Orthogonally oriented scaffolds with aligned fibers for engineering intestinal smooth muscle

Controlling cellular alignment is critical in engineering intestines with desired structure and function. Although previous studies have examined the directional alignment of cells on the surface (x–y plane) of parallel fibers, quantitative analysis of the cellular alignment inside implanted scaffolds with oriented fibers has not been reported. This study examined the cellular alignment in the x–z and y–z planes of scaffolds made with two layers of orthogonally oriented fibers. The cellular orientation inside implanted scaffolds was evaluated with immunofluorescence. Quantitative analysis of coherency between cell orientation and fiber direction confirmed that cells aligned along the fibers not only on the surface (x–y plane) but also inside the scaffolds (x–z & y–z planes). Our study demonstrated that two layers of orthogonally aligned scaffolds can generate the histological organization of cells similar to that of intestinal circular and longitudinal smooth muscle.     

甲壳素短纤维增强聚己内酯复合材料的制备及生物学评价

用共混法制备甲壳察短纤维增强聚己内酯复合材料，并对纯聚己内酯和甲壳察短纤维增强聚己内酯复合材料进行体外细胞毒性和生物相容性评价，为临床应用提供有价值的实验依据。对该两种材料进行体外细胞毒性试验、急性全身毒性试验、溶血试验、热源试验和过敏试验。结果显示受试材料最终细胞毒性级为0级，对细胞生长和增殖无明显抑制作用，材料中不存在潜在致敏性物质，浸提液无溶血反应和急性全身毒性反应，无热源反应，表明该复合材料具有良好的细胞和组织相容性，其作为胸壁缺损修补材料应用于临床具有可行性和安全性。     

Assessment of static and perfusion methods for decellularization of PCL membrane-supported periodontal ligament cell sheet constructs

ObjectivesDecellularization aims to harness the regenerative properties of native extracellular matrix. The objective of this study was to evaluate different methods of decellularization of periodontal ligament cell sheets whilst maintaining their structural and biological integrity.DesignHuman periodontal ligament cell sheets were placed onto melt electrospun polycaprolactone (PCL) membranes that reinforced the cell sheets during the various decellularization protocols. These cell sheet constructs (CSCs) were decellularized under static/perfusion conditions using a) 20 mM ammonium hydroxide (NH4OH)/Triton X-100, 0.5% v/v; and b) sodium dodecyl sulfate (SDS, 0.2% v/v), both +/− DNase besides Freeze–thaw (F/T) cycling method. CSCs were assessed using a collagen quantification assay, immunostaining and scanning electron microscopy. Residual fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were assessed with Bio-plex assays.ResultsDNA removal without DNase was higher under static conditions. However, after DNase treatment, there were no differences between the different decellularization methods with virtually 100% DNA removal. DNA elimination in F/T was less efficient even after DNase treatment. Collagen content was preserved with all techniques, except with SDS treatment. Structural integrity was preserved after NH4OH/Triton X-100 and F/T treatment, while SDS altered the extracellular matrix structure. Growth factor amounts were reduced after decellularization with all methods, with the greatest reduction (to virtually undetectable amounts) following SDS treatment, while NH4OH/Triton X-100 and DNase treatment resulted in approximately 10% retention.ConclusionsThis study showed that treatment with NH4OH/Triton X-100 and DNase solution was the most efficient method for DNA removal and the preservation of extracellular matrix integrity and growth factors retention.     

Polycaprolactone scaffold and reduced rhBMP-7 dose for the regeneration of critical-sized defects in sheep tibiae

The transplantation of autologous bone graft as a treatment for large bone defects has the limitation of harvesting co-morbidity and limited availability. This drives the orthopaedic research community to develop bone graft substitutes. Routinely, supra-physiological doses of bone morphogenetic proteins (BMPs) are applied perpetuating concerns over undesired side effects and cost of BMPs. We therefore aimed to design a composite scaffold that allows maintenance of protein bioactivity and enhances growth factor retention at the implantation site. Critical-sized defects in sheep tibiae were treated with the autograft and with two dosages of rhBMP-7, 3.5 mg and 1.75 mg, embedded in a slowly degradable medical grade poly(ε-caprolactone) (PCL) scaffold with β-tricalcium phosphate microparticles (mPCL–TCP). Specimens were characterised by biomechanical testing, microcomputed tomography and histology. Bridging was observed within 3 months for the autograft and both rhBMP-7 treatments. No significant difference was observed between the low and high rhBMP-7 dosages or between any of the rhBMP-7 groups and autograft implantation. Scaffolds alone did not induce comparable levels of bone formation compared to the autograft and rhBMP-7 groups. In summary, the mPCL–TCP scaffold with the lower rhBMP-7 dose led to equivalent results to autograft transplantation or the high BMP dosage. Our data suggest a promising clinical future for BMP application in scaffold-based bone tissue engineering, lowering and optimising the amount of required BMP.     

Enhanced antibacterial nanocomposite mats by coaxial electrospinning of polycaprolactone fibers loaded with Zn-based nanoparticles

ZnO and Zn acetate nanoparticles were embedded in polycaprolactone coaxial-fibers and uniaxial-fibers matrices to develop potential antibacterial nanocomposite wound dressings (mats). Morphology, composition, wettability, crystallinity and fiber structure of mats were characterized. Antibacterial properties of mats were tested against E. coli and S. aureus by turbidity and MTT assays. The effect of UVA illumination (prior to bacteria inoculation) on mats’ antibacterial activity was also studied. Results showed that a coaxial-fibers design maintained nanoparticles distributed in the outer-shell of fibers and, in general, enhanced the antibacterial effect of the mats, in comparison to conventional uniaxial-fibers mats. Results indicated that mats simultaneously inhibited planktonic and biofilm bacterial growth by, probably, two main antibacterial mechanisms; 1) release of Zn²⁺ ions (mainly from Zn acetate nanoparticles) and 2) photocatalytic oxidative processes exerted by ZnO nanoparticles. Antibacterial properties of mats were significantly improved by coaxial-fibers design and exposure to UVA-light prior to bacteria inoculation.     

Polymeric stent materials dysregulate macrophage and endothelial cell functions: Implications for coronary artery stent

Background

Biodegradable polymers have been applied as bulk or coating materials for coronary artery stents. The degradation of polymers, however, could induce endothelial dysfunction and aggravate neointimal formation. Here we use polymeric microparticles to simulate and demonstrate the effects of degraded stent materials on phagocytic activity, cell death and dysfunction of macrophages and endothelial cells.

Methods

Microparticles made of low molecular weight polyesters were incubated with human macrophages and coronary artery endothelial cells (ECs). Microparticle-induced phagocytosis, cytotoxicity, apoptosis, cytokine release and surface marker expression were determined by immunostaining or ELISA. Elastase expression was analyzed by ELISA and the elastase-mediated polymer degradation was assessed by mass spectrometry.

Results

We demonstrated that poly(D,L-lactic acid) (PLLA) and polycaprolactone (PCL) microparticles induced cytotoxicity in macrophages and ECs, partially through cell apoptosis. The particle treatment alleviated EC phagocytosis, as opposed to macrophages, but enhanced the expression of vascular cell adhesion molecule (VCAM)-1 along with decreased nitric oxide production, indicating that ECs were activated and lost their capacity to maintain homeostasis. The activation of both cell types induced the release of elastase or elastase-like protease, which further accelerated polymer degradation.

Conclusions

This study revealed that low molecule weight PLLA and PCL microparticles increased cytotoxicity and dysregulated endothelial cell function, which in turn enhanced elastase release and polymer degradation. These indicate that polymer or polymer-coated stents impose a risk of endothelial dysfunction after deployment which can potentially lead to delayed endothelialization, neointimal hyperplasia and late thrombosis.     

The influence of Gelatin/PCL ratio and 3-D construct shape of electrospun membranes on cartilage regeneration

Scaffolds play an important role in directing three-dimensional (3-D) cartilage regeneration. Our recent study reported the potential advantages of electrospun gelatin/polycaprolactone (GT/PCL) membranes in regenerating 3-D cartilage. However, it is still unknown whether the changes of GT/PCL ratio have significant influence on 3-D cartilage regeneration. To address this issue, the current study prepared three kinds of electrospun membranes with different GT/PCL ratios (70:30, 50:50, 30:70). Adhesion and proliferation of chondrocytes on the membranes were examined to evaluate biocompatibility of the membranes. Cartilage with different 3-D shapes was engineered to further evaluate the influences of GT/PCL ratio on cartilage regeneration. The current results demonstrated that all the membranes with different GT/PCL ratios presented good biocompatibility with chondrocytes. Nevertheless, the high PCL content in the membranes significantly hampered early 3-D cartilage formation at 3 weeks in vivo. Unexpectedly, at 12 weeks, all the cylinder-shaped constructs formed mature cartilage-like tissue with no statistical differences among groups. To our surprise, ear-shaped cartilage regeneration obtained quite different results again: the high PCL content completely disrupted cartilage regeneration even at 12 weeks, and only the least PCL content group formed homogeneous and continuous cartilage with a satisfactory shape and elasticity similar to human ear. All these results indicated that the high PCL content was unfavorable for 3-D cartilage regeneration, especially for the cartilage with a complicated shape, and that GT/PCL 70:30 might be a relatively suitable ratio for ear-shaped cartilage regeneration. The research models established in the current study provide detailed information for cartilage and other tissue regeneration based on electrospun GT/PCL membranes.     

Uncoupling angiogenesis and inflammation in peripheral artery disease with therapeutic peptide-loaded microgels

Peripheral artery disease (PAD) is characterized by vessel occlusion and ischemia in the limbs. Treatment for PAD with surgical interventions has been showing limited success. Moreover, recent clinical trials with treatment of angiogenic growth factors proved ineffective as increased angiogenesis triggered severe inflammation in a proportionally coupled fashion. Hence, the overarching goal of this research was to address this issue by developing a biomaterial system that enables controlled, dual delivery of pro-angiogenic C16 and anti-inflammatory Ac-SDKP peptides in a minimally-invasive way. To achieve the goal, a peptide-loaded injectable microgel system was developed and tested in a mouse model of PAD. When delivered through multiple, low volume injections, the combination of C16 and Ac-SDKP peptides promoted angiogenesis, muscle regeneration, and perfusion recovery, while minimizing detrimental inflammation. Additionally, this peptide combination regulated inflammatory TNF-α pathways independently of MMP-9 mediated pathways of angiogenesis in vitro, suggesting a potential mechanism by which angiogenic and inflammatory responses can be uncoupled in the context of PAD. This study demonstrates a translatable potential of the dual peptide-loaded injectable microgel system for PAD treatment.     

Characterization and osteogenic activity of a silicatein/biosilica-coated chitosan-graft-polycaprolactone

Several attempts have been made in the past to fabricate hybrid materials that display the complementary properties of the polyester polycaprolactone (PCL) and the polysaccharide chitosan (CHS) for application in the field of bone regeneration and tissue engineering. However, such composites generally have no osteogenic activity per se. Here we report the synthesis of a chitosan-graft-polycaprolactone (CHS-g-PCL) and its subsequent characterization, including crystallinity, chemical structure and thermal stability. Upon surface-functionalization of CHS-g-PCL with osteogenic biosilica via the surface-immobilized enzyme silicatein, protein adsorption, surface morphology and wettability were assessed. Finally, the cultivation of osteoblastic SaOS-2 cells on the surface-functionalized CHS-g-PCL was followed by analyses of cell viability, mineral deposition and alkaline phosphatase activity. These characterizations revealed a composite that combines the versatile properties of CHS-g-PCL with the osteogenic activity of the silicatein/biosilica coating and, hence, represents an innovative alternative to conventionally used CHS/PCL composites for biomedical applications, where stable bone–material interfaces are required.     

Cytotoxic and genotoxic effects of matrices for cartilage tissue engineering

Customizing auricles with biodegradable polyurethane colonized with autologous chondrocytes as an approach for tissue engineering cartilage transplants has been suggested for the reconstruction of the external ear to repair auricular deformities. Dextrose, triethanolamine and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG) are matrices of an open-pored polyurethane three-dimensional scaffold. After release from the polymer, these compounds can be absorbed into the human organism. Therefore, cytotoxic effects on human chondrocytes and lymphocytes and genotoxic effects on human lymphocytes were determined. Propidium iodide and fluoresceine diacetate staining as well as quantitative proliferations testing with EZ4U served to detect cytotoxic effects on chondrocytes. In lymphocytes cytotoxicity was checked by trypan blue staining and the alkaline single cell microgel electrophoresis (Comet) assay was used to study genotoxic effects. Dose-dependent cytotoxicity and genotoxicity of the matrices could be shown. Concentrations up to 4.25 mg/ml for dextrose, 0.15 mg/ml for PEG-PPG-PEG and 0.9 mg/ml for triethanolamine did not show cytotoxic effects in chondrocytes or genotoxic effects in lymphocytes. These data suggest that dextrose, triethanolamine and PEG-PPG-PEG could be safely used if scaffolds made of open-pored polyurethane do not release these compounds at a rate giving higher concentrations at the site of implantation or in body fluids, respectively.