Stem cell differentiation on electrospun nanofibrous substrates for vascular tissue engineering

Nanotechnology has enabled the engineering of a variety of materials to meet the current challenges and requirements in vascular tissue regeneration. In our study, poly-l-lactide (PLLA) and hybrid PLLA/collagen (PLLA/Coll) nanofibers (3:1 and 1:1) with fiber diameters of 210 to 430 nm were fabricated by electrospinning. Their morphological, chemical and mechanical characterizations were carried out using scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared (ATR-FTIR), and tensile instrument, respectively. Bone marrow derived mesenchymal stem cells (MSCs) seeded on electrospun nanofibers that are capable of differentiating into vascular cells have great potential for repair of the vascular system. We investigated the potential of MSCs for vascular cell differentiation in vitro on electrospun PLLA/Coll nanofibrous scaffolds using endothelial differentiation media. After 20 days of culture, MSC proliferation on PLLA/Coll(1:1) scaffolds was found 256% higher than the cell proliferation on PLLA scaffolds. SEM images showed that the MSC differentiated endothelial cells on PLLA/Coll scaffolds showed cobblestone morphology in comparison to the fibroblastic type of undifferentiated MSCs. The functionality of the cells in the presence of ‘endothelial induction media’, was further demonstrated from the immunocytochemical analysis, where the MSCs on PLLA/Coll (1:1) scaffolds differentiated to endothelial cells and expressed the endothelial cell specific proteins such as platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) and Von Willebrand factor (vWF). From the results of the SEM analysis and protein expression studies, we concluded that the electrospun PLLA/Coll nanofibers could mimic the native vascular ECM environment and might be promising substrates for potential application towards vascular regeneration.     

Design of novel three-phase PCL/TZ–HA biomaterials for use in bone regeneration applications

The design of bioactive scaffold materials able to guide cellular processes involved in new-tissue genesis is key determinant in bone tissue engineering. The aim of this study was the design and characterization of novel multi-phase biomaterials to be processed for the fabrication of 3D porous scaffolds able to provide a temporary biocompatible substrate for mesenchymal stem cells (MSCs) adhesion, proliferation and osteogenic differentiation. The biomaterials were prepared by blending poly(ε-caprolactone) (PCL) with thermoplastic zein (TZ), a thermoplastic material obtained by de novo thermoplasticization of zein. Furthermore, to bioactivate the scaffolds, microparticles of osteoconductive hydroxyapatite (HA) were dispersed within the organic phases. Results demonstrated that materials and formulations strongly affected the micro-structural properties and hydrophilicity of the scaffolds and, therefore, had a pivotal role in guiding cell/scaffold interaction. In particular, if compared to neat PCL, PCL–HA composite and PCL/TZ blend, the three-phase PCL/TZ–HA showed improved MSCs adhesion, proliferation and osteogenic differentiation capability, thus demonstrating potential for bone regeneration.     

不同类型多孔结构生物材料支架制备及其性能优化

多孔支架是组织工程应用中的关键环节,类似细胞外基质的作用,支撑细胞的粘附和随后细胞向组织的衍化。虽然目前已采用多种制备技术研发出大量的多孔支架,但是多孔生物材料支架的制备和性能优化,仍然是组织工程支架领域的研究热点。结合实验室工作,综述了多种制备不同类型多孔结构生物材料支架的制备技术,主要包括颗粒和纤维堆积型支架、泡沫浸渍法支架和颗粒制孔支架等的制备技术,并阐述了这些制备技术对多孔结构支架的孔结构、贯通性和力学性能的改善效果。其目的旨在提供满足组织工程需求的多孔生物材料支架。     

Selective laser sintering of porous tissue engineering scaffolds from poly(<Emphasis Type="SmallCaps">l</Emphasis>-lactide)/carbonated hydroxyapatite nanocomposite microspheres

This study focuses on the use of bio-nanocomposite microspheres, consisting of carbonated hydroxyapatite (CHAp) nanospheres within a poly(L: -lactide) (PLLA) matrix, to produce tissue engineering (TE) scaffolds using a modified selective laser sintering (SLS) machine. PLLA microspheres and PLLA/CHAp nanocomposite microspheres were prepared by emulsion techniques. The resultant microspheres had a size range of 5-30 mum, suitable for the SLS process. Microstructural analyses revealed that the CHAp nanospheres were embedded throughout the PLLA microsphere, forming a nanocomposite structure. A custom-made miniature sintering platform was installed in a commercial Sinterstation((R)) 2000 SLS machine. This platform allowed the use of small quantities of biomaterials for TE scaffold production. The effects of laser power; scan spacing and part bed temperature were investigated and optimized. Finally, porous scaffolds were successfully fabricated from the PLLA microspheres and PLLA/CHAp nanocomposite microspheres. In particular, the PLLA/CHAp nanocomposite microspheres appeared to be promising for porous bone TE scaffold production using the SLS technique.     

Poly(L-lactic acid) nanocylinders as nanofibrous structures for macroporous gelatin scaffolds

Electrospun Nanofiber sheets have been shown to mimic the structure of extracellular matrix (ECM). Although these nanofibers have shown great potential for use as tissue engineering scaffolds, it is difficult for the electrospun nanofiber based sheets to be shaped into the desired three-dimensional structure. In this study, poly(L-lactic acid) (PLLA), a biodegradable and biocompatible polyester, was electrospun to produce nanofibers that were treated with an amino group containing base in order to fabricate polymeric nanocylinders. The aspect ratio of the PLLA nanocylinders was tunable by varying the aminolysis time and density of the amino group containing base. The effects of changes in nanofibrous morphology of the PLLA nanocylinders/macro-porous gelatin scaffolds on cell adhesion and proliferation were evaluated. The results revealed different cell morphology, adhesion, and proliferation in the nanocylinders composite gelatin scaffold versus gelatin scaffold alone. Confocal laser scanning microscopy observation showed more spreading and a more flattened cell morphology after NIH3T3 cells were cultured on PLLA nanocylinders/gelatin scaffolds for 10 hours and 4 days. These results indicate that the gelatin/PLLA nanocylinder composite is a promising way to fabricate 3D nanofibrous scaffolds that accelerates cell adhesion and proliferation for tissue engineering.     

Effect of collagen II coating on mesenchymal stem cell adhesion on chitosan and on reacetylated chitosan fibrous scaffolds

The biocompatibility and biomimetic properties of chitosan make it attractive for tissue engineering but its use is limited by its cell adhesion properties. Our objectives were to produce and characterize chitosan and reacetylated-chitosan fibrous scaffolds coated with type II collagen and to evaluate the effect of these chemical modifications on mesenchymal stem cell (MSC) adhesion. Chitosan and reacetylated-chitosan scaffolds obtained by a wet spinning method were coated with type II collagen. Scaffolds were characterized prior to seeding with MSCs. The constructs were analyzed for cell binding kinetics, numbers, distribution and viability. Cell attachment and distribution were improved on chitosan coated with type II collagen. MSCs adhered less to reacetylated-chitosan and collagen coating did not improve MSCs attachment on those scaffolds. These findings are promising and encourage the evaluation of the differentiation of MSCs in collagen-coated chitosan scaffolds. However, the decreased cell adhesion on reacetylated chitosan scaffold seems difficult to overcome and will limit its use for tissue engineering.     

Fabrication and characterization of nano composite scaffold of poly(l-lactic acid)/hydroxyapatite

To mimic the nano-fibrous structure of the natural extracellular matrix, a nano composite scaffold of poly(l-lactic acid)/hydroxyapatite(PLLA/HAP) was fabricated by a thermally induced phase separation method. The characterization of the composite scaffold showed that the scaffold had a nano-fibrous PLLA network (fiber size 100–750 nm), an interconnective microporous structure (1–10 μm) and high porosity (>90%). HAP was homogeneously distributed in the scaffold, as a result, the compressive modulus of PLLA/HAP (80:20, w/w) increased to 3.15-fold compared with that of a pure PLLA scaffold. Incorporating HAP into PLLA network also buffered the pH decline in vitro degradation and enhanced the protein adsorption of the composite scaffold significantly. The new nano composite scaffold is potentially a very promising scaffold for tissue engineering.     

RGD-modified acellular bovine pericardium as a bioprosthetic scaffold for tissue engineering

Acellular biological tissues, including bovine pericardia (BP), have been proposed as natural biomaterials for tissue engineering. However, small pore size, low porosity and lack of extra cellular matrix (ECM) after native cell extraction directly restrict the seed cell adhesion, migration and proliferation and which is a vital problem for ABP’s application in the tissue engineered heart valve (TEHV). In the present study, we treated acellular BP with acetic acid, which increased the scaffold pore size and porosity and conjugated RGD polypeptides to ABP scaffolds. After 10 days of culture in vitro, the human mesenchymal stem cells (hMSCs) attached the best and proliferated the fastest on RGD-modified acellular scaffolds, and the cell has grown deep into the scaffold. In contrast, a low density of cells attached to the unmodified scaffolds, with few infiltrating into the acellular tissues. These findings support the potential use of modified acellular BP as a scaffold for tissue engineered heart valves.     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

Abstract

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

Formation of bone-like apatite on poly(L-lactide) to improve osteoblast-like compatibility in vitro and in vivo

The biomimetic apatite coating process was adopted to modify poly(L-lactide) (PLLA) surfaces with osteoblasts-like cell compatibility. The apatite coating was formed on the pre-hydrolyzed PLLA film and scaffold surfaces by incubating in simulated body fluid (SBF). Scanning electron microscopy and energy dispersive X-ray analyzer were utilized to characterize the composition and the structure of the apatite coating. The cytocompatibility of the modified PLLA films was investigated by testing osteoblast-like attachment, proliferation, alkaline phosphatase (ALP) activity, and cell cycle. Subsequently, the modified PLLA scaffolds were co-cultured with the osteoblasts-like in vitro and subcutaneously implanted into nude mice. The experimental results showed that the formed apatite had a nano-sized particle and matrix configuration. The surface modification of PLLA with apatite coating significantly promoted osteoblast-like compatibility. After a four-week culture in vivo, no significant inflammatory signs were observed in the implanted regions and osteoblast-like congeries with bone-like structure began to form in the scaffolds. The positive results of this study suggest a good way to produce desirable PLLA biomaterials for bone tissue engineering.     

Formation of bone-like apatite on poly(L-lactide) to improve osteoblast-like compatibility in vitro and in vivo

The biomimetic apatite coating process was adopted to modify poly(L-lactide) (PLLA) surfaces with osteoblasts-like cell compatibility. The apatite coating was formed on the pre-hydrolyzed PLLA film and scaffold surfaces by incubating in simulated body fluid (SBF). Scanning electron microscopy and energy dispersive X-ray analyzer were utilized to characterize the composition and the structure of the apatite coating. The cytocompatibility of the modified PLLA films was investigated by testing osteoblast-like attachment, proliferation, alkaline phosphatase (ALP) activity, and cell cycle. Subsequently, the modified PLLA scaffolds were co-cultured with the osteoblasts-like in vitro and subcutaneously implanted into nude mice. The experimental results showed that the formed apatite had a nano-sized particle and matrix configuration. The surface modification of PLLA with apatite coating significantly promoted osteoblast-like compatibility. After a four-week culture in vivo, no significant inflammatory signs were observed in the implanted regions and osteoblast-like congeries with bone-like structure began to form in the scaffolds. The positive results of this study suggest a good way to produce desirable PLLA biomaterials for bone tissue engineering.     

相分离法构建聚乳酸/纳米羟基磷灰石复合纳米纤维支架及性能

通过液.液相分离法构建纳米纤维聚左旋乳酸/蚋米羟基磷灰石(NF-PLLA/nHA)仿生复合支架,利用扫描电镜、压缩测试、微量二喹啉甲酸(BCA)法、X射线衍射及差示扫描量热等手段对其进行表征.结果显示,nHA均匀馕嵌在PLLA纳米纤维间隙中,不影响其纳米纤维结构且明显提高力学性能.同时,nHA的引入还能增加对牛血清白蛋...     

Low frequency magnetic force augments hepatic differentiation of mesenchymal stem cells on a biomagnetic nanofibrous scaffold

Liver tissue engineering using polymeric nanofibrous scaffold and stem cells holds great promises for treating end-stage liver failures. The aim of this study was to evaluate hepatic trans-differentiation potential of human mesenchymal stem cells (hMSCs) on a biomagnetic electrospun nanofibrous scaffold fabricated from a blend of poly-l-lactide (PLLA), collagen and fibrin-rich blood clot, under the influence of a low frequency magnetic field. The scaffold was characterized for surface properties, biochemical and biomechanical parameters and bio-magnetic behaviour. Cell proliferation assay revealed that the scaffold was suitable for hMSCs adhesion and proliferation. Hepatic trans-differentiation potential of hMSCs was augmented on nanofibrous scaffold in magnetic field exposure group compared to control groups, as evident by strong expression of hepatocyte specific markers, albumin release, urea synthesis and presence of an inducible cytochrome P450 system. Our results conclude that biomagnetic scaffold of PLLA/collagen/blood clot augments hepatic trans-differentiation of hMSCs under magnetic field influence.     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

Nanoscale tissue engineering: spatial control over cell-materials interactions

Cells interact with the surrounding environment by making tens to hundreds of thousands of nanoscale interactions with extracellular signals and features. The goal of nanoscale tissue engineering is to harness these interactions through nanoscale biomaterials engineering in order to study and direct cellular behavior. Here, we review two- and three-dimensional (2- and 3D) nanoscale tissue engineering technologies, and provide a holistic overview of the field. Techniques that can control the average spacing and clustering of cell adhesion ligands are well established and have been highly successful in describing cell adhesion and migration in 2D. Extension of these engineering tools to 3D biomaterials has created many new hydrogel and nanofiber scaffold technologies that are being used to design in vitro experiments with more physiologically relevant conditions. Researchers are beginning to study complex cell functions in 3D. However, there is a need for biomaterials systems that provide fine control over the nanoscale presentation of bioactive ligands in 3D. Additionally, there is a need for 2- and 3D techniques that can control the nanoscale presentation of multiple bioactive ligands and that can control the temporal changes in the cellular microenvironment.     

Effect of lecithin content blend with poly (L-lactic acid) on viability and proliferation of mesenchymal stem cells

Lecithin constitutes a natural mixture of phospholipids and neutral lipids and plays critical roles in cellular membrane structure and cellular signaling. In this study, lecithin was blended with poly (L-lactic acid) (PLLA) for modifying the surface of PLLA because it might obtain appropriate hydrophilicity and biocompatibility. The modified PLLA films were manufactured using conventional solvent-casting technique. The hydrophilicity clearly increased with an increase of lecithin content in the polymer blends, as determined by measuring the water contact angle (WCA). The cytocompatibility and any potential cytotoxic effects were studied over 7 days by seeding mesenchymal stem cells (MSCs) on the films of PLLA containing 0–15% lecithin (wt.%), in comparison with tissue culture plates (TCPs). Cell viability and proliferation were assessed using WST-8, lactate dehydrogenase (LDH) and cell morphology was studied by toluidine blue and propidium iodide staining. This results obtained above suggested that 5%lecithin-containing PLLA films could possess the optimal hydrophilicity, higher adhesion and proliferation of MSCs for a prolonged period and did not demonstrate any significant toxic effects to cells. The study showed that the hydrophilicity and biocompatibility of the modified PLLA were markedly improved by directly introducing lecithin into the polymer without the use of multiple synthetic steps. The information obtained should be useful for future research in vascular tissue engineering (VTE).     

A study on the in vitro degradation of poly(L-lactide)/chitosan microspheres scaffolds

Recent research shows that the addition of chitosan microspheres (CMs) to poly(L-lactide) (PLLA) can result in a composite scaffold material with improved biocompatibility and mechanical properties for tissue engineering applications. However, research regarding the influence of CMs on scaffold degradation is absent in the literature. This paper presents a study on the in vitro degradation of scaffolds made from PLLA with CMs. In this study, the PLLA/CMs scaffolds with a 25% ratio of CMs to PLLA were immersed in phosphate-buffered saline (PBS) solution at 37°C for 8 weeks. The in vitro degradation of the scaffolds was investigated using micro-computed tomography (μCT), weight loss analysis, Raman spectroscopy, and differential scanning calorimetry (DSC). Microstructure changes during degradation were monitored using μCT. The μCT results were consistent with the results obtained from Raman spectra and DSC analysis, which reflected that adding CMs into PLLA can decrease the degradation rate compared with pure PLLA scaffolds. The results suggest that PLLA/CMs scaffold degradation can be regulated and controlled to meet requirements imposed a given tissue engineering application.     

CPP/PLLA软骨组织工程支架复合材料初步研究

采用溶媒投放、颗粒滤取技术制备出CPP/PLLA软骨组织工程支架复合材料,测试了该复合材料的物理力学性能和降解性能。研究结果表明,CPP/PLLA软骨组织工程支架复合材料具有高的孔隙率(90%)、良好的生物降解性能和物理力学性能,以及三维连通、微孔、网状微观结构,故该复合材料有希望成为软骨组织工程支架材料之一。     

Electrospun poly(vinylidene fluoride-trifluoroethylene)/zinc oxide nanocomposite tissue engineering scaffolds with enhanced cell adhesion and blood vessel formation

Piezoelectric materials that generate electrical signals in response to mechanical strain can be used in tissue engineering to stimulate cell proliferation.Poly (vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)),a piezoelectric polymer,is widely used in biomaterial applications.We hypothesized that incorporation of zinc oxide (ZnO) nanoparticles into the P(VDF-TrFE) matrix could promote adhesion,migration,and proliferation of cells,as well as blood vessel formation (angiogenesis).In this study,we fabricated and comprehensively characterized a novel electrospun P(VDF-TrFE)/ZnO nanocomposite tissue engineering scaffold.We analyzed the morphological features of the polymeric matrix by scanning electron microscopy,and utilized Fourier transform infrared spectroscopy,X-ray diffraction,and differential scanning calorimetry to examine changes in the crystalline phases of the copolymer due to addition of the nanoparticles.We detected no or minimal adverse effects of the biomaterials with regard to blood compatibility in vitro,biocompatibility,and cytotoxicity,indicating that P(VDF-TrFE)/ZnO nanocomposite scaffolds are suitable for tissue engineering applications.Interestingly,human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells cultured on the nanocomposite scaffolds exhibited higher cell viability,adhesion,and proliferation compared to cells cultured on tissue culture plates or neat P(VDF-TrFE) scaffolds.Nanocomposite scaffolds implanted into rats with or without hMSCs did not elicit immunological responses,as assessed by macroscopic analysis and histology.Importantly,nanocomposite scaffolds promoted angiogenesis,which was increased in scaffolds pre-seeded with hMSCs.Overall,our results highlight the potential of these novel P(VDF-TrFE)/ZnO nanocomposites for use in tissue engineering,due to their biocompatibility and ability to promote cell adhesion and angiogenesis.     

Development of an early estimation method for predicting later osteogenic differentiation activity of rat mesenchymal stromal cells from their attachment areas

Abstract

Cell morphology has received considerable attention in recent years owing to its possible relationship with cell functions, including proliferation, differentiation, and migration. Recent evidence suggests that extracellular environments can also mediate cell functions, particularly cell adhesion. The aims of this study were to investigate the correlation between osteogenic differentiation activity and the morphology of rat mesenchymal stromal cells (MSCs), and to develop a method of estimating osteogenic differentiation capability of MSCs on biomaterials. We measured the attachment areas of MSCs on substrates with various types of surface after 2 h of seeding, and quantified the amount of osteocalcin secreted from MSCs after 3 weeks of culture under osteogenic differentiation conditions. MSCs with small attachment areas showed a high osteogenic differentiation activity. These findings indicate that cell attachment areas correlate well with the osteogenic differentiation activity of MSCs. They also suggest that the measurement of cell attachment areas is useful for estimating the osteogenic differentiation activity of MSCs and is a practical tool for applications of MSCs in regenerative medicine.