depends on pre-existing, embedded, functional, vascular-like structures that are able to integrate with the host vasculature. Bioprinting was employed to build perfusable vascular-like networks within thick constructs. However, the improvement of oxygen transportation facilitated by these vascular-like networks was directly quantified. Using an optical fiber oxygen sensor, we measured the oxygen content at different positions within 3D bioprinted constructs with and without perfusable microchannel networks. Perfusion was found to play an essential role in maintaining relatively high oxygen content in cell-laden constructs and, consequently, high cell viability. The concentration of oxygen changes following switching on and off the perfusion. Oxygen concentration depletes quickly after pausing perfusion but recovers rapidly after resuming the perfusion. The quantification of oxygen levels within cell-laden hydrogel constructs could provide insight into channel network design and cellular responses.

An Engineered Infected Epidermis Model for In Vitro Study of the Skin's Pro-Inflammatory Response.

Wound infection is a major clinical challenge that can significantly delay the healing process, can create pain, and requires prolonged hospital stays. Pre-clinical research to evaluate new drugs normally involves animals. However, ethical concerns, cost, and the challenges associated with interspecies variation remain major obstacles. Tissue engineering enables the development of in vitro human skin models for drug testing. However, existing engineered skin models are representative of healthy human skin and its normal functions. This paper presents a functional infected epidermis model that consists of a multilayer epidermis structure formed at an air-liquid interface on a hydrogel matrix and a three-dimensionally (3D) printed vascular-like network. The function of the engineered epidermis is evaluated by the expression of the terminal differentiation marker, filaggrin, and the barrier function of the epidermis model using the electrical resistance and permeability across the epidermal layer. The results showed that the multilayer structure enhances the electrical resistance by 40% and decreased the drug permeation by 16.9% in the epidermis model compared to the monolayer cell culture on gelatin. We infect the model with Escherichia coli to study the inflammatory response of keratinocytes by measuring the expression level of pro-inflammatory cytokines (interleukin 1 beta and tumor necrosis factor alpha). After 24 h of exposure to Escherichia coli, the level of IL-1β and TNF-α in control samples were 125 ± 78 and 920 ± 187 pg/mL respectively, while in infected samples, they were 1429 ± 101 and 2155.5 ± 279 pg/mL respectively. However, in ciprofloxacin-treated samples the levels of IL-1β and TNF-α without significant difference with respect to the control reached to 246 ± 87 and 1141.5 ± 97 pg/mL respectively. The robust fabrication procedure and functionality of this model suggest that the model has great potential for modeling wound infections and drug testing.

Engineering of human brain organoids with a functional vascular-like system.

Human cortical organoids (hCOs), derived from human embryonic stem cells (hESCs), provide an excellent platform to study human brain development and diseases in complex 3D tissue. However, current hCOs lack microvasculature, resulting in limited oxygen and nutrient delivery to the inner-most parts of hCOs. Here, we engineered hESCs to ectopically express human ETS variant 2 (hETV2) to create in vitro vasculature in hCOs, namely vhCOs (vascularized hCOs). hETV2-expressing cells in hCOs contributed to forming a complex vascular-like network in hCOs. Importantly, the presence of vascularization resulted in enhanced functional maturation of organoids. We found that vhCOs acquired several blood-brain barrier (BBB) characteristics, including an increase in the expression of tight junctions, nutrient transporters, and trans-endothelial electrical resistance. Finally, hETV2-induced endothelium supported the formation of perfused blood vessels in vivo. These vhCOs form vasculature that resemble the early prenatal brain, and present a robust model to study brain disease in vitro.

P5384Postnatal mouse aorta contains yolk sac-derived haemangioblasts with myeloid and endothelial plasticity and vasculogenic capacity

Abstract Background Macrophages and endothelial cells share an intimate relationship during neovessel formation in different pathophysiological conditions. Recent studies have determined that in some tissues, both cell types are derived embryonically from yolk sac (YS) progenitor cells and are maintained postnatally without contribution from circulating sources. The mechanism by which this local “self-maintenance” occurs is unknown. Purpose We previously identified that mouse arteries contain macrophage and endothelial progenitor cells in their adventitial Sca-1+CD45+ compartment. Here we investigated at a clonal level for the existence of postnatal adventitial haemangioblasts and studied their developmental origins. Methods and results Single cell digests were prepared from murine aortas to perform colony-forming unit (CFU) assays in methylcellulose. Aortic cells from C57BL/6J mice selectively generated macrophage colonies (CFU-M) which contained progenitor cells that displayed >95% positive for expression of CD45, Sca-1, c-Kit, CX3CR1 and CSF1R, but negative for Lineage markers, as well as mature monocyte/macrophage (CD11b, F4/80) and endothelial (CD144) markers. Secondary replating of CFU-M progenitors from adult aortas revealed their self-renewal capacity, with 1 in 10 cells forming new CFU-M. Lineage mapping using Flt3CrexRosamT/mG mice demonstrated that aortic CFU-M progenitors were FLT3-ve, indicating that they were not derived from definitive bone marrow haematopoiesis. CFU-M prevalence in C57BL/6J aortas was highest in neonatal mice and diminished progressively with increasing age (∼100 per 105 cells at P1, ∼15 at 12w, ∼5 at 52w, P<0.01, n>4/gp), consistent with prenatal seeding. Embryonic profiling determined that CFU-M progenitors first appeared in extra-embryonic yolk sac around E9.5 and in aorta-gonad-mesonephros at E10.5, before the emergence of definitive haematopoietic stem cells. Inducible fate-mapping then confirmed that aortic CFU-M progenitors originated from CX3CR1+ and CSF1R+ cells in E9.5 yolk sac. Both yolk sac and postnatal aortic CFU-M progenitors generated vascular-like networks when cultured in Matrigel in vitro, containing M2-like macrophages (CD11b+F4/80+CD206+) and endothelial cells (CD31+CD144+). They produced similar progeny and rescued adventitial vascular sprouting when seeded around aortic rings whose adventitia had been stripped. Finally, adoptive transfer of CFU-M progenitors into a mouse model of hindlimb ischaemia resulted in 80% augmentation in hindlimb perfusion compared to cell-free control, with de novo transformation of donor cells into macrophages, endothelial cells and perfused neovessels (n=6). Conclusion To the best of our knowledge, this is the first ever definitive proof at a clonal level for the existence of haemangioblasts in postnatal tissue. Adventitial haemangioblasts originate from extra-embryonic YS and are a source of vasculogenesis in the arterial wall, relevant to vasa vasorum formation. Acknowledgement/Funding NHMRC of Australia (GNT1086796, CDF1161506), NHFA (FLF100412, FLF102056) Royal Australasian College of Physicians

Expression profiling of long noncoding RNAs associated with vasculogenic mimicry in osteosarcoma.

Osteosarcoma (OS) is the most common highly malignant bone tumor in teens. Vasculogenic mimicry (VM) is defined as de novo extracellular matrix-rich vascular-like networks formed by highly aggressive tumor cells. We previously reported the presence of VM and it is an unfavorable prognostic factor in OS patients. Long noncoding RNAs (lncRNAs) are aberrantly expressed in OS and involved in cancer cell VM. However, lncRNAs in VM formation of OS have not been investigated. We, therefore, profiled the expression of lncRNAs in highly aggressive OS cell line 143B compared with its parental poorly aggressive cell line HOS. The differentially expressed (DE) lncRNAs and messenger RNA (mRNAs) were subjected to constructed lncRNA-mRNA coexpressed network. The top-ranked hub gene lncRNA n340532 knockdown 143B cells were used for in vitro and in vivo VM assays. The annotation of DE lncRNAs was performed according to the coexpressed mRNAs by Gene Ontology and pathway analysis. A total of 1360 DE lncRNAs and 1353 DE mRNAs were screened out. lncRNA MALAT1 and FTX, which have known functions related to VM formation and tumorigenesis were identified in our data. The coexpression network composed of 226 lncRNAs and 118 mRNAs in which lncRNA n340532 had the highest degree number. lncRNA n340532 knockdown reduced VM formation in vitro. The suppression of n340532 also exhibited potent anti-VM and antimetastasis effect in vivo, suggesting its potential role in OS VM and metastasis. Furthermore, n340532 coexpressed with 10 upregulation mRNAs and 3 downregulation mRNAs. The enriched transforming growth factor-β signaling pathway, angiogenesis and so forth were targeted by those coexpressed mRNAs, implying n340532 may facilitate VM formation in OS through these pathways and gene functions. Our findings provide evidence for the potential role of lncRNAs in VM formation of OS that could be used in the clinic for anti-VM therapy in OS.

Fabrication of Stand-Alone Cell-Laden Collagen Vascular Network Scaffolds Using Fugitive Pattern-Based Printing-Then-Casting Approach.

Vascular networks are of great significance in tissue engineering and viewed as the first step to fabricate human tissues. Although various techniques have been investigated to create vascular and vascular-like networks, the fabrication of stand-alone pure collagen-based vascular constructs is still a challenge because of the poor extrudability, weak mechanical property, and long cross-linking time of pure collagen solutions. In this study, a fugitive pattern-based printing-then-casting approach is investigated. The proposed alginate-based fugitive ink has excellent mechanical strength (by adding Laponite nanoclay), printability (by adding Laponite nanoclay), and controllable gelation rate (by adding disodium hydrogen phosphate). Using this fugitive ink, complex vascular-like structures can be easily printed and cross-linked in Laponite EP bath as fugitive vascular tree patterns. Each fugitive vascular tree pattern is then embedded in a gelatin bath to make a gelatin mold with the tree patterns. With the help of sodium citrate, the fugitive vascular tree pattern is liquefied and removed to create the gelatin mold with vascular channels. Finally, a stand-alone collagen vascular network scaffold embedded with fibroblasts can be fabricated by casting the cell-laden collagen suspension into the gelatin mold and releasing it from the mold at 37 °C. The cell-related investigations indicate that the cells grow and spread well in the pure collagen vascular network scaffold. The proposed hybrid printing-then-casting approach also provides a feasible technology to fabricate with materials having low viscosity, long gelation time, and poor mechanical property.

TGF-β1/CD105 signaling controls vascular network formation within growth factor sequestering hyaluronic acid hydrogels.

Cell-based strategies for the treatment of ischemic diseases are at the forefront of tissue engineering and regenerative medicine. Cell therapies purportedly can play a key role in the neovascularization of ischemic tissue; however, low survival and poor cell engraftment with the host vasculature following implantation limits their potential to treat ischemic diseases. To overcome these limitations, we previously developed a growth factor sequestering hyaluronic acid (HyA)-based hydrogel that enhanced transplanted mouse cardiosphere-derived cell survival and formation of vasculature that anastomosed with host vessels. In this work, we examined the mechanism by which HyA hydrogels presenting transforming growth factor beta-1 (TGF-β1) promoted proliferation of more clinically relevant human cardiosphere-derived cells (hCDC), and their formation of vascular-like networks in vitro. We observed hCDC proliferation and enhanced formation of vascular-like networks occurred in the presence of TGF-β1. Furthermore, production of nitric oxide (NO), VEGF, and a host of angiogenic factors were increased in the presence of TGF-β1. This response was dependent on the co-activity of CD105 (Endoglin) with the TGF-βR2 receptor, demonstrating its role in the process of angiogenic differentiation and vascular organization of hCDC. These results demonstrated that hCDC form vascular-like networks in vitro, and that the induction of vascular networks by hCDC within growth factor sequestering HyA hydrogels was mediated by TGF-β1/CD105 signaling.

Microfibrous Scaffolds Enhance Endothelial Differentiation and Organization of Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) are a promising source of endothelial cells (iPSC-ECs) for engineering three-dimensional (3D) vascularized cardiac tissues. To mimic cardiac microvasculature, in which capillaries are oriented in parallel, we hypothesized that endothelial differentiation of iPSCs within topographically aligned 3D scaffolds would be a facile one-step approach to generate iPSC-ECs as well as induce aligned vascular organization. Human iPSCs underwent endothelial differentiation within electrospun 3D polycaprolactone (PCL) scaffolds having either randomly oriented or parallel-aligned microfibers. Using transcriptional, protein, and endothelial functional assays, endothelial differentiation was compared between conventional two-dimensional (2D) films and 3D scaffolds having either randomly oriented or aligned microfibers. Furthermore, the role of parallel-aligned microfiber patterning on the organization of vessel-like networks was assessed. The cells in both the randomly oriented and aligned 3D scaffolds demonstrated an 11-fold upregulation in gene expression of the endothelial phenotypic marker, CD31, compared to cells on 2D films. This upregulation corresponded to >3-fold increase in CD31 protein expression in 3D scaffolds, compared to 2D films. Concomitantly, other endothelial phenotypic markers including CD144 and endothelial nitric oxide synthase also showed significant transcriptional upregulation in 3D scaffolds by >7-fold, compared to 2D films. Nitric oxide production, which is characteristic of endothelial function, was produced 4-fold more abundantly in 3D scaffolds, compared to on 2D PCL films. Within aligned scaffolds, the iPSC-ECs displayed parallel-aligned vascular-like networks with 70% longer branch length, compared to cells in randomly oriented scaffolds, suggesting that fiber topography modulates vascular network-like formation and patterning. Together, these results demonstrate that 3D scaffold structure promotes endothelial differentiation, compared to 2D substrates, and that aligned topographical patterning induces anisotropic vascular network organization.

Maturation of human pluripotent stem cell derived cardiomyocytes is improved in cardiovascular construct

In order to translate preclinical data into the clinical studies, relevant in vitro models with structure and key functional properties similar to native human tissue should be used. In vitro cardiac models with vascular structures mimic the highly vascularized myocardium and provide interactions between endothelial cells, stromal cells and cardiomyocytes. Currently, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been shown to present immature morphology and fetal-like electrophysiological properties that may limit their use as physiological test platform. The aim of this study was to develop multicellular in vitro cardiovascular construct modeling human heart tissue. In the cardiovascular construct, hPSC-CMs were cultured with a vascular-like network formed by human foreskin fibroblasts and human umbilical vein endothelial cells that served as a platform in the construct. Cardiomyocyte orientation, maturation, electrophysiological properties and drug responses of the cardiovascular construct were characterized and compared to CM monoculture. hPSC-CMs in cardiovascular construct showed elongated morphology and aligned with the vascular-like network. Electrophysiological properties and calcium metabolism of hPSC-CMs as well as response to E-4031 and adrenaline demonstrated normal physiological behavior. Increased expression of cardiac structural proteins and ion channels in cardiovascular construct compared to CM monoculture were detected. In conclusion, vascular-like network supports the structural and functional maturation of hPSC-CMs. Our results suggest that cardiovascular construct presents more mature in vitro cardiac model compared to CM monoculture and could therefore serve as an advanced test system for cardiac safety and efficacy assessment as well as a model system for biomedical research.

Human and Swine Dental Pulp Stem Cells Form a Vascularlike Network after Angiogenic Differentiation in Comparison with Endothelial Cells: A Quantitative Analysis

IntroductionThe aim of this study was to quantify vascular network formation capacity after angiogenic induction of human and swine dental pulp stem cells (DPSCs) in comparison with endothelial cells. MethodsPrimary human DPSCs or swine DPSCs were induced in endothelial growth medium for 7 days. The expression of the endothelial marker von Willebrand factor was determined by immunostaining. Induced DPSCs (iDPSCs) and noninduced DPSCs (niDPSCs) were seeded at different cell numbers onto Matrigel (BD Biosciences, San Jose, CA) for vascular network formation assays and analyzed after 4, 8, 12, and 18 hours in comparison with human microvascular endothelial cells (hMECs). Quantitative analysis of vascular tubule formation was performed using ImageJ software (National Institutes of Health, Bethesda, MD). The vascular network formation was also conducted by coculturing of niDPSCs and iDPSCs. ResultsVon Willebrand factor was detected by immunofluorescence in both niDPSCs and iDPSCs (human and swine). Time-lapse microscopic observation showed that the vascular network was formed by iDPSCs but not niDPSCs. After 4 hours, iDPSCs showed vascular network formation, whereas niDPSCs started to aggregate and formed clusters. Human iDPSCs displayed a similar capacity to form vascular networks in Matrigel compared with hMECs based on quantitative analysis. Swine iDPSCs had a higher capacity compared with human iDPSCs or hMECs (P < .05) in forming the network structures including segments, nodes, and mesh. A coculture experiment showed that human niDPSCs colocalized on the angiogenic tubules and vascular networks that were formed by human iDPSCs. ConclusionsOur findings indicate that iDPSCs in combination with their noninduced counterparts may be used as a future clinical strategy for enhancing angiogenesis during the process of pulp-dentin regeneration.

Engineering Microvascularized 3D Tissue Using Alginate-Chitosan Microcapsules.

Construction of vascularized tissues is one of the major challenges of tissue engineering. The goal of this study was to engineer 3D microvascular tissues by incorporating the HUVEC-CS cells with a collagen/alginate-chitosan (AC) microcapsule scaffold. In the presence of AC microcapsules, a 3D vascular-like network was clearly observable. The results indicated the importance of AC microcapsules in engineering microvascular tissues -- providing support and guiding alignment of HUVEC-CS cells. This approach provides an alternative and promising method for constructing vascularized tissues.

ハイドロゲルの局所加熱を用いた多層細胞構造体の作製

We propose a new method to fabricate channels inside cell structures for tissue engineering applications. A local melting of cell embedded hydrogel is employed to make channels inside cell structures. The fabricated channels can be used as vascular-like networks to supply nutrition and oxygen to cells, or patterning another types of cells. Microelectrodes were prepared as heaters on a glass substrate by photolithography techniques. Channel patterning were conducted using the microelectrodes. The channel width and height are controlled by the heating time when the applied voltage to the microelectrodes is constant. The multi-layered channel fabrication was achieved by repeating melting-filling hydrogel. The results indicate that the proposed method can be used to making vascular-like networks inside cell structures to construct in vitro 3D cell systems.

A Versatile Method for Fabricating Tissue Engineering Scaffolds with a Three-Dimensional Channel for Prevasculature Networks.

Despite considerable advances in tissue engineering over the past two decades, solutions to some crucial problems remain elusive. Vascularization is one of the most important factors that greatly influence the function of scaffolds. Many research studies have focused on the construction of a vascular-like network with prevascularization structure. Sacrificial materials are widely used to build perfusable vascular-like architectures, but most of these fabricated scaffolds only have a 2D plane-connected network. The fabrication of three-dimensional perfusable branched networks remains an urgent issue. In this work, we developed a novel sacrificial molding technique for fabricating biocompatible scaffolds with a three-dimensional perfusable branched network. Here, 3D-printed poly(vinyl alcohol) (PVA) filament was used as the sacrificial material. The fused PVA was deposited on the surface of a cylinder to create the 3D branched solid network. Gelatin was used to embed the solid network. Then, the PVA mold was dissolved after curing the hydrogel. The obtained architecture shows good perfusability. Cell experiment results indicated that human umbilical vein endothelial cells (HUVECs) successfully attached to the surface of the branched channel and maintained high viability after a few days in culture. In order to prevent deformation of the channel, paraffin was coated on the surface of the printed structure, and hydroxyapatite (HA) was added to gelatin. In conclusion, we demonstrate a novel strategy toward the engineering of prevasculature thick tissues through the integration of the fused PVA filament deposit. This approach has great potential in solving the issue of three-dimensional perfusable branched networks and opens the way to clinical applications.

Hyperglycemia Induces Bioenergetic Changes in Adipose-Derived Stromal Cells While Their Pericytic Function Is Retained.

Diabetic retinopathy (DR) is a hyperglycemia (HG)-mediated microvascular complication. In DR, the loss of pericytes and subsequently endothelial cells leads to pathologic angiogenesis in retina. Adipose-derived stromal cells (ASC) are a promising source of therapeutic cells to replace lost pericytes in DR. To date, knowledge of the influence of HG on the bioenergetics and pericytic function of ASC is negligible. Human ASC were cultured in normoglycemia medium (5 mM d-glucose) or under HG (30 mM d-glucose) and assessed. Our data showed that HG increased the level of apoptosis and reactive oxygen species production in ASC, yet their proliferation rate was not affected. HG induced alterations in mitochondrial function and morphology in ASC. HG also strongly affected the bioenergetic status of ASC in which both the maximum oxygen consumption rate and extracellular acidification rate were decreased. This was corroborated by a reduced uptake of glucose under HG. In spite of these observations, in vitro, ASC promoted the formation of vascular-like networks of human umbilical vein endothelial cells on monolayers of ASC under HG with minimally affected.

Melanoma educates mesenchymal stromal cells towards vasculogenic mimicry.

Accumulating evidence suggests that mesenchymal stromal cells (MSCs) are recruited to the tumor, and promote tumor development and growth. The present study was performed to investigate the communication between aggressive melanoma and MSCs in vasculogenic mimicry (VM). Normal human MSCs plated on Matrigel were unable to form capillary-like structures (CLSs). By contrast, MSCs co-cultured with aggressive melanoma cell lines, namely, Mel Cher, Mel Kor and Mel P, generated CLSs. Significantly, MSCs co-cultured with poorly aggressive melanoma cells, namely, Mel Me, failed to form CLSs. To identify factors responsible for VM, the effects of vascular endothelial growth factor A (VEGFA), pro-epidermal growth factor, basic fibroblast growth factor and stromal cell-derived factor 1α on the formation of CLSs by MSCs were tested. VM was induced by the addition of VEGFA, whereas other cytokines were inefficient. To confirm the hypothesis that aggressive tumor cells can increase the vasculogenic ability of MSCs, a standard B16/F10 mouse melanoma test system was used. MSCs isolated from the adipose tissues of C57BL/6 mice with melanoma formed a vascular-like network on Matrigel, whereas MSCs from healthy mice failed to form such structures. This study provides the first direct evidence that melanoma tumors educate MSCs to engage in VM. The education may occur distantly. These findings offer promise for novel therapeutic directions in the treatment of metastatic melanoma.

Preliminary observations on scleral ossicles in performing functionalized 3D vascularized scaffolds for “critical-size” bone defect healing

The problem of “critical-size” bone defects occurs when a severe lesion is difficult to be self-recovered. Many strategies of regenerative medicine were used in the last decade, with translational approaches, to mimic both structure and function of the native bone tissue, making use of synthetic materials, nanotechnologies, bio/synthetic constructs or some of their combination. The main obstacle to engineering strategies is mostly due to the lack of a proper vascularization of the construct used. In this feasibility study, our attention is directed towards the main tissue engineering items: scaffolds, cells and conditioning factors. We propose the use of scleral ossicles of lower vertebrates (1), as natural scaffolds which will be functionalized to allow the best adhesion of endothelial cells along a geometrically controlled pattern on the bony surface of the construct; successively, on the functionalized scaffold, osteogenic cell lines will be cultured. In the preliminary phases of the study, the ossicles were scratched to remove soft tissue residues, variously flattened with different methods to reach a regular morphology on both sides, and finally autoclaved to eliminate cellular remnants and to annul antigenic properties. Ossicles were observed under SEM and subjected to micro-assay, to establish the best scaffold preparation and to characterize morphological properties more suitable for engineering phases. Functionalization will be made by immobilizing on the engineered ossicles specific growth factors for endothelial cells; later, mouse primary lung endothelial cells (ECs) and immortalized osteogenic cells (IDG-SW3) will be used. As expected results ECs should adhere to the ossicle surface and organize to form lumenized microvascular-like structures; later, supported by the vascular-like network, the osteogenic lineage should produce bone matrix on the construct. The production of newly-formed bone around vascular-like buds will be verify. 3D tissue constructs generated in vitro will be used in successive in vivo study for the healing of “critical-size” bone defects experimentally induced in mice.

局所的熱融解による生体親和性ゲルの選択的チャネルパターニング

We propose a new method to construct microchannels inside cell structures. Recently, three-dimensional cell structures have been actively developed such as cell sheets stacking or inkjet bio-printing method. However, those methods have some disadvantages such as stacking rate or non-biodegradable materials. In this paper, local melting of cell embedded hydrogel is employed to make microchannels inside cell structures. The fabricated microchannels can be used as vascular-like networks to supply nutrition and oxygen to cells. A microchannel was experimentally fabricated using an electrode on a glass substrate. The results indicate that the proposed method can be used to making vascularlike networks inside cell structures to construct in vitro 3D cell systems.