Regulation Of Smooth Muscle Cells Research Articles

Fabric-Enhanced Vascular Graft with Hierarchical Structure for Promoting the Regeneration of Vascular Tissue.

Natural blood vessels have completed functions, including elasticity, compliance, and excellent antithrombotic properties because of their mature structure. To replace damaged blood vessels, vascular grafts should perform these functions by simulating the natural vascular structures. Although the structures of natural blood vessels are thoroughly explored, constructing a small-diameter vascular graft that matches the mechanical and biological properties of natural blood vessels remains a challenge. A hierarchical vascular graft is fabricated by Electrospinning, Braiding, and Thermally induced phase separation (EBT) processes, which could simulate the structure of natural blood vessels. The internal electrospun structure facilitates the adhesion of endothelial cells, thereby accelerating endothelialization. The intermediate PLGA fabric exhibits excellent mechanical properties, which allow it to maintain its shape during long-term transplantation and prevent graft expansion. The external macroporous structure is beneficial for cell growth and infiltration. Blood vessel remodeling aims to combine a structure that promotes tissue regeneration with anti-inflammatory materials. The results in vitro demonstrated that it EBT vascular graft (EBTVG) has matched the mechanical properties, reliable cytocompatibility, and the strongest endothelialization in situ. The results in vitro and replacement of the resected artery in vivo suggest that the EBTVG combines different structural advantages with biomechanical properties and reliable biocompatibility, significantly promoting the stabilization and regeneration of vascular endothelial cells and vascular smooth muscle cells, as well as stabilizing the blood microenvironment.

Small-diameter vascular graft composing of core-shell structured micro-nanofibers loaded with heparin and VEGF for endothelialization and prevention of neointimal hyperplasia

Despite the significant progress made in recent years, clinical issues with small-diameter vascular grafts related to low mechanical strength, thrombosis, intimal hyperplasia, and insufficient endothelialization remain unresolved. This study aims to design and fabricate a core-shell fibrous small-diameter vascular graft by co-axial electrospinning process, which will mechanically and biologically meet the benchmarks for blood vessel replacement. The presented graft (PGHV) comprised polycaprolactone/gelatin (shell) loaded with heparin-VEGF and polycaprolactone (core). This study hypothesized that the shell structure of the fibers would allow rapid degradation to release heparin-VEGF, and the core would provide mechanical strength for long-term application. Physico-mechanical evaluation, in vitro biocompatibility, and hemocompatibility assays were performed to ensure safe in vivo applications. After 25 days, the PGHV group released 79.47 ± 1.54% of heparin and 86.25 ± 1.19% of VEGF, and degradation of the shell was observed but the core remained pristine. Both the control (PG) and PGHV groups demonstrated robust mechanical properties. The PGHV group showed excellent biocompatibility and hemocompatibility compared to the PG group. After four months of rat aorta implantation, PGHV exhibited smooth muscle cell regeneration and complete endothelialization with a patency rate of 100%. The novel core-shell structured graft could be pivotal in vascular tissue regeneration application.

Fate mapping RNA-sequencing reveal Malat1 regulates Sca1+ progenitor cells to vascular smooth muscle cells transition in vascular remodeling

Regeneration of smooth muscle cells (SMCs) is vital in vascular remodeling. Sca1+ stem/progenitor cells (SPCs) can generate de novo smooth muscle cells after severe vascular injury during vessel repair and regeneration. However, the underlying mechanisms have not been conclusively determined. Here, we reported that lncRNA Metastasis-associated lung adenocarcinoma transcript 1 (Malat1) was down-regulated in various vascular diseases including arteriovenous fistula, artery injury and atherosclerosis. Using genetic lineage tracing mice and veingraft mice surgery model, we found that suppression of lncRNA Malat1 promoted Sca1+ cells to differentiate into SMCs in vivo, resulting in excess SMC accumulation in neointima and vessel stenosis. Genetic ablation of Sca1+ cells attenuated venous arterialization and impaired vascular structure normalization, and thus, resulting in less Malat1 down-regulation. Single cell sequencing further revealed a fibroblast-like phenotype of Sca1+ SPCs-derived SMCs. Protein array sequencing and in vitro assays revealed that SMC regeneration from Sca1+ SPCs was regulated by Malat1 through miR125a-5p/Stat3 signaling pathway. These findings delineate the critical role of Sca1+ SPCs in vascular remodeling and reveal that lncRNA Malat1 is a key regulator and might serve as a novel biomarker or potential therapeutic target for vascular diseases.

Developing small-diameter vascular grafts with human amniotic membrane: long-term evaluation of transplantation outcomes in a small animal model

While current clinical utilization of large vascular grafts for vascular transplantation is encouraging, tissue engineering of small grafts still faces numerous challenges. This study aims to investigate the feasibility of constructing a small vascular graft from decellularized amniotic membranes (DAMs). DAMs were rolled around a catheter and each of the resulting grafts was crosslinked with (a) 0.1% glutaraldehyde; (b) 1-ethyl-3-(3-dimethylaminopropyl) crbodiimidehydro-chloride (20 mM)-N-hydroxy-succinimide (10 mM); (c) 0.5% genipin; and (d) no-crosslinking, respectively. Our results demonstrated the feasibility of using a rolling technique followed by lyophilization to transform DAM into a vessel-like structure. The genipin-crosslinked DAM graft showed an improved integrated structure, prolonged stability, proper mechanical property, and superior biocompatibility. After transplantation in rat abdominal aorta, the genipin-crosslinked DAM graft remained patent up to 16 months, with both endothelial and smooth muscle cell regeneration, which suggests that the genipin-crosslinked DAM graft has great potential to be implemented as a small tissue engineered graft for future vascular transplantation.

VEGF combined with DAPT promotes tissue regeneration and remodeling in vascular grafts.

Previous research on tissue-engineered blood vessels (TEBVs) has mainly focused on the intima or adventitia unilaterally, neglecting the equal importance of both layers. Meanwhile, the efficacy of grafts modified with vascular endothelial growth factor (VEGF) merely has been limited. Here, we developed a small-diameter graft that can gradually release VEGF and γ secretase inhibitor IX (DAPT) to enhance tissue regeneration and remodeling in both the intima and adventitia. In vitro, experiments revealed that the combination of VEGF and DAPT had superior pro-proliferation and pro-migration effects on endothelial cells. In vivo, the sustained release of VEGF and DAPT from the grafts resulted in improved regeneration and remodeling. Specifically, in the intima, faster endothelialization and regeneration of smooth muscle cells led to higher patency rates and better remodeling. In the adventitia, a higher density of neovascularization, M2 macrophages and fibroblasts promoted cellular ingrowth and replacement of the implant with autologous neo-tissue. Furthermore, western blot analysis confirmed that the regenerated ECs were functional and the effect of DAPT was associated with increased expression of vascular endothelial growth factor receptor 2. Our study demonstrated that the sustained release of VEGF and DAPT from the graft can effectively promote tissue regeneration and remodeling in both the intima and adventitia. This development has the potential to significantly accelerate the clinical application of small-diameter TEBVs.

Endothelialization and smooth muscle cell regeneration capabilities of a bi-layered small diameter vascular graft for blood vessel reconstruction

Despite the tremendous advances in the past few decades, the clinical problems associated with low mechanical strength, thrombosis, and inadequate endothelialization still have not been improved in small diameter vascular grafts. The objective of this study is to design and fabricate a dual layered small-diameter vascular graft by electrospinning process which will mechanically and biologically match the gold standard of blood vessel substitution. The presented graft was composed of polycaprolactone/gelatin (inner layer) and polyurethane/polycaprolactone (outer layer) and fabricated by sequential electrospinning process. Physico-mechanical evaluation, in vitro biocompatibility and hemocompatibility assays were performed for both layers to ensure safe in vivo applications Then, the vascular grafts were successfully implanted into a rat abdominal aorta model. Tubular vascular graft had a nanofibrous and porous outer structure, which provided a conducive micro-environment for smooth muscle cell migration as well as proliferation but the lumen wall had a smooth surface to reduce thrombogenicity and improve endothelialization. Both layers showed excellent biocompatibility and hemocompatibility in all in vitro assays. After three months, the harvested grafts exhibited smooth muscle cell regeneration and complete endothelialization on the graft lumen. The dual layered graft could therefore play a pivotal role in the arena of vascular tissue regeneration application.

Role of miRNAs in vascular development.

The development of the vertebrate vascular system is an extremely important and complex process. The circulatory system is the first organ system to develop during embryogenesis. The development of the vasculature into highly branched canals must occur clearly in many places in order to supply oxygen and nutrients to the rapidly developing embryo. This process is mediated by a coordinated response of vascular endothelial and parietal cells to heterogeneous angiogenic signals provided by tissues and organs. MicroRNAs regulate gene expression at the transcriptional and post-transcriptional levels and participate in many important physiological and pathological processes. MicroRNAs mainly play an important role in the developmental regulation of vascular smooth muscle cells and vascular endothelial cells. This article summarizes the research progress of microRNAs in vascular development in recent years, focusing on the regulatory mechanism of miR-126 and miR-17/92 families in vascular endothelial cells, as well as the miR-143/145 family, miR-21 in vascular smooth muscle cell's regulation. The research prospects of the role of microRNAs in vascular development are also presented in this article.

The loading of C-type natriuretic peptides improved hemocompatibility and vascular regeneration of electrospun poly(ε-caprolactone) grafts

As a result of thrombosis or intimal hyperplasia, synthetic artificial vascular grafts had a low success rate when they were used to replace small-diameter arteries (inner diameter < 6 mm). C-type natriuretic peptides (CNP) have anti-thrombotic effects, and can promote endothelial cell (EC) proliferation and inhibit vascular smooth muscle cell (SMC) over-growth. In this study, poly(ε-caprolactone) (PCL) vascular grafts loaded with CNP (PCL-CNP) were constructed by electrospinning. The PCL-CNP grafts were able to continuously release CNP at least 25 days in vitro. The results of scanning electron microscopy (SEM) and mechanical testing showed that the loading of CNP did not change the microstructure and mechanical properties of the PCL grafts. In vitro blood compatibility analysis displayed that PCL-CNP grafts could inhibit thrombin activity and reduce platelet adhesion and activation. In vitro cell experiments demonstrated that PCL-CNP grafts activated ERK1/2 and Akt signaling in human umbilical vein endothelial cells (HUVECs), as well as increased cyclin D1 expression, enhanced proliferation and migration, and increased vascular endothelial growth factor (VEGF) secretion and nitric oxide (NO) production. The rabbit arteriovenous (AV)-shunt ex vitro indicated that CNP loading significantly improved the antithrombogenicity of PCL grafts. The assessment of vascular grafts in rat abdominal aorta implantation model displayed that PCL-CNP grafts promoted the regeneration of ECs and contractile SMCs, modulated macrophage polarization toward M2 phenotype, and enhanced extracellular matrix remodeling. These findings confirmed for the first time that loading CNP is an effective approach to improve the hemocompatibility and vascular regeneration of synthetic vascular grafts. Statement of significanceSmall-diameter (< 6 mm) vascular grafts (SDVGs) have not been made clinically available due to their prevalence of thrombosis, limited endothelial regeneration and intimal hyperplasia. The incorporation of bioactive molecules into SDVGs serves as an effective solution to improve hemocompatibility and endothelialization. In this study, for the first time, we loaded C-type natriuretic peptides (CNP) into PCL grafts by electrospunning and confirmed the effectiveness of loading CNP on improving the hemocompatibility and vascular regeneration of artificial vascular grafts. Regenerative advantages included enhancement of endothelialization, modulation of macrophage polarization toward M2 phenotypes, and improved contractile smooth muscle cell regeneration. Our investigation brings attention to CNP as a valuable bioactive molecule for modifying cardiovascular biomaterial.

The Role of Gap Junctions in the Generation of Smooth Muscle Cells from Bone Marrow Mesenchymal Stem Cells.

Background Studies have shown that stem cell transplantation can improve smooth muscle cell (SMC) regeneration and remodelling. Gap junctions can enhance the cytoprotective effects of neighbouring cells. We investigated the effect of gap junctions on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into SMCs. Materials and Methods Rat BMSCs and SMCs were obtained from the bone marrow and bladder of Sprague-Dawley rats, respectively. Flow cytometry and multilineage differentiation were performed to assess the characteristics of these cells. BMSCs and SMCs were incubated together in cocultures in the presence and absence of heptanol, an uncoupler of gap junctions. Cocultures were divided into three groups consisting of a contact coculture, noncontact coculture, and contact coculture plus heptanol groups. The expression of BMSC-specific markers and the effect of gap junctions on the differentiation of BMSCs were evaluated by performing real-time reverse transcription-polymerase chain reaction, immunofluorescence analysis, and western blotting after cocultures. Results CD90 and CD44 were markedly expressed, and CD31 and CD45 were weakly or not expressed in BMSCs. The cells also showed good osteogenic and adipogenic differentiation ability. Compared with the noncontact coculture group, the SMC markers such as α-SMA, calponin, and connexin43 increased in the contact coculture group. The effect of contact in the coculture group was significantly weakened by heptanol. Conclusions The results suggested that gap junctions play an important role in the generation of SMCs from BMSCs. The formation of SMCs can potentially be used to repair the sphincter muscle of patients with stress urinary incontinence.

Oligoglycine and fluoropolymer functionalized enzyme-responsive gene delivery surface for rapid in situ endothelialization of vascular grafts

The treatment success of small-caliber artificial vascular grafts is seriously limited by high thrombogenesis and low endothelialization. Gene transfection can effectively enhance the proliferation and migration of endothelial cells (ECs) and accelerating the formation of endothelium layer. How to efficiently and temporarily immobilize the gene complexes on the material surface, how to intelligently release them and effectively transfect ECs are still the challenges for in situ endothelialization. Herein, a matrix-metalloproteinase (MMP)-responsive gene delivery system is constructed for in situ smart release of genes from the graft surface. The fluorinated cationic polymer (F-ssPEI) is served as high-efficient and low-cytotoxic gene vector. The gene complexes are temporarily immobilized via a MMP-cleavable peptide (MCP) linker which contains a MMP-susceptible sequence as the principal part and oligoglycine sequence (Gm) with different units (m = 0, 4 and 8) as the flexible spacer arm. These gene delivery surfaces achieve the temporary immobilization, smart release and high internalization of gene complexes. The MCP(G8) group shows the highest transfection efficacy and the optimal HUVEC proliferation/migration/function. The enhanced endothelialization and contractile smooth muscle cell regeneration of grafts are achieved in vivo. The MMP-responsive gene delivery system provides an effective platform to promote regeneration of vascular graft.

Photooxidation and Pentagalloyl Glucose Cross-Linking Improves the Performance of Decellularized Small-Diameter Vascular Xenograft In Vivo.

Small-diameter vascular grafts have a significant need in peripheral vascular surgery and procedures of coronary artery bypass graft (CABG); however, autografts are not always available, synthetic grafts perform poorly, and allografts and xenografts dilate, calcify, and induce inflammation after implantation. We hypothesized that cross-linking of decellularized xenogeneic vascular grafts would improve the mechanical properties and biocompatibility and reduce inflammation, degradation, and calcification in vivo. To test this hypothesis, the bovine internal mammary artery (BIMA) was decellularized by detergents and ribozymes with sonication and perfusion. Photooxidation and pentagalloyl glucose (PGG) were used to cross-link the collagen and elastin fibers of decellularized xenografts. Modified grafts’ characteristics and biocompatibility were studied in vitro and in vivo; the grafts were implanted as transposition grafts in the subcutaneous of rats and the abdominal aorta of rabbits. The decellularized grafts were cross-linked by photooxidation and PGG, which improved the grafts’ biomechanical properties and biocompatibility, prevented elastic fibers from early degradation, and reduced inflammation and calcification in vivo. Short-term aortic implants in the rabbits showed collagen regeneration and differentiation of host smooth muscle cells. No occlusion and stenosis occurred due to remodeling and stabilization of the neointima. A good patency rate (100%) was maintained. Notably, implantation of non-treated grafts exhibited marked thrombosis, an inflammatory response, calcification, and elastin degeneration. Thus, photooxidation and PGG cross-linking are potential tools for improving grafts’ biological performance within decellularized small-diameter vascular xenografts.

Degeneration and Regeneration of Smooth Muscle Cells in Two Different Abdominal Aortic Aneurysm Models in Rabbits

We aimed to investigate the formation and self-healing process of rabbit abdominal aortic aneurysm (AAA) by focus on the degeneration and regeneration of smooth muscle cells (SMCs) in elastase-induced AAA model and enlarging AAA model in rabbits. Sixty rabbits were equally divided into 2 aneurysm groups (Group A and Group B). Rabbits received a 10-min incubation of elastase in Group A (10 units/µL) and Group B (1 unit/µL). Rabbits underwent aortic stenosis above the incubated segment in Group B. Aortic diameter was measured and rabbits were sacrificed for histopathological and immunohistochemical studies. The incubated aorta dilated immediately and ran up to maxima by day 21 in Group A. All aneurysms formed by day 21 and enlarged progressively in Group B. SMCs content, elastin content and intima-media thickness decreased significantly by day 0 in Group A. SMCs and elastic fibers were destroyed gradually in Group B, however, SMCs content was significantly lower than Group A by day 70. Intimal thickness increased significantly by day 70 in the Aneurysm groups. MMP2 maintained moderate expression in Group A, which decreased significantly by day 3 in Group B. MMP9 and RAM11 expressions were higher by day 1, but decreased significantly by day 3 in Group B. Irreversible degeneration of SMCs is critical to a rapid formation of elastase-induced rabbit AAA model, and SMCs excessive regeneration accounts for the selfhealing process. SMCs degradation and regeneration remain relatively stable in an enlarging AAA model. SMCs should be the key target for studying the mechanism of AAA and intervention therapy.

Resident CD34-positive cells contribute to peri-endothelial cells and vascular morphogenesis in salivary gland after irradiation

Salivary gland (SG) hypofunction is a common post-radiotherapy complication. Besides the parenchymal damage after irradiation (IR), there are also effects on mesenchymal stem cells (MSCs) which were shown to contribute to regeneration and repair of damaged tissues by differentiating into stromal cell types or releasing vesicles and soluble factors supporting the healing processes. However, there are no adequate reports about their roles during SG damage and regeneration so far. Using an irradiated SG mouse model, we performed certain immunostainings on tissue sections of submandibular glands at different time points after IR. Immunostaining for CD31 revealed that already one day after IR, vascular impairment was induced at the level of capillaries. In addition, the expression of CD44—a marker of acinar cells—diminished gradually after IR and, by 20 weeks, almost disappeared. In contrast, the number of CD34-positive cells significantly increased 4 weeks after IR and some of the CD34-positive cells were found to reside within the adventitia of arteries and veins. Laser confocal microscopic analyses revealed an accumulation of CD34-positive cells within the area of damaged capillaries where they were in close contact to the CD31-positive endothelial cells. At 4 weeks after IR, a fraction of the CD34-positive cells underwent differentiation into α-SMA-positive cells, which suggests that they may contribute to regeneration of smooth muscle cells and/or pericytes covering the small vessels from the outside. In conclusion, SG-resident CD34-positive cells represent a population of progenitors that could contribute to new vessel formation and/or remodeling of the pre-existing vessels after IR and thus, might be an important player during SG tissue healing.

ADAMTS-5 Decreases in Aortas and Plasma From Aortic Dissection Patients and Alleviates Angiotensin II-Induced Smooth Muscle-Cell Apoptosis.

Background: Acute aortic dissection (AAD) is associated with degeneration of the aortic media and accompanied by vascular extracellular matrix (ECM) remodeling. Recently, a disintegrin and metalloproteinase with thrombospondin type 1 motifs-5 (ADAMTS-5) has been reported to be involved in ECM remodeling and vascular diseases. The aim of this study was to examine ADAMTS-5 levels in AAD patients and investigate the underlying mechanisms.Methods: Aortic tissue samples were collected from normal donors and AAD patients, and the expression of ADAMTS-5 was analyzed in all aortic tissues. In addition, plasma levels of ADAMTS-5, matrix metalloproteinase (MMP)-2 and MMP-9, and tumor necrosis factor-α (TNF-α) were measured in repeated samples from AAD patients and compared to the non-AAD (NAD) group. In addition, we investigated the effects of ADAMTS-5 in smooth muscle cell (SMC) apoptosis.Results: The results showed that ADAMTS-5 expression was significantly reduced in the aortas of AAD patients and that SMCs were the main source of ADAMTS-5. In addition, the plasma ADAMTS-5 level was lower, but plasma MMP-2, MMP-9, and TNF-α levels were increased in the AAD patients. Multivariate linear regression analyses showed that a decreased ADAMTS-5 level in patients was independently associated with an increased risk of AAD. Furthermore, recombinant human ADAMTS-5 significantly ameliorated angiotensin (Ang II)-evoked SMC apoptosis.Conclusions: ADAMTS-5 shows promise as a novel potential biomarker for AAD, and regulation of SMC is a possible mechanism for the effects of ADAMTS-5.

Dynamic Changes in Erectile Function and Histological Architecture After Intracorporal Injection of Human Placental Stem Cells in a Pelvic Neurovascular Injury Rat Model

IntroductionThe human placenta provides a bountiful and noncontroversial source of stem cells which have the potential for regeneration of injured tissue. These cells may restore erectile function after neurovascular tissue injury such as that seen in radical pelvic surgeries and pelvic trauma. AimTo determine the effect of human placenta–derived stem cells on erectile function recovery and histological changes at various time points in a cavernous nerve injury rat model and to study the fate of injected stem cells throughout the regenerative process. MethodsHuman placental stem cells (PSCs) were dual labeled with monomeric Katushka far red fluorescent protein (mKATE)-renLUC using a lentivirus vector. A pelvic neurovascular injury–induced erectile dysfunction model was established in male, athymic rats by crushing the cavernous nerves and ligating the internal pudendal neurovascular bundles, bilaterally. At the time of defect creation, nonlabeled PSCs were injected into the corpus cavernosum at a concentration of 2.5 × 106 cells/0.2 mL. The phosphate-buffered saline–treated group served as the negative control group, and age-matched rats (age-matched controls) were used as the control group. Erectile function, histomorphological analyses, and Western blot were assessed at 1, 6, and 12 weeks after model creation. The distribution of implanted, dual-labeled PSCs was monitored using an in vivo imaging system (IVIS). Implanted cells were further tracked by detection of mKATE fluorescence in histological sections. Main Outcome MeasureThe main outcome measure includes intracavernous pressure/mean arterial pressure ratio, neural, endothelial, smooth muscle cell regeneration, mKATE fluorescence, and IVIS imaging. ResultsThe ratio of intracavernous pressure to mean arterial pressure significantly increased in PSC-injected rats compared with phosphate-buffered saline controls (P < 0.05) at the 6- and 12-week time points, reaching 72% and 68% of the age-matched control group, respectively. Immunofluorescence staining and Western blot analysis showed significant increases in markers of neurons (84.3%), endothelial cells (70.2%), and smooth muscle cells (70.3%) by 6 weeks in treatment groups compared with negative controls. These results were maintained through 12 weeks. IVIS analysis showed luminescence of implanted PSCs in the injected corpora immediately after injection and migration of cells to the sites of injury, including the incision site and periprostatic vasculature by day 1. mKATE fluorescence data revealed the presence of PSCs in the penile corpora and major pelvic ganglion at 1 and 3 days postoperatively. At 7 days, immunofluorescence of penile PSCs had disappeared and was diminished in the major pelvic ganglion. Clinical ImplicationsPlacenta-derived stem cells may represent a future “off-the-shelf” treatment to mitigate against development of erectile dysfunction after radical prostatectomy or other forms of pelvic injury. Strength & LimitationsSingle dose injection of PSCs after injury resulted in maximal functional recovery and tissue regeneration at 6 weeks, and the results were maintained through 12 weeks. Strategies to optimize adult stem cell therapy might achieve more effective outcomes for human clinical trials. ConclusionHuman PSC therapy effectively restores the erectile tissue and function in this animal model. Thus, PSC therapy may provide an attractive modality to lessen the incidence of erectile dysfunction after pelvic neurovascular injury. Further improvement in tissue regeneration and functional recovery may be possible using multiple injections or systemic introduction of stem cells.Gu X, Thakker PU, Matz EL, et al. Dynamic Changes in Erectile Function and Histological Architecture After Intracorporal Injection of Human Placental Stem Cells in a Pelvic Neurovascular Injury Rat Model. J Sex Med 2020;17:400–411.

Tethering transforming growth factor β1 to soft hydrogels guides vascular smooth muscle commitment from human mesenchymal stem cells

Mesenchymal stem cells (MSCs) hold great promise for vascular smooth muscle regeneration. However, most studies have mainly relied on extended supplementation of sophisticated biochemical regimen to drive MSC differentiation towards vascular smooth muscle cells (vSMCs). Herein we demonstrate a concomitant method that exploits the advantages of biomimetic matrix stiffness and tethered transforming growth factor β1 (TGF-β1) to guide vSMC commitment from human MSCs. Our designed poly(ethylene glycol) hydrogels, presenting a biomimetic stiffness and tethered TGF-β1, provide an instructive environment to potently upregulate smooth muscle marker expression in vitro and in vivo. Importantly, it significantly enhances the functional contractility of vSMCs derived from MSCs within 3 days. Interestingly, compared to non-tethered one, tethered TGF-β1 enhanced the potency of vSMC commitment on hydrogels. We provide compelling evidence that combining stiffness and tethered TGF-β1 on poly(ethylene glycol) hydrogels can be a promising approach to drastically enhance maturation and function of vSMCs from stem cell differentiation in vitro and in vivo. Statement of SignificanceA fast, reliable and safe regeneration of vascular smooth muscle cells (vSMCs) from stem cell differentiation is promising for vascular tissue engineering and regenerative medicine applications, but remains challenging. Herein, a photo-click hydrogel platform is devised to recapitulate the stiffness of vascular tissue and appropriate presentation of transforming growth factor β1 (TGF-β1) to guide vSMC commitment from mesenchymal stem cells (MSCs). We demonstrate that such concomitant method drastically enhanced regeneration of mature, functional vSMCs from MSCs in vitro and in vivo within only a 3-days span. This work is not only of fundamental scientific importance, revealing how physiochemical factors and the manner of their presentation direct stem cell differentiation, but also attacks the long-standing difficulty in regenerating highly functional vSMCs within a short period.

Polycaprolactone/gelatin degradable vascular grafts simulating endothelium functions modified by nitric oxide generation.

Aim: Host remolding with scaffolds degradation and rapid formation of a complete endothelium, are prospective solutions for improving performance of small diameter vascular grafts. Materials & methods: For this purpose, microfibrous polycaprolactone (PCL)/gelatin scaffolds were prepared by electrospinning and subsequently functionalized with heparin and organoselenium-immobilized polyethyleneimine for nitric oxide generation through layer-by-layer self-assembly. Results: Our results showed that modified PCL/gelatin grafts had strong catalytic nitric oxide generation capacity and facilitated the enhanced attachment of endothelial cells, compared with control scaffold groups. Meanwhile, the modified grafts exhibited good hemocombatility, rapid endothelialization and smooth muscle cell regeneration. Conclusion: Modification of biodegradable scaffolds, proposed in this work, could enhance biological functions of vascular grafts and provides new strategies for the construction of small diameter vascular grafts.

Hypoxia-Preconditioned Adipose-Derived Endothelial Progenitor Cells Promote Bladder Augmentation.

Tissue engineering technology provides an alternative for bladder reconstruction, which remains confronted with challenges, such as insufficient vascularization in regenerated bladder tissue. Short-term hypoxic preconditioning has been reported to be an effective method to enhance the angiogenic effect of stem cells. This study evaluated the effect and mechanism of hypoxia-preconditioned adipose-derived endothelial progenitor cells (hp-ADEPCs) on the vascularization and smooth muscle regeneration of tissue-engineered bladders. Rats were randomly divided into the following four groups: hp-ADEPC, ADEPC, bladder acellular matrix (BAM), and cystotomy groups. A partial cystectomy was performed to remove 50% of the bladder, which was augmented with hp-ADEPC-BAM, ADEPC-BAM, or BAM. Histological and functional assessments of the engineered neobladder were performed at 1 and 3 months after surgery, while the mechanism of hp-ADEPCs on vascularization was also investigated. Immunohistochemical analysis revealed that hp-ADEPC-BAM significantly promoted urothelium, blood vessel, smooth muscle, and nerve cell regeneration. Animals in the hp-ADEPC-BAM group exhibited higher bladder compliance and a relatively normal micturition pattern compared with the ADEPC-BAM and BAM groups. In addition, compared with ADEPCs, hp-ADEPCs promoted ERK phosphorylation activation and hypoxia-inducible factor-1α expression, thereby secreting more vascular endothelial growth factor and basic fibroblast growth factor and significantly enhancing the migration and angiogenesis abilities of rat endothelial cells. This is the first study to demonstrate that a combination of ADEPCs and BAM with short-term hypoxic preconditioning enhances angiogenesis and promotes the functional recovery of tissue-engineered bladders. Impact Statement Insufficient vascularization in regenerated bladder tissue remains a challenge for bladder tissue engineering. We successfully isolated adipose-derived endothelial progenitor cells (ADEPCs) with high proliferative potential and angiogenic properties. Hypoxic preconditioning was confirmed to effectively enhance stem cell activity. In this study, a porcine bladder acellular matrix (BAM) was established, and hypoxia-preconditioned autologous ADEPCs were simultaneously introduced for bladder reconstruction in a rat model, followed by an assessment of their feasibility and potential for bladder vascularization. For the first time, we demonstrated that hypoxic preconditioning of ADEPCs improves angiogenesis and the functional recovery of tissue-engineered bladders.

Optimization of Electrospun Poly(caprolactone) Fiber Diameter for Vascular Scaffolds to Maximize Smooth Muscle Cell Infiltration and Phenotype Modulation.

Due to the morphological resemblance between the electrospun nanofibers and extracellular matrix (ECM), electrospun fibers have been widely used to fabricate scaffolds for tissue regeneration. Relationships between scaffold morphologies and cells are cell type dependent. In this study, we sought to determine an optimum electrospun fiber diameter for human vascular smooth muscle cell (VSMC) regeneration in vascular scaffolds. Scaffolds were produced using poly(caprolactone) (PCL) electrospun fiber diameters of 0.5, 0.7, 1, 2, 2.5, 5, 7 or 10 μm, and VSMC survivals, proliferations, infiltrations, and phenotypes were recorded after culturing cells on these scaffolds for one, four, seven, or 10 days. VSMC phenotypes and macrophage infiltrations into scaffolds were evaluated by implanting scaffolds subcutaneously in a mouse for seven, 14, or 28 days. We found that human VSMC survival was not dependent on the electrospun fiber diameter. In summary, increasing fiber diameter reduced VSMC proliferation, increased VSMC infiltration and increased macrophage infiltration and activation. Our results indicate that electrospun PCL fiber diameters of 7 or 10 µm are optimum in terms of VSMC infiltration and macrophage infiltration and activation, albeit at the expense of VSMC proliferation.

Bladder smooth muscle cell differentiation of the human induced pluripotent stem cells on electrospun Poly(lactide-co-glycolide) nanofibrous structure

Smooth muscle cell (SMC) regeneration plays an important role in retrieving the bladder-wall functionality and it can be achieved by a proper cell-co-polymer constructed by tissue engineering. Human induced pluripotent stem cells (iPSCs), which can be specifically prepared for the patient, was considered as cells in this study, and Poly(lactide-co-glycolide) (PLGA) as a most interesting polymer in biomedical applications was applied to the scaffold fabrication by electrospinning. After scaffold characterization, SMC differentiation potential of the human iPSCs was investigated while cultured on the PLGA nanofibrous scaffold by evaluation of the SMC related important gene and protein markers. Alpha-smooth muscle actin (ASMA), Smooth muscle 22 alpha (SM-22a) as two early SMC markers were significantly up regulated either two and three weeks after differentiation induction in human iPSCs cultured on PLGA compared to those cells cultured on the tissue culture polystyrene (TCPS). But Calponin-1, Caldesmon1 and myosin heavy chain (MHC) expression differences in human iPSCs cultured on PLGA and TCPS were significant only three weeks after differentiation induction based on its lately expression in the differentiation process. ASMA and MHC proteins were also considered for evaluation by immunocytochemistry on differentiated iPSCs whereas results showed higher expression of these proteins in stem cells grown on PLGA compared to the TCPS. According to the results, human iPSCs demonstrated a great SMC differentiation potential when grown on PLGA and it could be considered as a promising cell-co-polymer for use in bladder tissue engineering.