Adventitial delivery of miR-145 to treat intimal hyperplasia post vascular injuries through injectable and in-situ self-assembling peptide hydrogels.

Intimal hyperplasia is a common lesion that can be observed in diverse vascular diseases. Drug-eluting stents and drug-coated balloons, which can release anti-proliferative agents to inhibit smooth muscle cell (SMC) proliferation, are developed to prevent intimal hyperplasia. However, these intervention devices still cannot achieve satisfactory clinical outcomes. In contrast to endovascular drug delivery, vascular adventitial drug delivery is a new strategy. To develop a vascular adventitial drug delivery system to treat intimal hyperplasia post vascular injuries, we loaded miR-145-5p-agomir (miR-145) into an injectable and in-situ self-assembling RAD peptide hydrogel. In vitro data showed that the miR-145 could be well incorporated into the RAD peptide hydrogels and released in a slow and controlled manner. The released miR-145 could transfect SMCs successfully, and the transfected SMCs exhibited a reduced migration capacity and higher expressions of SMC contractile biomarkers as compared to the non-transfected SMCs. In vivo data showed that the retention of the miR-145 was greatly elongated by the RAD peptide hydrogels. In addition, the application of the miR-145-loaded RAD peptide hydrogels surrounding injured arteries decreased the proliferative SMCs, promoted the regeneration of endothelium, reduced the macrophage infiltration, inhibited the neointimal formation and prevented adverse ECM remodeling via downregulation of KLF4 expression. The RAD peptide hydrogels loaded with miR-145 can successfully inhibit intimal hyperplasia after vascular injuries and thus hold great potential as an innovative extravascular drug delivery approach to treat vascular diseases. STATEMENT OF SIGNIFICANCE: Intimal hyperplasia is a common lesion that can be observed in diverse vascular diseases. Drug-eluting stents and drug-coated balloons, which can release anti-proliferative agents to inhibit smooth muscle cell (SMC) proliferation, are developed to prevent intimal hyperplasia. However, these intervention devices still cannot achieve satisfactory clinical outcomes. In contrast to endovascular drug delivery, vascular adventitial drug delivery is a new strategy. Our work here demonstrates that the RAD peptide hydrogels loaded with miR-145-5p-agomir (miR-145) can successfully reverse intimal hyperplasia after vascular injuries and thus hold great potential as an innovative vascular adventitial drug delivery approach to treat vascular diseases. Our work proposes a possible paradigm shift from endovascular drug delivery to extravascular drug delivery for vascular disorder treatment.

Polydopamine (PDA) coatings with endothelial vascular growth factor (VEGF) immobilization inhibiting neointimal formation post zinc (zn) wire implantation in rat aortas.

BACKGROUND: Bioresorbable stents are designed to provide temporary mechanical support to the coronary arteries and then slowly degrade in vivo to avoid chronic inflammation. Zinc (Zn) is a promising material for bioresorbable stents; However, it can cause inflammation and neointimal formation after being implanted into blood vessels. METHODS: To improve biocompatibility of Zn, we first coated it with polydopamine (PDA), followed by immobilization of endothelial vascular growth factor (VEGF) onto the PDA coatings. Adhesion, proliferation, and phenotype maintenance of endothelial cells (ECs) on the coated Zn were evaluated in vitro. Then, a wire aortic implantation model in rats mimicking endovascular stent implantation in humans was used to assess vascular responses to the coated Zn wires in vivo. Thrombosis in aortas post Zn wire implantation, degradation of Zn wires in vivo, neointimal formation surrounding Zn wires, and macrophage infiltration and extracellular matrix (ECM) remodeling in the neointimas were examined. RESULTS: In vitro data showed that the PDA-coated Zn encouraged EC adhesion, spreading, proliferation, and phenotype maintenance on its surfaces. VEGF functionalization on PDA coatings further enhanced the biocompatibility of Zn to ECs. Implantation of PDA-coated Zn wires into rat aortas didn't cause thrombosis and showed a faster blood flow than pure Zn or the Zn wires coated with VEGF alone. In addition, the PDA coating didn't affect the degradation of Zn wires in vivo. Besides, the PDA-coated Zn wires reduced neointimal formation, increased EC coverage, decreased macrophage infiltration, and declined aggrecan accumulation in ECM. VEGF immobilization onto PDA coatings didn't cause thrombosis and affect Zn degradation in vivo as well, and further increased the endothelization percentage as compared to PDA coating alone, thus resulting in thinner neointimas. CONCLUSION: These results indicate that PDA coatings with VEGF immobilization would be a promising approach to functionalize Zn surfaces to increase biocompatibility, reduce inflammation, and inhibit neointimal formation after Zn implantation in vivo.

A pDNA/rapamycin nanocomposite coating on interventional balloons for inhibiting neointimal hyperplasia.

Drug-coated balloon (DCB) is a therapeutic method that can effectively deliver antiproliferative drugs such as paclitaxel and rapamycin (RAPA) with no permanent implants left behind. However, delayed reendothelialization due to the toxicity of the delivered drugs leads to poor therapeutic effects. Here, we propose a new design of DCB coating, which incorporates both vascular endothelial growth factor (VEGF)-encoding plasmid DNA (pDNA) that can promote endothelial repair and RAPA into protamine sulfate (PrS). We demonstrate that the PrS/pDNA/RAPA coating had stability and good anticoagulation properties in vitro. We further show that the coating exhibited excellent transfer capacity from balloon substrates to vessel walls both in vitro and in vivo. Furthermore, the PrS/pDNA/RAPA coating effectively inhibited neointimal hyperplasia after balloon-induced vascular injuries through the down-regulation of the mammalian target of Rapamycin (mTOR) and promoted endothelium regeneration through increased expression of VEGF in vivo. These data indicate that our nanocomposite coating has great potential for use as a novel coating of DCB to treat neointimal hyperplasia after vascular injuries.

Blending with transition metals improves bioresorbable zinc as better medical implants.

Zinc (Zn) is a new class of bioresorbable metal that has potential for cardiovascular stent material, orthopedic implants, wound closure devices, etc. However, pure Zn is not ideal for these applications due to its low mechanical strength and localized degradation behavior. Alloying is the most common/effective way to overcome this limitation. Still, the choice of alloying element is crucial to ensure the resulting alloy possesses sufficient mechanical strength, suitable degradation rate, and acceptable biocompatibility. Hereby, we proposed to blend selective transition metals (i.e., vanadium-V, chromium-Cr, and zirconium-Zr) to improve Zn's properties. These selected transition metals have similar properties to Zn and thus are beneficial for the metallurgy process and mechanical property. Furthermore, the biosafety of these elements is of less concern as they all have been used as regulatory approved medical implants or a component of an implant such as Ti6Al4V, CoCr, or Zr-based dental implants. Our study showed the first evidence that blending with transition metals V, Cr, or Zr can improve Zn's properties as bioresorbable medical implants. In addition, three in vivo implantation models were explored in rats: subcutaneous, aorta, and femoral implantations, to target the potential clinical applications of bioresorbable Zn implants.

Inhibition of neointimal hyperplasia in balloon-induced vascular injuries in a rat model by miR-22 loading Laponite hydrogels.

Percutaneous coronary intervention (PCI) is the mainstream treatment to widen narrowed or obstructed coronary arteries due to pathological conditions. However, the post-operational neointimal hyperplasia occurs because of endothelium denudation during surgical procedures and the following inflammation. MicroRNAs (miRs) are new therapeutics of great potential for cardiovascular diseases. However, miRs easily degrade in vivo. A vehicle that can maintain their bioactivities and extend their retention at the site of delivery is prerequisite for miRs to play their roles as therapeutic reagents. Here, we reported the use of the Laponite hydrogels to deliver miR-22 that are modulators of phenotypes of smooth muscle cells (SMCs). The Laponite hydrogels allow a homogenous distribution of miR-22 within the gels, which had the capacity to transfect SMCs in vitro. Upon the injection of the miR-22 incorporated in the Laponite hydrogels in vivo, miR-22 could be well retained surrounding arteries for at least 7 days. Moreover, the miR-22 loading Laponite hydrogels inhibited the neointimal formation, reduced the infiltration of the macrophages, and reversed the adverse vascular ECM remodeling after the balloon-induced vascular injuries by upregulation of miR-22 and downregulation of its target genes methyl-CpG binding protein 2 (MECP2). The application of the Laponite hydrogels for miR local delivery may offer a novel strategy to treat cardiovascular diseases.

Association between electronic cigarettes use and whole blood cell among adults in the USA-a cross-sectional study of National Health and Nutrition Examination Survey analysis.

Electronic cigarettes (E-cigarettes) use is an emerging public health problem. Trying to assess the independent associations between E-cigarettes use and whole blood cell in a nationally representative sample of the US adults is very important for the smoking population. Using E-cigarettes data from NHANES (National Health and Nutrition Examination Survey) 2013-2018, 17,180 adults were included in this cross-sectional analysis. All participants were stratified into four different groups (non-smoke group N=10087, E-cigarettes group N=52, dual-smoke group N=249, cigarettes group N=6792) based on questions SMQ020 (smoked at least 100 cigarettes in life) and SMQ690H (used last 5 days E-cigarettes). Whole blood cell tests included white blood cell (WBC) with differentials, red blood cell (RBC) with characteristics, and platelet variables. With adjusted by age, gender, and race ethnicity, multivariate logistic regression analyses were used to assess independent associations between E-cigarettes group and other groups for different whole blood cell variables. A total of 17,180 participants were included in the study; 47.9% were males, with a mean age of 46.99 (±0.29). In WBC-related variables, non-smoke group had the lowest value in WBC counts (7.15±0.05), lymphocyte (2.15±0.02), and monocyte (0.57±0.01), among the four different groups. In RBC-related variables, non-smoke group had the lowest value in mean cell volume (MCV, 88.46±0.14, p<0.05) and mean cell hemoglobin (MCH, 29.73±0.06, p<0.05), among the four different groups. In adjusted analysis, WBC (OR = 0.97, 95% CI: 0.96-0.98, p<0.001), especially lymphocyte (OR = 0.97, 95% CI: 0.96-0.98, p<0.001) and monocyte (OR = 0.11, 95% CI: 0.02-0.66, p<0.001) of non-smoke group, showed negative significant effect for E-cigarettes group. Meanwhile, lower odds of MCV (OR = 0.91, 95% CI: 0.81-1.04, p<0.05) and MCH (OR = 0.81, 95% CI: 0.65-1.00, p<0.05) in non-smoke group were observed compared to E-cigarettes group. Conversely, for dual-smoke group and cigarette group, there was no significant results in all whole blood cell variables compared to E-cigarettes group. E-cigarettes use might be associated with a systemic response that could lead to an increase in WBC, especially lymphocytes and monocytes, in the US adults. Meanwhile, the properties of RBC might also be influenced simultaneously; MCV and MCH in E-cigarettes population were bigger than the non-smoke population.

Mir-22-incorporated polyelectrolyte coating prevents intima hyperplasia after balloon-induced vascular injury.

Drug-coated balloons (DCBs) offer potential to deliver drugs to treat coronary lesions but without leaving permanent implants behind. Paclitaxel and sirolimus are anti-proliferation drugs that are commonly used in commercially available DCBs. However, these drugs present significant cytotoxicity concern and low efficacy in vivo. Here, we use microRNA-22 (miR-22) as balloon loaded drugs and polyelectrolyte complexes (PECs) polyethyleneimine/polyacrylic acid (PEI/PAA) as balloon coatings to establish a new DCB system through the ultrasonic spray method. The PEI/PAA forms a stable and thin coating on the balloon, which resulted in a good transfer capacity to the vessel wall both in vitro and in vivo. miR-22 that could modulate smooth muscle cell (SMC) phenotype switching is incorporated into the PEI/PAA coating and shows a sustained release profile. The PEI/PAA/miR-22 coated balloon successfully inhibits intima hyperplasia after balloon-induced vascular injury in a rat model through decreasing proliferative SMCs via the miR-22-methyl-CpG binding protein 2 (MECP2) axis. Our findings indicate that balloons coated with PEI/PAA/miR-22 have great potential to be promising DCBs in the treatment of cardiovascular disease.

Improved mechanical, degradation, and biological performances of Zn-Fe alloys as bioresorbable implants.

Zinc (Zn) is a promising bioresorbable implant material with more moderate degradation rate compared to magnesium (Mg) and iron (Fe). However, the low mechanical strength and localized degradation behavior of pure Zn limit its clinical applications. Alloying is one of the most effective ways to overcome these limitations. After screening the alloying element candidates regarding their potentials for improvement on the degradation and biocompatibility, we proposed Fe as the alloying element for Zn, and investigated the in vitro and in vivo performances of these alloys in both subcutaneous and femoral tissues. Results showed that the uniformly distributed secondary phase in Zn-Fe alloys significantly improved the mechanical property and facilitated uniform degradation, which thus enhanced their biocompatibility, especially the Zn-0.4Fe alloy. Moreover, these Zn-Fe alloys showed outstanding antibacterial property. Taken together, Zn-Fe alloys could be promising candidates as bioresorbable medical implants for various cardiovascular, wound closure, and orthopedic applications.

Thick PCL Fibers Improving Host Remodeling of PGS-PCL Composite Grafts Implanted in Rat Common Carotid Arteries.

Vasculopathy and the consequential ischemia are major medical challenges. Grafting is an effective treatment to vascular occlusion. However, autologous grafting, despite scarcity, is the only choice for small diameter blood vessels. Synthetic grafts can fill the gap if they can work satisfactorily in arterial circulation. Electrospun polycaprolactone (PCL) sheathed porous poly(glycerol sebacate) (PGS) vascular grafts have good performances in arterial circulation in abdominal aortas and carotid arteries in rats. However, a major issue associated with the graft remodeling in vivo is limited neo-tissue formation inside PCL sheaths. Small pores of PCL sheaths inhibit cell infiltration and migration. To increase porosity of PCL sheaths of PGS-PCL composite grafts, diameters of electrospun PCL fibers are increased. The thick PCL fibers encourage cell migration and elicit a higher degree of CD206+ cells. In addition, some of the CD206+ cells co-express vascular cell markers in the thick-fiber grafts. The thick-fiber grafts also show improved mechanical properties and a higher elastin and collagen content. The data demonstrate the feasibility of improving graft vascular remodeling by increasing PCL fiber diameters and the critical role of CD206+ cells during graft vascular remodeling.

Slow degrading poly(glycerol sebacate) derivatives improve vascular graft remodeling in a rat carotid artery interposition model.

Porous synthetic grafts made of poly (glycerol sebacate) (PGS) can transform into autologous vascular conduits in vivo upon degradation of PGS. A long-held doctrine in tissue engineering is the necessity to match degradation of the scaffolds to tissue regeneration. Here, we tested the impact of degradation of PGS and its derivative in an interposition model of rat common carotid artery (CCA). Previous work indicates a complete degradation of PGS within approximately 2 weeks, likely at the fast end of the spectrum. Thus, the derivation of PGS focuses on delay degradation by conjugating the free hydroxy groups in PGS with a long chain carboxylic acid: palmitic acid, one of the most common lipid components. We evaluated two of the resultant palmitate-PGS (PPGS) in this study: one containing 9% palmitate (9-PPGS) and the other16% palmitate (16-PPGS). 16-PPGS grafts had the highest patency. Ultrasound imaging showed that the lumens of 16-PPGS grafts were similar to CCA and smaller than 9-PPGS and PGS grafts 12 weeks post-operation. Immunohistological and histological examination showed an endothelialized lumens in all three types of grafts within 4 weeks. Inflammatory responses to 16-PPGS grafts were limited to the adventitial space in contrast to a more diffusive infiltration in 9-PPGS and PGS grafts in week 4. Examination of calponin+ and αSMA+ cells revealed that 16-PPGS grafts remodeled into a distinctive bi-layered wall, while the walls of 9-PPGS grafts and PGS grafts only had one thick layer of smooth muscle-like cells. Correspondingly, the expression of collagen III and elastin displayed an identical layered structure in the remodeled 16-PPGS grafts, in contrast to a more spread distribution in 9-PPGS and PGS grafts. All the three types of grafts exhibited the same collagen content and burst pressure after 12 weeks of host remodeling. However, the compliance and elastin content of 16-PPGS grafts in week 12 were closest to those of CCA. Overall, placing the degradation of PGS derived elastomer to a window of 4-12 weeks results in vascular conduits closer to arteries in a rat carotid artery interposition model over a 12-week observation period.

Evolution of metallic cardiovascular stent materials: A comparative study among stainless steel, magnesium and zinc.

A cardiovascular stent is a small mesh tube that expands a narrowed or blocked coronary artery. Unfortunately, current stents, regardless metallic or polymeric, still largely fall short to the ideal clinical needs due to late restenosis, thrombosis and other clinical complications. Nonetheless, metallic stents are preferred clinically thanks to their superior mechanical property and radiopacity to their polymeric counterparts. The emergence of bioresorbable metals opens a window for better stent materials as they may have the potential to reduce or eliminate late restenosis and thrombosis. In fact, some bioresorbable magnesium (Mg)-based stents have obtained regulatory approval or under trials with mixed clinical outcomes. Some major issues with Mg include the too rapid degradation rate and late restenosis. To mitigate these problems, bioresorbable zinc (Zn)-based stent materials are being developed lately with the more suitable degradation rate and better biocompatibility. The past decades have witnessed the unprecedented evolution of metallic stent materials from first generation represented by stainless steel (SS), to second generation represented by Mg, and to third generation represented by Zn. To further elucidate their pros and cons as metallic stent materials, we systematically evaluated their performances in vitro and in vivo through direct side-by-side comparisons. Our results demonstrated that tailored Zn-based material with proper configurations could be a promising candidate for a better stent material in the future.

Co-culture of human umbilical vein endothelial cells and human bone marrow stromal cells into a micro-cavitary gelatin-methacrylate hydrogel system to enhance angiogenesis.

Vascular tissue engineering seeks to develop functional blood vessels that comprise of both endothelial cells and pericytes for translational medicine and is often faced with numerous challenges such as nutrients and wastes diffusion problem in the centre of the scaffolds. Various strategies have been adopted to solve the diffusion problem in thick engineered scaffolds. Typically, microchannels or dissolvable microspheres are introduced into three-dimensional (3D) scaffolds as an alternative way to improve the infiltration of scaffolds and endothelial cells are usually incorporated into the biomaterials. While some research groups now focus on finding supporting cells to build further vascularized structures in the scaffolds. In this study, a bioinspired 3D gelatin-methacrylate (Gel-MA) hydrogel with dissolvable microspheres was created to encapsulate human bone marrow stromal cells (HMSCs) and human umbilical vein endothelial cells (HUVECs) which was used to investigate whether HMSCs could play a pericytes-like role and enhance vascularization within the engineered scaffolds. The results showed co-culture of HMSCs and HUVECs demonstrated significantly improved vascularization when compared to either HUVECs or HMSCs monoculture. Angiogenic genes were expressed significantly higher in co-culture group. Moreover, when implanting the pre-vascularized scaffolds in vivo, co-culture system integrated more successfully with host tissue and showed higher host tissue invasion than any other groups. More importantly, both the qPCR and immunofluorescence results indicated MSCs differentiated towards pericytes to enhance vascularization in this study. This paper highlights the enhanced capability of 3D micro-cavitary Gel-MA hydrogel for co-culturing HUVECs and HMSCs to promote vascularization which presents a potential strategy for future tissue repair and regeneration.

Mechanical Strength, Biodegradation, and in Vitro and in Vivo Biocompatibility of Zn Biomaterials.

Zn-based biomaterials have emerged as promising new types of bioresorbable metallics applicable to orthopedic devices, cardiovascular stents, and other medical applications recently. Compared to other degradable metallic biomaterials (i.e., Mg- or Fe-based), Zn biomaterials have a more appropriate corrosion rate without hydrogen gas evolution. Here, we evaluated the potential of Zn-based metallics as medical implants, both in vitro and in vivo, alongside a standard benchmark Mg alloy, AZ31. The mechanical properties of the pure Zn were not strong enough but were significantly enhanced (microhardness > 70 kg/mm2, strength > 220 MPa, elongation > 15%) after alloying with Sr or Mg (1.5 at. %), surpassing the minimal design criteria for load-bearing device applications. The corrosion rate of Zn-based biomaterials was about 0.4 mm/year, significantly slower than that of AZ31. The measured cell viability and proliferation of three different human primary cells fared better for Zn-based biomaterials than AZ31 using both direct and indirect culture methods. Platelet adhesion and activation on Zn-based materials were minimal, significantly less than on AZ31. The hemolysis ratio of red cells (<0.5%) after incubation with Zn-based materials was also well below the ISO standard of 5%. Moreover, Zn-based biomaterials promoted stem cell differentiation to induce the extracellular matrix mineralization process. In addition, in vivo animal testing using subcutaneous, bone, and vascular implantations revealed that the acute toxicity and immune response of Zn-based biomaterials were minimal/moderate, comparable to that of AZ31. No extensive cell death and foreign body reactions were observed. Taken together, Zn-based biomaterials may have a great potential as promising candidates for medical implants.

In Situ Organ-Specific Vascularization in Tissue Engineering.

Other than a few avascular tissues, almost all human tissues are connected to the systemic circulation via blood vessels that promote metabolism and function. Accordingly, engineered vascularization is a vital goal in tissue engineering for regenerative medicine. Endothelial cells (ECs) play a central role in vascularization with two significant specificities: physical interfaces between vascular stroma and blood, and phenotypic organ-specificity. Biomaterial scaffolding technologies that address these unique properties of ECs have been developed to promote the vascularization of various engineered tissues, and these have advanced from mimicking vascular architectures ex situ towards promoting spontaneous angiogenic remodeling in situ. Simultaneously, endothelial progenitor cells (EPCs) and organ-specific ECs are attracting more and more attention with the increasing awareness of the diversity of ECs in different organs.

Respective Effects of Gelatin-Coated Polydimethylsiloxane (PDMS) Substrates on Self-renewal and Cardiac Differentiation of Induced Pluripotent Stem Cells (iPSCs).

The effects of substrate stiffness on the development of cardiomyocytes have been investigated extensively. Polydimethylsiloxane (PDMS) elastomer is one of biomaterials that are commonly used to explore the effects of substrate compliance on stem cell differentiation. Although the effects of substrate stiffness on cardiac differentiation of pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have been reported, whether the stiffness of PDMS-based substrates could enhance differentiation of iPSCs toward cardiomyocyte lineage or not remains unknown. In this study, we found that a denser gelatin distribution and a higher gelatin adsorption on the stiffer PDMS. In addition, nanotopographies on PDMS substrates with different stiffness were distinct. iPSCs on the stiffer PDMS substrates showed higher pluripotency marker but lower cardiac gene expressions. In contrast, iPSCs on the softer PDMS substrates revealed lower pluripotency marker but higher cardiac gene expressions. These results indicate that stiffer PDMS substrates with gelatin coating could be used to support iPSC self-renewal and softer PDMS substrates coated with gelatin could be used for enhanced cardiac differentiation of iPSCs.

Improvement of endothelial progenitor outgrowth cell (EPOC)-mediated vascularization in gelatin-based hydrogels through pore size manipulation.

In addition to chemical compositions, physical properties of scaffolds, such as pore size, can also influence vascularization within the scaffolds. A larger pore has been shown to improve host vascular tissue invasion into scaffolds. However, the influence of pore sizes on vascularization by endothelial cells directly encapsulated in hydrogels remains unknown. In this study, micro-cavitary hydrogels with different pore sizes were created in gelatin-methacrylate hydrogels with dissolvable gelatin microspheres (MS) varying in sizes. The effect of pore sizes on vascular network formation by endothelial progenitor outgrowth cells (EPOCs) encapsulated in hydrogels was then investigated both in vitro and in vivo. When cultured in vitro, vascular networks were formed around pore structures in micro-cavitary hydrogels. The middle pore size supported best differentiation of EPOCs and thus best hydrogel vascularization in vitro. When implantation in vivo, functional connections between encapsulated EPOCs and host vasculature micro-cavitary hydrogels were established. Vascularization in vivo was promoted best in hydrogels with the large pore size due to the increased vascular tissue invasion. These results highlight the difference between in vitro and in vivo culture conditions and indicate that pore sizes shall be designed for in vitro and in vivo hydrogel vascularization respectively. Pore sizes for hydrogel vascularization in vitro shall be middle ones and pore sizes for hydrogel vascularization in vivo shall be large ones. STATEMENT OF SIGNIFICANCE: This study reveals that the optimal pore size for hydrogel vascularization in vitro and in vivo is different. The middle pore size supported best differentiation of EPOCs and thus best hydrogel vascularization in vitro, while vascularization in vivo was promoted best in hydrogels with the large pore size due to the increased vascular tissue invasion. These results highlight the difference between in vitro and in vivo culture conditions and indicate that pore sizes shall be designed for in vitro and in vivo hydrogel vascularization respectively. Pore sizes for hydrogel vascularization in vitro shall be middle ones and pore sizes for hydrogel vascularization in vivo shall be large ones.

Optimization of a polydopamine (PD)-based coating method and polydimethylsiloxane (PDMS) substrates for improved mouse embryonic stem cell (ESC) pluripotency maintenance and cardiac differentiation.

Myocardiocyte derived from pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), is a promising cell source for cardiac tissue engineering. Combined with microfluidic technologies, a heart-on-a-chip is very likely to be developed and function as a platform for high throughput drug screening. Polydimethylsiloxane (PDMS) silicone elastomer is a widely-used biomaterial for the investigation of cell-substrate interactions and biochip fabrication. However, the intrinsic PDMS surface hydrophobicity inhibits cell adhesion on the PDMS surface, and PDMS surface modification is required for effective cell adhesion. Meanwhile, the formulation of PDMS also affects the behaviors of the cells. To fabricate PDMS-based biochips for ESC pluripotency maintenance and cardiac differentiation, PDMS surface modification and formulation were optimized in this study. We found that a polydopamine (PD) with gelatin coating greatly improved the ESC adhesion, proliferation and cardiac differentiation on its surface. In addition, different PDMS substrates varied in their surface properties, which had different impacts on ESCs, with the 40 : 1 PDMS substrate being more favorable for ESC adhesion and proliferation as well as embryoid body (EB) attachment than the other PDMS substrates. Moreover, the ESC pluripotency was best maintained on the 5 : 1 PDMS substrate, while the cardiac differentiation of the ESCs was optimal on the 40 : 1 PDMS substrate. Based on the optimized coating method and PDMS formulation, biochips with two different designs were fabricated and evaluated. Compared to the single channels, the multiple channels on the biochips could provide larger areas and accommodate more nutrients to support improved ESC pluripotency maintenance and cardiac differentiation. These results may contribute to the development of a real heart-on-a-chip for high-throughput drug screening in the future.

Real-time and non-invasive monitoring of embryonic stem cell survival during the development of embryoid bodies with smart nanosensor.

Embryonic stem cells (ESCs)-derived embryoid body (EB) is a powerful model for the study of early embryonic development and the discovery of therapeutics for tissue regeneration. This article reports a smart nanosensor platform for labeling and tracking the survival and distribution of ESCs during the EB development in a real-time and non-invasive way. Compared with the cell tracker (i.e. DiO) and the green fluorescent protein (GFP), nanosensors provide the homogenous and highly-efficient ESC labeling. Following the internalization, intracellular nanosensors gradually release the non-fluorescent molecules that become fluorescent only in viable cells. This allows a continuous monitoring of ESC survival and distribution during the process of EB formation. Finally, we confirm that nanosensor labeling does not cause the significant influences to biological properties of the ESCs and EBs. STATEMENT OF SIGNIFICANCE: The distribution pattern of viable embryonic stem cells (ESCs) within embryoid body (EB) is closely related with the maturation of EBs. Noninvasive and real-time monitoring of viable ESC distribution in EBs would allow researchers to optimize the culturing condition in time during the EB development and to select the suitable EBs for subsequent applications.

Enhancing vascularization of a gelatin-based micro-cavitary hydrogel by increasing the density of the micro-cavities.

The transport of nutrients and oxygen by vascular networks into engineered tissue constructs is critical to their successful integration into host tissues. Hydrogel has achieved some promising results as scaffolds for vascularization. However, the vascularization of hydrogel is still constrained by its inherent submicron- or nano-sized pores. In this study, two gelatin-based micro-cavitary gel (Gel-MCG) constructs with varying densities of micro-cavities were developed with a photocrosslinkable gelatin methacrylate (Gel-MA) precursor and porogenic gelatin microspheres (MS), and their functions in supporting vascularization within hydrogels were evaluated with endothelial progenitor outgrowth cells (EPOCs). The increase of cavitary density could enhance the vascularization of Gel-MCG constructs. After 14 d of culture in vitro, the vascularization of Gel-MCG constructs with higher cavitary density was significantly superior to that of gelatin spongy control and the fusion of vascularized cavities in the constructs could be observed. Further subcutaneous implantation of the Gel-MCG constructs with higher cavitary density into nude mice also showed obvious vascular invasion from host tissues. Taken together, these results indicate that the increase in cavitary density can efficiently facilitate the vascularization of Gel-MCG constructs both in vitro and in vivo and that such highly-porous Gel-MCG constructs have great potential to be a promising scaffold for the development of vascularized tissue constructs.

Murine pluripotent stem cells derived scaffold-free cartilage grafts from a micro-cavitary hydrogel platform.

By means of appropriate cell type and scaffold, tissue-engineering approaches aim to construct grafts for cartilage repair. Pluripotent stem cells especially induced pluripotent stem cells (iPSCs) are of promising cell candidates due to the pluripotent plasticity and abundant cell source. We explored three dimensional (3D) culture and chondrogenesis of murine iPSCs (miPSCs) on an alginate-based micro-cavity hydrogel (MCG) platform in pursuit of fabricating synthetic-scaffold-free cartilage grafts. Murine embryonic stem cells (mESCs) were employed in parallel as the control. Chondrogenesis was fulfilled using a consecutive protocol via mesoderm differentiation followed by chondrogenic differentiation; subsequently, miPSC and mESC-seeded constructs were further respectively cultured in chondrocyte culture (CC) medium. Alginate phase in the constructs was then removed to generate a graft only comprised of induced chondrocytic cells and cartilaginous extracellular matrix (ECMs). We found that from the mESC-seeded constructs, formation of intact grafts could be achieved in greater sizes with relatively fewer chondrocytic cells and abundant ECMs; from miPSC-seeded constructs, relatively smaller sized cartilaginous grafts could be formed by cells with chondrocytic phenotype wrapped by abundant and better assembled collagen type II. This study demonstrated successful creation of pluripotent stem cells-derived cartilage/chondroid graft from a 3D MCG interim platform. By the support of materials and methodologies established from this study, particularly given the autologous availability of iPSCs, engineered autologous cartilage engraftment may be potentially fulfilled without relying on the limited and invasive autologous chondrocytes acquisition. STATEMENT OF SIGNIFICANCE: In this study, we explored chondrogenic differentiation of pluripotent stem cells on a 3D micro-cavitary hydrogel interim platform and creation of pluripotent stem cells-derived cartilage/chondroid graft via a consecutive procedure. Our results demonstrated chondrogenic differentiation could be realized on the platform via mesoderm differentiation. The mESCs/miPSCs derived chondrocytic cells were further cultured to finally generate a pluripotent stem cells-derived scaffold-free construct based on the micro-cavitary hydrogel platform, in which alginate hydrogel could be removed finally. Our results showed that miPSC-derived graft could be formed by cells with chondrocytic phenotype wrapped by abundant and assembled collagen type II. To our knowledge, this study is the first study that initials from pluripotent stem cell seeding on 3D scaffold environment and ends with a scaffold-free chondrogenic micro-tissue. By the support of materials and methodologies established from this study, engineered autologous iPSC-derived cartilage engraftment may be potentially developed instead of autologous chondrocytes grafts that have limited source.