In Vitro Biocompatibility and Antibacterial Efficacy of a Degradable Poly(l-lactide-co-epsilon-caprolactone) Copolymer Incorporated with Silver Nanoparticles

Silver nanoparticles (Ag-nps) are currently used as a natural biocide to prevent undesired bacterial growth in clothing, cosmetics and medical products. The objective of the study was to impart antibacterial properties through the incorporation of Ag-nps at increasing concentrations to electrospun degradable 50:50 poly(l-lactide-co-epsilon-caprolactone) scaffolds for skin tissue engineering applications. The biocompatibility of the scaffolds containing Ag-nps was evaluated with human epidermal keratinocytes (HEK); cell viability and proliferation were evaluated using Live/Dead and alamarBlue viability assays following 7 and 14 days of cell culture on the scaffolds. Significant decreases in cell viability and proliferation were noted for the 1.0 mg(Ag) g(scaffold)^?1 after 7 and 14 days on Ag-nps scaffolds. After 14 days, scanning electron microscopy revealed a confluent layer of HEK on the surface of the 0.0 and 0.1 mg(Ag) g(scaffold)^?1. Both 0.5 and 1.0 mg(Ag) g(scaffold)^?1 were capable of inhibiting both Gram positive and negative bacterial strains. Uniaxial tensile tests revealed a significant (p < 0.001) decrease in the modulus of elasticity following Ag-nps incorporation compared to control. These findings suggest that a scaffold containing between 0.5 and 1.0 mg(Ag) g(scaffold)^?1 is both biocompatible and antibacterial, and is suitable for skin tissue engineering graft scaffolds.     

Human basic fibroblast growth factor fused with Kringle4 peptide binds to a fibrin scaffold and enhances angiogenesis

Appropriate three-dimensional (3D) scaffolds and signal molecules could accelerate tissue regeneration and wound repair. In this work, we targeted human basic fibroblast growth factor (bFGF), a potent angiogenic factor, to a fibrin scaffold to improve therapeutic angiogenesis. We fused bFGF to the Kringle4 domain (K4), a fibrin-binding peptide from human plasminogen, to endow bFGF with specific fibrin-binding ability. The recombinant K4bFGF bound specifically to the fibrin scaffold so that K4bFGF was delivered in a site-specific manner, and the fibrin scaffold provided 3D support for cell migration and proliferation. Subcutaneous implantation of the fibrin scaffolds bound with K4bFGF but not with bFGF induced neovascularization. Immunohistochemical analysis showed significantly more proliferation cells in the fibrin scaffolds incorporated with K4bFGF than in those with bFGF. Moreover, the regenerative tissues were integrated well with the fibrin scaffolds, suggesting its good biocompatibility. In summary, targeted delivery of K4bFGF could potentially improve therapeutic angiogenesis.     

In vivo cellular repopulation of tubular elastin scaffolds mediated by basic fibroblast growth factor

In vivo tissue engineering has been explored as a method to repopulate scaffolds with autologous cells to create a functional, living, and non-immunogenic tissue substitute. In this study, we describe an approach to in vivo cellular repopulation of a tissue-derived tubular elastin scaffold. Pure elastin scaffolds were prepared from porcine carotid arteries (elastin tubes). Elastin tubes were filled with agarose gel containing basic fibroblast growth factor (bFGF) to allow sustained release of growth factor. These tubes were implanted in subdermal pouches in adult rats. The elastin tubes with growth factor had significantly more cell infiltration at 28 days than those without growth factor. Immunohistochemical staining indicated that most of these cells were fibroblasts, of which a few were activated fibroblasts (myofibroblasts). Microvasculature was also observed within the scaffolds. Macrophage infiltration was seen at 7 days, which diminished by 28 days of implantation. None of the elastin tubes with bFGF calcified. These results demonstrated that the sustained release of bFGF brings about repopulation of elastin scaffolds in vivo while inhibiting calcification. Results showing myofibroblast infiltration and vascularization are encouraging since such an in vivo implantation technique could be used for autologous cell repopulation of elastin scaffolds for vascular graft applications.     

P38/AKT通路调控骨髓间充质干细胞在不同空间结构纳米纤维环支架中的定向分化

文题释义： 拓扑结构：泛指组织工程支架的一系列细微的结构差异,涵盖多方面因素,如支架质地、硬度等材料特性相关的属性;支架表面的光滑程度、支架内部的孔隙率等结构特性等;实验主要指静电纺丝纤维环支架中纤维排列方式。 差异分化：实验中用于描述骨髓间充质干细胞在不同条件下(主要指支架拓扑结构的不同),接受外界信息后发生一系列变化,通过细胞通路等机制的调节分化为不同细胞的现象,如实验中骨髓间充质干细胞在不同支架中分别向成纤维细胞或成软骨细胞分化。对差异分化的机制进行研究,有助于今后通过改变条件诱导细胞向所需方向分化、发挥组织工程技术的优势更好修复组织的目的。 背景：以往研究表明多种材料可用于组织工程支架的构建,支架表面的拓扑结构特征对干细胞增殖、分化等生物学行为有调节作用,但其具体机制尚不明确。 目的：研究骨髓间充质干细胞在纳米结构支架中定向分化过程中P38、AKT通路的作用。 方法：构建3种结构不同的纳米纤维支架,即取向纳米纤维支架AFS、取向纳米纱支架AYS、三维多孔纳米纤维支架3-DPS;将大鼠骨髓间充质干细胞分别接种于3种纳米纤维支架表面,成软骨诱导培养后,分别进行细胞形态、黏附、增殖检测,利用qRT-PCR检测细胞关键表型分子(Ⅱ型胶原α1、Ⅰ型胶原α1、蛋白聚糖、Sox-9)mRNA的表达水平,Western blot测定细胞内P38、AKT、ERK1/2、JNK的蛋白表达水平。 结果与结论：①3-DPS支架组培养4,8 h的细胞黏附率高于其余两组支架(P < 0.05),培养7 d的细胞增殖快于其余两组支架(P < 0.05);②骨髓间充质干细胞在3种材料表面均贴附牢固,生长状态良好,其中在AFS、AYS支架上呈类成纤维细胞样生长,在3-DPS支架上呈类软骨细胞样生长;③成软骨诱导3周后,3-DPS支架组Ⅱ型胶原α1、蛋白聚糖、Sox9 mRNA表达高于其余两组支架(P < 0.05),Ⅰ型胶原α1 mRNA表达低于其余两组支架(P < 0.05);AFS支架组、AYS支架组中Ⅱ型胶原α1、蛋白聚糖、Sox9 mRNA表达无明显差异(P > 0.05);④成软骨诱导3周后,3-DPS支架组p-AKT、p-P38蛋白相对表达量高于其余两组支架(P < 0.05),3组间AKT总蛋白表达量及ERK1/2、JNK、P38、p-ERK1/2、p-JNK、p-P38蛋白表达量比较均无显著性意义;⑤结果表明,不同结构支架形貌可通过Integrin-FAK信号通路下游的P38、AKT通路诱导骨髓间充质干细胞的的定向分化。 ORCID: 0000-0003-3571-8840(王爽) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

The effect of anisotropic collagen-GAG scaffolds and growth factor supplementation on tendon cell recruitment, alignment, and metabolic activity

Current surgical and tissue engineering approaches for treating tendon injuries have shown limited success, suggesting the need for new biomaterial strategies. Here we describe the development of an anisotropic collagen-glycosaminoglycan (CG) scaffold and use of growth factor supplementation strategies to create a 3D platform for tendon tissue engineering. We fabricated cylindrical CG scaffolds with aligned tracks of ellipsoidal pores that mimic the native physiology of tendon by incorporating a directional solidification step into a conventional lyophilization strategy. By modifying the freezing temperature, we created a homologous series of aligned CG scaffolds with constant relative density and degree of anisotropy but a range of pore sizes (55-243?μm). Equine tendon cells showed greater levels of attachment, metabolic activity, and alignment as well as less cell-mediated scaffold contraction, when cultured in anisotropic scaffolds compared to an isotropic CG scaffold control. The anisotropic CG scaffolds also provided critical contact guidance cues for cell alignment. While tendon cells were randomly oriented in the isotropic control scaffold and the transverse (unaligned) plane of the anisotropic scaffolds, significant cell alignment was observed in the direction of the contact guidance cues in the longitudinal plane of the anisotropic scaffolds. Scaffold pore size was found to significantly influence tendon cell viability, proliferation, penetration into the scaffold, and metabolic activity in a manner predicted by cellular solids arguments. Finally, the addition of the growth factors PDGF-BB and IGF-1 to aligned CG scaffolds was found to enhance tendon cell motility, viability, and metabolic activity in dose-dependent manners. This work suggests a composite strategy for developing bioactive, 3D material systems for tendon tissue engineering.     

Intestinal smooth muscle cell maintenance by basic fibroblast growth factor

Intestinal tissue engineering is a potential therapy for patients with short bowel syndrome. Tissue engineering scaffolds that promote smooth muscle cell proliferation and angiogenesis are essential toward the regeneration of functional smooth muscles for peristalsis and motility. Since basic fibroblast growth factor (bFGF) can stimulate smooth muscle proliferation and angiogenesis, the delivery of bFGF was employed to stimulate proliferation and survival of primary intestinal smooth muscle cells. Two methods of local bFGF delivery were examined: the incorporation of bFGF into the collagen coating and the encapsulation of bFGF into poly(D,L-lactic-co-glycolic acid) microspheres. Cell-seeded scaffolds were implanted into the omentum and were retrieved after 4, 14, and 28 days. The seeded cells proliferated from day 4 to day 14 in all implants; however, at 28 days, significantly higher density of implanted cells and blood vessels was observed, when 10 microg of bFGF was incorporated into the collagen coating of scaffolds as compared to scaffolds with either no bFGF or 1 microg of bFGF in collagen. Microsphere encapsulation of 1 microg of bFGF produced similar effects as 10 microg of bFGF mixed in collagen and was more effective than the delivery of 1 microg of bFGF by collagen incorporation. The majority of the implanted cells also expressed alpha-smooth muscle actin. Scaffolds coated with microsphere-encapsulated bFGF and seeded with smooth muscle cells may be a useful platform for the regeneration of the intestinal smooth muscle.     

Fibrin-based scaffold incorporating VEGF- and bFGF-loaded nanoparticles stimulates wound healing in diabetic mice

Diabetic skin ulcers are difficult to heal spontaneously due to the reduced levels and activity of endogenous growth factors. Recombinant human vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are known to stimulate cell proliferation and accelerate wound healing. Direct delivery of VEGF and bFGF at the wound site in a sustained and controllable way without loss of bioactivity would enhance their biological effects. The aim of this study was to develop a poly(ether)urethane–polydimethylsiloxane/fibrin-based scaffold containing poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with VEGF and bFGF (scaffold/GF-loaded NPs) and to evaluate its wound healing properties in genetically diabetic mice (db/db). The scaffold application on full-thickness dorsal skin wounds significantly accelerated wound closure at day 15 compared to scaffolds without growth factors (control scaffold) or containing unloaded PLGA nanoparticles (scaffold/unloaded NPs). However, the closure rate was similar to that observed in mice treated with scaffolds containing free VEGF and bFGF (scaffold/GFs). Both scaffolds containing growth factors induced complete re-epithelialization, with enhanced granulation tissue formation/maturity and collagen deposition compared to the other groups, as revealed by histological analysis. The ability of the scaffold/GF-loaded NPs to promote wound healing in a diabetic mouse model suggests its potential use as a dressing in patients with diabetic foot ulcers.     

Cell distribution in a scaffold with random architectures under the influence of fluid dynamics

Fluid dynamic environment and scaffold architectures have an important influence on cell growth and distribution inside the scaffold. A porous cylindrical scaffold with a central channel is seeded with the sheep mesenchymal stem cells (MSCs) in this study. Then the cell seeded scaffold is continuously perfused with alpha-MEM medium by a peristaltic pump for 7, 14, and 28 days. Histological study shows that the cell proliferation rates are different throughout the whole scaffolds. The different cell coverage is shown in various positions of the scaffold. A computational fluid dynamics (CFD) modeling is used to simulate the flow conditions within perfused cell-seeded scaffolds to give insight into the mechanisms of these cell growth phenomena. Relating the simulation results to perfusion experiments, the even fluid velocity (approximately 0.26-0.64 mm/s) and shear stress (approximately 0.0029-0.027 Pa) are found to correspond to increased cell proliferation within the cell-scaffold constructs. This method exhibits novel capabilities to compare results obtained for different perfusion rates or different scaffold microarchitectures and may allow specific fluid velocities and shear stresses to be determined that optimize the perfusion flow rate, porous scaffold architecture, and distribution of in vitro tissue growth.     

Novel apatite fiber scaffolds can promote three-dimensional proliferation of osteoblasts in rodent bone regeneration models

We have successfully synthesized hydroxyapatite fibers via a homogenous precipitation method. Using these hydroxyapatite fibers, we have produced the apatite fiber scaffolds (AFS) with well-controlled pore sizes (porosity above 95%). The AFS is relatively simple to synthesize, and its porosity and pore size are controllable. The usefulness of AFS as a scaffold for bone regeneration was evaluated by (1) seeding and culturing cells in the AFS in vitro, (2) implanting the AFS seeded with cells inside the subcutaneous tissue of mice. The AFS had biocompatibility to support cell adhesion, proliferation, and differentiation. Ectopic bone formation could be formed in the AFS at 12 weeks after implantation into the subcutaneous tissue. Because of its high interpore connection, pore diameters, and porosity, it was believed that AFS was an effective scaffold that provided a three-dimensional cell culture environment. In both in vitro and in vivo environments, the more porous AFS was more advantageous in cell proliferation, cell adhesion, proliferating capacity, robust cell differentiation, ultimately inducing bone ingrowth inside the scaffolds.     

Flow perfusion culture of human fetal bone cells in large beta-tricalcium phosphate scaffold with controlled architecture

One unsolved problem in bone tissue engineering is how to enable the survival and proliferation of osteoblastic cells in large scaffolds. In this work, large beta-tricalcium phosphate scaffolds with tightly controlled channel architectures were fabricated and a custom-designed perfusion bioreactor was developed. Human fetal bone cells in third passage were seeded onto the scaffolds and cultured in static or flow perfusion conditions for up to 16 days. Compared with nonperfused constructs, flow perfused constructs demonstrated improved cells proliferation and differentiation according to cell viability, glucose consumption, alkaline phosphatase activity, and osteopontin. Moreover, after 16 days of perfusion culture, a homogenous layer composed of cells and mineralized matrix throughout the whole scaffold was observed by scanning electron microscopy and histological study. In contrast, cells were located only along the scaffold perimeter in static culture. These results demonstrated the feasibility and benefit of perfusion culture in conjunction with well-defined three-dimensional environment for large bone graft construction. Porous scaffold with controlled architecture can be a potential tool to evaluate the effects of scaffold specific geometry on fluid flow configuration and cell behavior under perfusion culture.     

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.     

Enhancing the vascularization of three-dimensional porous alginate scaffolds by incorporating controlled release basic fibroblast growth factor microspheres

Site-specific delivery of angiogenic growth factors from tissue-engineered devices should provide an efficient means of stimulating localized vessel recruitment to the cell transplants and would ensure cell survival and function. In the present article, we describe the construction of a novel porous alginate scaffold that incorporates tiny poly (lactic-co-glycolic acid) microspheres capable of controlling the release of angiogenic factors, such as basic fibroblast growth factor (bFGF). The microspheres are an integral part of the solid alginate matrix, and their incorporation does not affect the scaffold porosity or pore size. In vitro, bFGF was released from the porous composite scaffolds in a controlled manner and it was biologically active as assessed by its ability to induce the proliferation of cardiac fibroblasts. The controlled delivery of bFGF from the three-dimensional scaffolds accelerated the matrix vascularization after implantation on the mesenteric membrane in rat peritoneum. The number of penetrating capillaries into the bFGF-releasing scaffolds was nearly fourfold higher than into the control scaffolds (those incorporating microspheric BSA and heparin but not bFGF). At day 10 posttransplantation, capillary density in the composite scaffolds was 45 +/- 3/mm(2) and it increased to 70 +/- 7/mm(2) by day 21. The released bFGF induced the formation of large and matured blood vessels, as judged by the massive layer of mural cells surrounding the endothelial cells. The control over bFGF delivery and localizing its effects to areas of need, may aid in the wider application of bFGF in therapeutic angiogenesis as well as in tissue engineering.     

Rapid and complete cellularization of hydroxyapatite for bone tissue engineering

Using a tissue construct generated by cells in a scaffold in reconstructive surgery, as a substitute for autografts, is still challenging. Routine methods of incorporating cells into scaffolds are either passive, i.e. by gravity, or forced, as in a bioreactor. Extensive use of these methods is obstructed by tissue formation around the scaffold, hindrance in cell penetration and time required for cell coverage within the scaffold. In this study, human osteoblast cells as cell sheet structures were seeded to porous and dense hydroxyapatite with the hypothesis that preservation of native extracellular structures and cell-cell contacts would facilitate the cellularization process. Cellularization was assessed by fluorescence, confocal and scanning electron microscopy at intervals of 1 h, 2 days and 7 days. Cell patches with intact cell-cell and cell-extra cellular matrix contact attached and adhered on a scaffold within 1 h. The patches formed a monolayer within 2 days and complete cellularization of the scaffold was attained in 7 days. Cell viability, proliferation and function were assessed to understand the application of cell patch transfer to bone substitute. This novel approach for application in bone tissue engineering was successful in uniform distribution of intact osteoblast cell sheet structures on to bone substitute materials for rapid and complete cellularization without altering material characteristics.     

Use of human mesenchymal cells to improve vascularization in a mouse model for scaffold-based dermal regeneration

All engineered bioartificial structures developed for tissue regeneration require oxygen and nutrients to establish proper physiological functions. Aiming to improve vascularization during dermal regeneration, we combined the use of a bioartificial collagen scaffold and a defined human mesenchymal cell (MC) line. This cell line, termed V54/2, exhibits typical morphologic and immunohistochemical characteristics of MC. V54/2 cells seeded in the scaffold were able to survive, proliferate, and secrete significant amounts of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) during 2 weeks in vitro. To induce dermal regeneration, scaffolds with or without cells were transplanted in a nude mice full skin defect model. After 2 weeks of transplantation, scaffolds seeded with V54/2 cells showed more vascularization during the dermal regeneration process than controls, and the presence of human cells in the regenerating tissue was detected by immunohistochemistry. To confirm if local presence of angiogenic growth factors is sufficient to induce neovascularization, scaffolds were loaded with VEGF and bFGF and used to induce dermal regeneration in vivo. Results showed that scaffolds supplemented with growth factors were significantly more vascularized than control scaffolds (scaffolds without growth factors). The present work suggests that combined use of MC and bioartificial scaffolds induces therapeutic angiogenesis during the scaffold-based dermal regeneration process.     

Osteoblast activity on collagen-GAG scaffolds is affected by collagen and GAG concentrations

Optimization of a tissue engineering scaffold for use in bone tissue engineering requires control of many factors such as pore size, porosity, permeability and, as this study shows, the composition of the matrix. The collagen-glycosaminoglycan (GAG) scaffold variants were fabricated by varying the collagen and GAG content of the scaffold. Scaffolds were seeded with MC3T3 osteoblasts and cultured for up to 7 days. During the culture period, osteoblastic activity was evaluated by measuring metabolic activity, cell number, and spatial distribution. Collagen and GAG concentrations both affected osteoblast viability, proliferation, and spatial distribution within the scaffold. Scaffolds containing 1% collagen (w/v) and 0.088% GAG (w/v) were found to have a porosity of approximately 99%, high cell metabolic activity and cell number, and good cell infiltration over the 7 days in culture. Taken together, these results indicate the need to tailor the parameters of a biological substrate for use in a specific tissue application, in this case bone tissue engineering.     

Heparin-modified gelatin scaffolds for human corneal endothelial cell transplantation

Although one of the most transplanted tissues, a shortage of cadaveric corneas for transplantation still exists in the western society and elsewhere. The goal of this study was to develop a biological scaffold to support transfer of cultured human corneal endothelial cells (HCECs) into the anterior chamber of the eye, potentially a replacement for cadaveric donor tissue. A series of transparent scaffolds were fabricated from gelatin and modified with heparin. Mechanical parameters of the scaffolds, such as stiffness, affected cell proliferation, phenotype and cell surface marker expression were determined. The heparin-modified scaffolds had a greater capacity to absorb basic fibroblast growth factor (bFGF) and showed better release kinetics for up to 20 days. The release of bFGF from the scaffolds improved HCECs survival and reduced cellular loss. The scaffolds were flexible and could be folded and implanted in rabbits' eyes, through a small incision in the cornea. The scaffolds adhered to the inner surface of the corneal stroma and gradually integrated with the surrounding tissue. These results indicate that gelatin based corneal scaffolds modified to absorb and release growth factors and seeded with HCECs, might be a suitable alternative for cadaveric cornea transplantation.     

Liver tissue engineering within alginate scaffolds: effects of cell-seeding density on hepatocyte viability,morphology, and function

Tissue engineering with three-dimensional biomaterials represents a promising approach for developing hepatic tissue to replace the function of a failing liver. Herein, we address cell seeding and distribution within porous alginate scaffolds, which represent a new type of porous biomaterial for tissue engineering. The hydrophilic nature of the alginate scaffold as well as its pore structure and interconnectivity enabled the efficient seeding of hepatocytes into the scaffolds, that is, 70-90% of the initial cells depending on the seeding method. Utilization of centrifugal force during seeding enhanced cell distribution in the porous scaffolds, consequently enabling the seeding of concentrated cell suspensions (>1 x 10(7) cells/mL). Cell density in scaffolds affected hepatocyte viability as judged by MTT assay. At a cell density of 0.28 x 10(6) cells/cm3 scaffold, the number of viable hepatocytes decreased to 33% of its initial value within 7 days, whereas at the denser cultures, 5.7 x 10(6) cells/cm3 scaffold and higher, the cells maintained higher viability while forming a network of connecting spheroids. In the high-density cellular constructs, hepatocellular functions such as albumin and urea secretion, and detoxification (cytochrome P-450 and phase II conjugating enzyme activities), remained high during the 7-day culture. Collectively, the results of the present study highlight the importance of cell density on the hepatocellular functions of three-dimensional hepatocyte constructs as well as the advantages of alginate matrices as scaffoldings.     

In vitro cell performance on hydroxyapatite particles/poly(L-lactic acid) nanofibrous scaffolds with an excellent particle along nanofiber orientation

Highly porous hydroxyapatite (HA)/poly(L-lactide) (PLLA) nanofibrous scaffolds were prepared by incorporating needle-shaped nano- or micro-sized HA particles into PLLA nanofibers using electrospinning. The scaffolds had random or aligned fibrous assemblies and both types of HA particles were perfectly oriented along the fiber long axes. The biocompatibility and cell signaling properties of these scaffolds were evaluated by in vitro culture of rat osteosarcoma ROS17/2.8 cells on the scaffold surface. Cell morphology, viability and alkaline phosphatase (ALP) activity on each scaffold were examined at different time points. The HA/PLLA scaffolds exhibited higher cell viability and ALP activity than a pure PLLA scaffold. In addition, micro-sized HA particles supported cell proliferation and differentiation better than nano-sized ones in random scaffolds through a 10 day culture period and in aligned scaffolds at an early culture stage. The fibrous assembly of the scaffold had a pronounced impact on the morphology of the cells in direct contact with the scaffold surface, but not on cell proliferation and differentiation. Thus, HA/PLLA nanofibrous scaffolds could be good candidates for bone tissue engineering.     

Vascular endothelial growth factor immobilized in collagen scaffold promotes penetration and proliferation of endothelial cells

A key challenge in engineering functional tissues in vitro is the limited transport capacity of oxygen and nutrients into the tissue. Inducing vascularization within engineered tissues is a key strategy to improving their survival in vitro and in vivo. The presence of vascular endothelial growth factor (VEGF) in a three-dimensional porous collagen scaffold may provide a useful strategy to promote vascularization of the engineered tissue in a controlled manner. To this end, we investigated whether immobilized VEGF could promote the invasion and assembly of endothelial cells (ECs) into the collagen scaffolds. We conjugated VEGF onto collagen scaffolds using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride chemistry, and measured the concentrations of immobilized VEGF in collagen scaffolds by direct VEGF enzyme-linked immunosorbent assay. We demonstrated that immobilized VEGF (relative to soluble VEGF) promoted the penetration and proliferation of ECs in the collagen scaffold, based on results of cell density analysis in histological sections, immunohistochemistry, XTT proliferation assay, glucose consumption and lactate production. Furthermore, we observed increased viability of ECs cultured in scaffolds with immobilized VEGF relative to soluble VEGF. This research demonstrates that immobilization of VEGF is a useful strategy to promote the invasion and proliferation of ECs into a scaffold, which may in turn lead to a vascularized scaffold.     

Interaction of human valve interstitial cells with collagen matrices manufactured using rapid prototyping

Rapid prototyping is a novel process for the production of scaffolds of predetermined size and three-dimensional shape. The aim of the study was to determine the feasibility of this technology for producing scaffolds for tissue engineering an aortic valve and the optimal concentration of collagen processed in this manner that would maintain viability and promote proliferation of human valve interstitial cells. Scaffolds of 1%, 2% and 5% w/v bovine type-I collagen were manufactured using rapid prototyping. Valve interstitial cells isolated from three human aortic valves were seeded on the scaffolds and cultured for up to 4 weeks. Cell viability was assessed using the CellTiter 96 Aq(ueous) One Solution Cell Proliferation Assay and cell death by lactate dehydrogenase (LDH) measurement. Valve interstitial cells remained viable and proliferated within the collagen scaffolds. Cells consistently proliferated to a greater extent on 1% collagen scaffolds rather than either 2% or 5% collagen and after 4 weeks reached 212+/-33.1%, 139+/-25.9% and 129+/-38.3% (mean+/-SD) of their initial seeding density on 1%, 2% and 5% collagen scaffolds, respectively. LDH analysis demonstrated that there was minimal cell death indicating that the collagen scaffold was not toxic to human valve interstitial cells. Rapid prototyping provides a route to optimize biological scaffold designs for tissue engineering cardiac valves. This technology has the versatility to create scaffolds that are compatible with the specific needs of the valve interstitial cells and should enhance cell viability, proliferation and function.