Engineering stem cell cardiac patch with microvascular features representative of native myocardium

The natural myocardium is a highly aligned tissue with an oriented vasculature. Its characteristic cellular as well as nanoscale extracellular matrix (ECM) organization along with an oriented vascular network ensures appropriate blood supply and functional performance. Although significant efforts have been made to develop anisotropic cardiac structure, currently neither an ideal biomaterial nor an effective vascularization strategy to engineer oriented and high-density capillary-like microvessels has been achieved for clinical cardiovascular therapies. A naturally derived oriented ECM nanofibrous scaffold mimics the physiological structure and components of tissue ECM and guides neovascular network formation. The objective of this study was to create an oriented and dense microvessel network with physiological myocardial microvascular features. Methods: Highly aligned decellularized human dermal fibroblast sheets were used as ECM scaffold to regulate physiological alignment of microvascular networks by co-culturing human mesenchymal stem cells (hMSCs) and endothelial cells (ECs). The influence of topographical features on hMSC and EC interaction was investigated to understand underlying mechanisms of neovasculature formation. Results: Results demonstrate that the ECM topography can be translated to ECs via CD166 tracks and significantly improved hMSC-EC crosstalk and vascular network formation. The aligned ECM nanofibers enhanced structure, length, and density of microvascular networks compared to randomly organized nanofibrous ECM. Moreover, hMSC-EC co-culture promoted secretion of pro-angiogenic growth factors and matrix remodeling via metalloprotease-2 (MMP-2) activation, which resulted in highly dense vascular network formation with intercapillary distance (20 μm) similar to the native myocardium. Conclusion: HMSC-EC co-culture on the highly aligned ECM generates physiologically oriented and dense microvascular network, which holds great potential for cardiac tissue engineering

Polydopamine and collagen coated micro-grated polydimethylsiloxane for human mesenchymal stem cell culture

Natural tissues contain highly organized cellular architecture. One of the major challenges in tissue engineering is to develop engineered tissue constructs that promote cellular growth in physiological directionality. To address this issue, micro-patterned polydimethylsiloxane (PDMS) substrates have been widely used in cell sheet engineering due to their low microfabrication cost, higher stability, excellent biocompatibility, and most importantly, ability to guide cellular growth and patterning. However, the current methods for PDMS surface modification either require a complicated procedure or generate a non-uniform surface coating, leading to the production of poor-quality cell layers. A simple and efficient surface coating method is critically needed to improve the uniformity and quality of the generated cell layers. Herein, a fast, simple and inexpensive surface coating method was analyzed for its ability to uniformly coat polydopamine (PD) with or without collagen on micro-grated PDMS substrates without altering essential surface topographical features. Topographical feature, stiffness and cytotoxicity of these PD and/or collagen based surface coatings were further analyzed. Results showed that the PD-based coating method facilitated aligned and uniform cell growth, therefore holds great promise for cell sheet engineering as well as completely biological tissue biomanufacturing

Intracellular mechanisms underlying the nicotinic enhancement of LTP in the rat dentate gyrus

We have previously shown that activation of nicotinic acetylcholine receptors (nAChRs) enhanced long-term potentiation (LTP) in the rat dentate gyrus in vitro via activation of α7 nAChR. In the present studies, mechanisms underlying the acute and chronic nicotinic enhancement of LTP were examined. In particular, the involvement of activation of intracellular kinases was examined using selective kinase antagonists, and the effects of enhancing cholinergic function with positive allosteric modulators of the α7 nAChR and with acetylcholinesterase (AChE) inhibitors were also investigated. Activation of extracellular signal-regulated kinase (ERK) and cAMP-dependent protein kinase (PKA) was found to be involved in the induction of the acute nicotinic enhancement of LTP, although not control LTP. In contrast, activation of the tyrosine kinase Src, Ca2+-calmodulin-dependent protein kinase II, Janus kinase 2 and p38 mitogen-activated protein kinase was not involved in the acute nicotinic enhancement of LTP, although Src activation was necessary for control LTP. Moreover, activation of phosphoinositide 3-kinase was involved in the acute nicotinic enhancement of LTP to a much lesser extent than in control LTP. Chronic nicotine enhancement of LTP was found to be dependent on PKA, ERK and Src kinases. Acute nicotinic enhancement of LTP was occluded by chronic nicotine treatment. The positive allosteric modulator PNU-120596 was found to strongly reduce the threshold for nicotinic enhancement of LTP, an affect mediated via the α7 nAChR as it was blocked by the selective antagonist methyllycaconitine. The AChE inhibitors tacrine and physostigmine enhanced control LTP

Antiallodynic Effects of Electroacupuncture Combined with MK-801 Treatment through the Regulation of p35/p25 in Experimental Diabetic Neuropathy

The anti-allodynic effect of NMDA receptor antagonist and acupuncture treatments were explored through spinal p35 regulation of diabetic neuropathic rat. We evaluated the change over time of p35/p25 protein levels in the spinal cord compared with behavioral responses to thermal and mechanical stimulation in streptozotocin (STZ)-induced diabetic rats. Additionally, we studied p35 expression when electroacupuncture (EA) and a sub-effective dose of NMDA (N-methyl-D-aspartate) receptor antagonist (MK-801) were used to treat hyperalgesia in the diabetic neuropathic pain (DNP). Thermal paw withdrawal latency (PWL) and mechanical paw withdrawal threshold (PWT) were significantly decreased in the early stage of diabetes in rats. p35 expression after STZ injection gradually decreased from 1 week to 4 weeks compared to normal controls. p25 expression in 4-week diabetic rats was significantly higher than that of 2-week diabetic rats, and thermal PWL in 4-week diabetic rats showed delayed responses to painful thermal stimulation compared with those at 2 weeks. EA applied to the SP-9 point (2 Hz frequency) significantly prevented the thermal and mechanical hyperalgesia in the DNP rat. Additionally, EA combined with MK-801 prolonged anti-hyperalgesia, increased p35 expression, and decreased the cleavage of p35 to p25 during diabetic neuropathic pain. In this study we show EA combined with a sub-effective dose of MK-801 treatment in DNP induced by STZ that is related to p35/p25 expression in spinal cord

Molecular Pathology of Neuro-AIDS (CNS-HIV)

The cognitive deficits in patients with HIV profoundly affect the quality of life of people living with this disease and have often been linked to the neuro-inflammatory condition known as HIV encephalitis (HIVE). With the advent of more effective anti-retroviral therapies, HIVE has shifted from a sub-acute to a chronic condition. The neurodegenerative process in patients with HIVE is characterized by synaptic and dendritic damage to pyramidal neurons, loss of calbindin-immunoreactive interneurons and myelin loss. The mechanisms leading to neurodegeneration in HIVE might involve a variety of pathways, and several lines of investigation have found that interference with signaling factors mediating neuroprotection might play an important role. These signaling pathways include, among others, the GSK3β, CDK5, ERK, Pyk2, p38 and JNK cascades. Of these, GSK3β has been a primary focus of many previous studies showing that in infected patients, HIV proteins and neurotoxins secreted by immune-activated cells in the brain abnormally activate this pathway, which is otherwise regulated by growth factors such as FGF. Interestingly, modulation of the GSK3β signaling pathway by FGF1 or GSK3β inhibitors (lithium, valproic acid) is protective against HIV neurotoxicity, and several pilot clinical trials have demonstrated cognitive improvements in HIV patients treated with GSK3β inhibitors. In addition to the GSK3β pathway, the CDK5 pathway has recently been implicated as a mediator of neurotoxicity in HIV, and HIV proteins might activate this pathway and subsequently disrupt the diverse processes that CDK5 regulates, including synapse formation and plasticity and neurogenesis. Taken together, the GSK3β and CDK5 signaling pathways are important regulators of neurotoxicity in HIV, and modulation of these factors might have therapeutic potential in the treatment of patients suffering from HIVE. In this context, the subsequent sections will focus on reviewing the involvement of the GSK3β and CDK5 pathways in neurodegeneration in HIV

EFFECTS OF TOPOGRAPHICAL FEATURES ON MICROVASCULAR NETWORK FORMATION

Current advancements in tissue engineering and biomaterials have inspired researchers to develop highly biomimetic tissues including vascular grafts, cardiac patches, skin substitutes, bone tissues etc. All of these tissues in their natural form contains highly organized microfluidic network composed of blood vessels, capillaries and lymphatics. Vascular network is one the crucial component in tissue architecture that maintains exchange of gases, nutrients and metabolic byproducts. Importantly, engineered tissues having thickness greater than 150-200 μm cannot rely upon diffusive nutrient transport after implantation. Therefore, one of the biggest obstacle in current scenario is to develop vascular networks in engineered tissues that can perfuse with host vasculature. Several approaches have been tested in past to develop microvacular network in vitro. These include external provision of angiogenic growth factors or appropriate extracellular matrix (ECM) proteins that stimulate endothelial cells (ECs) to form pro-angiogenic vascular networks. Interestingly, a completely fascinating avenue of topographical stimulation is still needs to be explored in order to determine its potential to stimulate angiogenesis. The objective of this study was to develop de novo microvascular network in vitro by Human mesenchymal stem cell (hMSC)- EC co-culture and investigate influence of micron scale topographical features on characteristics of this microvascular network. To achieve this goal, we tested mussel inspired polydopamine (PD) based coating to improve polydimethylsiloxane (PDMS) surface properties for optimum cell attachment and spreading. Our results indicated that micron scale topographical features have significant influence on properties of microvascular network including vessel alignment, intercapillary distance and vessel length

Mesenchymal stem cells for pre-vascularization of engineered tissues

Engineered tissues with a thickness larger than 150μm require an embedded functional vascular network to support cell survival and integration with host vasculature after in vivo implantation. Mesenchymal stem cells (MSCs) can secrete trophic factors to promote angiogenesis and function as pericytes to stabilize endothelial cell engineered neo-capillary structures. Recently, studies have shown that co-culturing MSCs with endothelial cells (ECs) could develop a pre-capillary network in tissue scaffolds. To realize such an outcome, several factors need to be considered, including MSC source, cell seeding order, oxygen concentration, and extra cellular matrix features. The present mini review summarizes these crucial considerations and will provide beneficial references for successful development of functional pre-vascularized tissues

Constructing biomimetic cardiac tissues: a review of scaffold materials for engineering cardiac patches

Engineered cardiac patches (ECPs) hold great promise to repair ischemia-induced damages to the myocardium. Recent studies have provided robust technological advances in obtaining pure cardiac cell populations as well as various novel scaffold materials to generate engineered cardiac tissues that can significantly improve electrical and contractile functions of damaged myocardium. Given the significance in understanding the cellular and extracellular structural as well as compositional details of native human heart wall, in order to fabricate most suitable scaffold material for cardiac patches, herein, we have reviewed the structure of the human pericardium and heart wall as well as the compositional details of cardiac extracellular matrix (ECM). Moreover, several strategies to obtain cardiac-specific scaffold materials have been reviewed, including natural, synthetic and hybrid hydrogels, electrospun fibers, decellularized native tissues or whole organs, and scaffolds derived from engineered cell sheets. This review provides a comprehensive analysis of different scaffold materials for engineering cardiac tissues

Fabrication of a Completely Biological and Anisotropic Human Mesenchymal Stem Cell-Based Vascular Graft

Tissue-engineered small-diameter vascular grafts are required to match mechanical properties as well as cellular and extracellular architecture of native blood vessels. Although various engineering technologies have been developed, the most reliable strategy highlights the needs for incorporating completely biological components and anisotropic cellular and biomolecular organization into the tissue-engineered vascular graft (TEVG). Based on the antithrombogenic, immunoregulatory, and regenerative properties of human mesenchymal stem cells (hMSCs), this chapter provides a step-by-step protocol for generating a completely biological and anisotropic TEVG that comprises of hMSCs and highly aligned extracellular matrix (ECM) nanofibers. The hMSCs were grown on an aligned nanofibrous ECM scaffold derived from an oriented human dermal fibroblast (hDF) sheet and then wrapped around a temporary mandrel to form a tubular assembly, followed by a maturation process in a rotating wall vessel (RWV) bioreactor. The resulting TEVG demonstrates anisotropic structural and mechanical properties similar to that of native blood vessels. A completely biological, anisotropic, and mechanically strong TEVG that incorporates immunoregulatory hMSCs is promising to meet the urgent needs of a surgical intervention for bypass grafting

Preservation of microvascular integrity and immunomodulatory property of prevascularized human mesenchymal stem cell sheets

Prevascularization is essential to ensure the viability, functionality, and successful integration of tissue-engineered three-dimensional (3D) constructs with surrounding host tissues after transplantation. Human mesenchymal stem cell (hMSC) sheet can be prevascularized by coculturing with endothelial cells (ECs), and then be further used as building blocks for engineering 3D complex tissues. In addition, predifferentiation of hMSCs into a tissue-specific lineage in vitro has been proven to promote graft engraftment and regeneration. However, it is unclear if the prevascularized hMSC sheets can still maintain their microvascular integrity as well as the immune-regulatory properties after their tissue-specific differentiation. The objective of this study was to investigate the effects of differentiation cues on the microvascular structure, angiogenic factor secretion, and immunogenic responses of prevascularized hMSC sheets. The results showed that upon coculturing with ECs, hMSC sheets successfully formed microvascular network, while maintaining hMSCs\u27 multi-lineage differentiation capability. The next step, osteogenic and adipogenic induction, damaged the preformed microvascular structures and compromised the angiogenic factor secretion ability of hMSCs. Nonetheless, this effect was mitigated by adjusting the concentration of differentiation factors. The subcutaneous transplantation in an immunocompetent rat model demonstrated that the osteogenic differentiated prevascularized hMSC sheet preserved its microvascular structure and immunomodulatory properties comparable to the undifferentiated prevascularized hMSC sheets. This study suggested that a balanced and optimal differentiation condition can effectively promote the tissue-specific predifferentiation of prevascularized hMSC sheet while maintaining its immunomodulatory and tissue integration properties