Fabrication and properties of porous scaffold of zein/PCL biocomposite for bone tissue engineering

The aim of this study was to fabricate porous scaffolds of zein/poly(ε-caprolactone) (PCL) biocomposite by solvent casting–particulate leaching method using sodium chloride particles as the porogen. Porous biocomposite scaffolds with porosity around 70% and well-interconnected network were obtained. The incorporation of zein into PCL led to the improvement of hydrophilicity as indicated by the results of water contact angle measurement. After immersion in phosphate buffered saline (PBS) in vitro for 28 days, it was observed that the degradation rate of the zein/PCL biocomposite scaffold was faster than the PCL scaffold and that the rate could be tailored by adjusting the amount of zein in the composite. The results demonstrate the potential of the zein/PCL biocomposite scaffolds to be used in tissue engineering strategies to regenerate bone defects.     

Fabrication and characterization of scaffold from cadaver goat-lung tissue for skin tissue engineering applications

The present study aims to fabricate scaffold from cadaver goat-lung tissue and evaluate it for skin tissue engineering applications. Decellularized goat-lung scaffold was fabricated by removing cells from cadaver goat-lung tissue enzymatically, to have cell-free 3D-architecture of natural extracellular matrix. DNA quantification assay and Hematoxylin and eosin staining confirmed the absence of cellular material in the decellularized lung-tissue. SEM analysis of decellularized scaffold shows the intrinsic porous structure of lung tissue with well-preserved pore-to-pore interconnectivity. FTIR analysis confirmed non-denaturation and well maintainance of collagenous protein structure of decellularized scaffold. MTT assay, SEM analysis and H&E staining of human skin-derived Mesenchymal Stem cell, seeded over the decellularized scaffold, confirms stem cell attachment, viability, biocompatibility and proliferation over the decellularized scaffold. Expression of Keratin18 gene, along with CD105, CD73 and CD44, by human skin-derived Mesenchymal Stem cells over decellularized scaffold signifies that the cells are viable, proliferating and migrating, and have maintained their critical cellular functions in the presence of scaffold. Thus, overall study proves the applicability of the goat-lung tissue derived decellularized scaffold for skin tissue engineering applications.     

Fabrication and in vitro evaluation of a sponge-like bioactive-glass/gelatin composite scaffold for bone tissue engineering

In this work a bioactive composite scaffold, comprised of bioactive-glass and gelatin, is introduced. Through direct foaming a sponge-like composite of a sol–gel derived bioactive-glass (70S30C; 70% SiO₂, 30% CaO) and porcine gelatin was developed for use as a biodegradable scaffold for bone tissue engineering. The composite was developed to provide a suitable alternative to synthetic polymer based scaffolds, allowing directed regeneration of bone tissue. The fabricated scaffold was characterised through X-ray microtomography, scanning electron and light microscopy demonstrating a three dimensionally porous and interconnected structure, with an average pore size (170 μm) suitable for successful cell proliferation and tissue ingrowth. Acellular bioactivity was assessed through apatite formation during submersion in simulated body fluid (SBF) whereby the rate and onset of apatite nucleation was found to be comparable to that of bioactive-glass. Modification of dehydrothermal treatment parameters induced varying degrees of crosslinking, allowing the degradation of the composite to be tailored to suit specific applications and establishing its potential for a wide range of applications. Use of genipin to supplement crosslinking by dehydrothermal treatment provided further means of modifying degradability. Biocompatibility of the composite was qualified through successful cultures of human dental pulp stem cells (HDPSCs) on samples of the composite scaffold. Osteogenic differentiation of HDPSCs and extracellular matrix deposition were confirmed through positive alkaline phosphatase staining and immunohistochemistry.     

骨组织工程多孔支架材料性质及制备技术

多孔性生物可降解支架的选择和制备是组织工程技术成功运用的关键。从骨架的材料要求、常用的骨架材料、骨架的制备技术等几个方面对组织工程和生物降解支架的研究进行了综述 ,并对该研究的前景进行了展望     

Behaviour of MG-63 osteoblast-like cells on wood-based biomorphic SiC ceramics coated with bioactive glass

The aim of this study was to test the in vitro cytotoxicity of wood-based biomorphic Silicon Carbide (SiC) ceramics coated with bioactive glass, using MG-63 human osteoblast-like cells, with a view to their application in bone implantology. To better understand the scope of this study, it should be taken into account that biomorphic SiC ceramics have only recently been developed and this innovative product has important properties such as interconnected porosity, high strength and toughness, and easy shaping. In the solvent extraction test, all the extracts had almost no effect on cellular activity even at 100% concentration, and cells incubated in the bioactive glass-coated SiC ceramics extracts showed a proliferation rate similar to that of the Thermanox control. There were no significant differences when the cellular attachment response of the cells on the wood-based biomorphic SiC ceramics, uncoated or coated with bioactive glass, was compared to the one exhibited by reference materials like Ti6Al4V and bulk bioactive glass. This fact looks very promising for biomedical applications.     

New paradigms in hierarchical porous scaffold design for tissue engineering

This paper presented an effective method for the three-dimensional (3D) hierarchical porous scaffold design for tissue engineering. To achieve such a hierarchical porous structure with accurately controlled internal pore architectures, the recursive intersection Boolean operation (RIBO) was proposed in order to satisfy computational efficiency and biological function requirements of a porous scaffold. After generating the distance field (DF) for the given anatomic model and required pore architectures, the recursive DF modifications enable us to design hierarchical porous scaffolds with complex combinations of pore morphologies. A variety of experimental results showed that the proposed hierarchical porous scaffold design method has the potential benefits for accurately controlling both the porosity and the pore architecture gradients while preserving the advantages of triply periodic minimal surface pore geometries.     

Synthesis of organic derived hydroxyapatite scaffold from pig bone waste for tissue engineering applications

Micro porous hydroxyapatite (HAp) had drawn great attention in the field of tissue engineering due to its numerous applications such as tissue regeneration, dental, drug delivery, and adsorption and desorption of substances etc. The chemical synthesis of HAp is often faced with the high cost of starting materials and often lacks the presence of beneficial ions which can promote biological reactions. This paper examined a novel application of pig bone waste for the synthesis of HAp via heat treatment between 600 and 1000 °C. Thus synthesized powder was characterized by X-ray diffraction (XRD), Fourier transform infra-red spectroscopy (FT-IR), Thermogravimetric analysis (TGA), Scanning electron microscopy (SEM), Energy dispersive X-ray analysis (EDX) and Transmission electron microscopy (TEM). XRD results revealed the main characteristic peaks of single phase HAp powder, while the presence of various functional groups such as PO₄^3?, CO₃^2? and OH^? corresponding to HAp were observed by FT-IR analysis. The elemental composition of as-synthesized HAp powder as observed by EDX showed the presence of Ca and P in addition to some beneficial metals such as Na, K, Mg and Si which plays vital roles in biological applications. SEM and TEM observation confirmed the microscopic sctructure of the as-synthesized HAp to be rod-like morphology with 38–52 nm in length. Porous HAp scaffold up to 65% porosity could be prepared using ammonium bicarbonate as pore forming agent. Therefore, bio-waste such as pig bones can be utilized in the synthesis of porous hydroxyapatite scaffold which can serve as an alternative for the conventional chemical method.     

Fabrication and characterization of a biphasic scaffold for osteochondral tissue engineering

A biphasic scaffold with a stratified structure for osteochondral tissue engineering was developed. The chondral phase was a collagen-chitosan composite. The osseous phase was a composite of bioactive glass and collagen. Collagen integrated in the two respective phases was connected by cross-linking agents. Both layers of the scaffold showed interconnected porous structures. After being immersed into stimulated body fluid, precipitation of spherulitic grains could be found on the surface of the osseous phase and this precipitation was proved to be hydroxyapatite by X-ray diffraction and Fourier transform infrared spectroscopy. Inversion, fluorescence and scanning electron microscopy further confirmed that bone marrow stromal cells could anchor on this scaffold with healthy spreading. As the consequence, this biphasic scaffold may have significant potential as an alternative for osteochondral tissue engineering.     

Fabrication and characterization of PCL/gelatin composite nanofibrous scaffold for tissue engineering applications by electrospinning method

In the present study, composite nanofibrous tissue engineering-scaffold consisting of polycaprolactone and gelatin, was fabricated by electrospinning method, using a new cost-effective solvent mixture: chloroform/methanol for polycaprolactone (PCL) and acetic acid for gelatin. The morphology of the nanofibrous scaffold was investigated by using field emission scanning electron microscopy (FE-SEM) which clearly indicates that the morphology of nanofibers was influenced by the weight ratio of PCL to gelatin in the solution. Uniform fibers were produced only when the weight ratio of PCL/gelatin is sufficiently high (10:1). The scaffold was further characterized by Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric (TG) analysis, and X-ray diffraction (XRD). FT-IR and TG analysis indicated some interactions between PCL and gelatin molecules within the scaffold, while XRD results demonstrated crystalline nature of PCL/gelatin composite scaffold. Cytotoxicity effect of scaffold on L929 mouse fibroblast cells was evaluated by MTT assay and cell proliferation on the scaffold was confirmed by DNA quantification. Positive results of MTT assay and DNA quantification L929 mouse fibroblast cells indicated that the scaffold made from the combination of natural polymer (gelatin) and synthetic polymer (PCL) may serve as a good candidate for tissue engineering applications.     

Fabrication and characterization of PCL/gelatin/chitosan ternary nanofibrous composite scaffold for tissue engineering applications

In the present study, we have fabricated a ternary composite nanofibrous scaffold from PCL/gelatin/chitosan, by electrospinning technique, using a solvent system—chloroform/methanol for polycaprolactone (PCL) and acetic acid for gelatin and chitosan, for tissue engineering applications. Field emission scanning electron microscopy (FE-SEM) was used to investigate the fiber morphology of the scaffold and it was found that the fiber morphology was influenced by the concentrations of PCL, gelatin, and chitosan in polymer solution during electrospinning. X-ray diffraction, Fourier transform infrared, and thermogravimetric (TG) analysis results showed some interactions among the molecules of PCL, gelatin, and chitosan within the scaffold. In-vitro cell culture studies were done by seeding L929 mouse fibroblasts on fabricated composite scaffold, which confirmed the cell viability, high cell proliferation rate, and cell adhesion on composite scaffold as indicated by MTT assay, DNA quantification, and FE-SEM analysis of cell-scaffold construct. Thus, the ternary composite scaffold made from the combination of PCL (synthetic polymer), gelatin, and chitosan (natural polymer) may find potential application in tissue engineering.     

Morphology-controllable modeling approach for a porous scaffold structure in tissue engineering

Heterogeneous structures represent an important new frontier for twenty-first-century engineering. In this paper, based on the shape function in the finite element method, a morphology-controllable modelling approach for constructing tissue engineering (TE) bone scaffold with various irregular pores is presented. The modelling approach consists of both irregular element modelling and the whole bone scaffold modelling. Accepting the elements’ information after all-hex mesh generation as inputs, the basic pore-making element can be mapped into various irregular elements based on the shape function. In the bone scaffold modelling, the Boolean difference between the contour model of the solid entity and the pore model which can be constructed by the Boolean operation union would generate a porous bone scaffold model. Compared to the stochastic geometry method and the discrete element packing method, the bone scaffold model obtained in this paper has a continuous, smooth contour and various irregular pores. Moreover, a decrease in computational complexity is achieved in this paper.     

In vitro evaluation of electrospun PCL/nanoclay composite scaffold for bone tissue engineering

Polycaprolactone (PCL) is a widely accepted synthetic biodegradable polymer for tissue engineering, however its use in hard tissue engineering is limited because of its inadequate mechanical strength and low bioactivity. In this study, we used halloysite nanoclay (NC) as an inorganic filler material to prepare PCL/NC fibrous scaffolds via electrospinning technique after intercalating NC within PCL by solution intercalation method. The obtained nanofibrous mat was found to be mechanically superior to PCL fibrous scaffolds. These scaffolds allowed greater protein adsorption and enhanced mineralization when incubated in simulated body fluid. Moreover, our results indicated that human mesenchymal stem cells (hMSCs) seeded on these scaffolds were viable and could proliferate faster than in PCL scaffolds as confirmed by fluorescence and scanning electron microscopic observations. Further, osteogenic differentiation of hMSCs on nanoclay embedded scaffolds was demonstrated by an increase in alkaline phosphatase activity when compared to PCL scaffold without nanoclay. All of these results suggest the potential of PCL/NC scaffolds for bone tissue engineering.     

骨组织工程用纳米羟基磷灰石/ 壳聚糖多孔支架材料的制备及性能表征

以16.7%(质量分数)的柠檬酸水溶液作溶剂,通过粒子沥滤法制备了 n HA/CS多孔材料,并对其进行了IR、XRD、SEM、孔隙率及力学性能测试。结果表明n HA/CS复合材料中羟基磷灰石呈弱结晶状态,复合前后两组分的化学组成未发生显著变化,但两相间发生了相互作用。多孔材料呈高度多孔结构,孔壁上富含微孔,孔间贯通性高;复合材料/致孔剂质量比为1时,多孔材料的孔隙率为 53%,其抗压强度可达17 MPa左右,可以满足组织工程支架材料的要求。     

Fabrication of nano-structured electrospun collagen scaffold intended for nerve tissue engineering

Nerve tissue engineering is one of the most promising methods in nerve tissue regeneration. The development of blended collagen and glycosaminoglycan scaffolds can potentially be used in many soft tissue engineering applications. In this study an attempt was made to develop two types of random and aligned electrospun, nanofibrous scaffold using collagen and a common type of glycosaminoglycan. Ion chromatography test, MTT and attachment assays were conducted respectively to trace the release of glycosaminoglycan, and to investigate the biocompatibility of the scaffold. Cell cultural tests showed that the scaffold acted as a positive factor to support connective tissue cell outgrowth. The positive effect of fiber orientation on cell outgrowth organization was traced through SEM images. Porosity percentage calculation and tensile strength measurement of the webs specified analogous properties to the native neural matrix tissue. These results suggested that nanostructured porous collagen-glycosaminoglycan scaffold is a potential cell carrier in nerve tissue engineering.     

Attachment and proliferation of human osteoblast-like cells (MG-63) on laser-ablated titanium implant material

Demand is increasing for shortening the long (3–6 months) osseointegration period to rehabilitate patients' damaged chewing apparatus in as short a time as possible. For dental implants, as for biomaterials in general, the bio- and osseointegration processes can be controlled at molecular and cellular levels by modification of the implant surface. One of the most promising of such surface modifications is laser ablation, as demonstrated by our previous results [46]. Commercially pure (CP4) sand-blasted, acid-etched titanium disks (Denti® System Ltd., Hungary) were irradiated with a KrF excimer laser (248 nm, fluence 0.4 J/cm², FWHM 18 ns, 2000 pulses), or with a Nd:YAG laser (532 nm, 1.3 J/cm², 10 ns, 200 pulses) then examined by SEM, AFM, and XPS. In vitro attachment (24 h) and proliferation (72 h) of MG-63 osteoblast cells were investigated via dimethylthiazol-diphenyl tetrazolium bromide (MTT), alamarBlue (AB) assays alkaline phosphatase quantification (ALP) and SEM. SEM and AFM revealed significant changes in morphology and roughness. XPS confirmed the presence of TiO₂ on each sample; after Nd:YAG treatment a reduced state of Ti (Ti^3 +) was also observed. MTT, AB and ALP measurements detected an increase in the number of cells between the 24- and 72 hour observations; however, laser treatment did not affect cell attachment and proliferation significantly.     

A novel porous scaffold fabrication technique for epithelial and endothelial tissue engineering

Porous scaffolds have the ability to minimize transport barriers for both two- (2D) and three-dimensional tissue engineering. However, current porous scaffolds may be non-ideal for 2D tissues such as epithelium due to inherent fabrication-based characteristics. While 2D tissues require porosity to support molecular transport, pores must be small enough to prevent cell migration into the scaffold in order to avoid non-epithelial tissue architecture and compromised function. Though electrospun meshes are the most popular porous scaffolds used today, their heterogeneous pore size and intense topography may be poorly-suited for epithelium. Porous scaffolds produced using other methods have similar unavoidable limitations, frequently involving insufficient pore resolution and control, which make them incompatible with 2D tissues. In addition, many of these techniques require an entirely new round of process development in order to change material or pore size. Herein we describe “pore casting,” a fabrication method that produces flat scaffolds with deterministic pore shape, size, and location that can be easily altered to accommodate new materials or pore dimensions. As proof-of-concept, pore-cast poly(ε-caprolactone) (PCL) scaffolds were fabricated and compared to electrospun PCL in vitro using canine kidney epithelium, human colon epithelium, and human umbilical vein endothelium. All cell types demonstrated improved morphology and function on pore-cast scaffolds, likely due to reduced topography and universally small pore size. These results suggest that pore casting is an attractive option for creating 2D tissue engineering scaffolds, especially when the application may benefit from well-controlled pore size or architecture.     

Fabrication and in vivo osteogenesis of biomimetic poly(propylene carbonate) scaffold with nanofibrous chitosan network in macropores for bone tissue engineering

A biomimetic poly(propylene carbonate) (PPC) porous scaffold with nanofibrous chitosan network within macropores (PPC/CSNFs) for bone tissue engineering was fabricated by a dual solid–liquid phase separation technique. PPC scaffold with interconnected solid pore wall structure was prepared by the first phase separation, which showed a high porosity of 91.9% and a good compressive modulus of 14.2 ± 0.56 MPa, respectively. By the second phase separation, nanofibrous chitosan of 50–500 nm in diameter was formed in the macropores with little influence on the pore structure and the mechanical properties of PPC scaffold. The nanofibrous chitosan content was calculated to be 9.78% by elemental analysis. After incubation in SBF for 14 days, more apatite crystals were deposited on the pore surface as well as the nanofibrous chitosan surface of PPC/CSNFs scaffold compared with PPC scaffold. The in vitro culture of bone mesenchymal stem cells showed that PPC/CSNFs scaffold exhibited a better cell viability than PPC scaffold. After implantation in rabbits for 16 weeks, the defect was entirely repaired by PPC/CSNFs scaffold, as opposed to the incomplete healing for PPC scaffold. It indicated that PPC/CSNFs scaffold showed a faster in vivo osteogenesis rate than PPC scaffold. Hereby, PPC/CSNFs scaffold will be a potential candidate for bone tissue engineering.     

一种新型的骨组织工程用支架材料的制备与性能表征

以硼硅酸盐玻璃粉为原料,采用有机泡沫浸渍工艺,制备了高孔隙率的网眼多孔支架.应用XRD、SEM及ICP-AES等对硼酸盐生物玻璃粉末在生理模拟液中的降解性能、生物活性等进行了测试分析.结果表明,硼硅酸盐生物玻璃的降解性和生物活性与材料的组成配比有关,因此,可以通过调整玻璃的组成有效控制材料的降解性和表面形成的羟基磷灰石晶体的形态.硼硅酸盐生物活性玻璃作为硬组织工程支架材料的研究具有重要的意义和广泛的应用前景.     

Characterization of decellularized scaffold derived from porcine meniscus for tissue engineering applications

Menisci are fundamental fibrocartilaginous organs in knee joints. The injury in meniscus can impair normal knee function and predisposes patients to osteoarthritis. This study prepared decellularized meniscus scaffolds using a 1% (w/w) sodium dodecyl sulfate solution and sufficient rinsing steps. Complete cell removal was verified by hematoxylin and eosin staining and DNA content assay. Decellularized menisci had accordant tension properties to intact ones, but with declined compression properties. This occurred because the collagen fiber was not damaged but glycosaminoglycans was significantly lost during the decellularization process, which was confirmed by biochemical assay and histology staining. In vitro cytotoxicity assay demonstrated that decellularized meniscus scaffolds have no toxicity on L929 murine fibroblasts and porcine chondrocytes. Further experiment showed that porcine chondrocytes could adhere and proliferate on the scaffold surface, and some cells even could infiltrate into the scaffold. All results showed the potential of this decellularized meniscus to be the scaffolds in tissue engineering.     

Biodegradable and bioactive properties of a novel bone scaffold coated with nanocrystalline bioactive glass for bone tissue engineering

The development of the new technologies of bone tissue engineering requires the production of bioactive and biodegradable macroporous scaffolds. Hydroxyapatite (HA) ceramics are useful bone substitutes, but they degrade minimally. Tricalcium phosphates also show poor ability of Ca-P formation both in-vitro and in-vivo, although they are degradable. The present study introduces a biodegradable, bioactive, and macroporous scaffold with suitable mechanical properties. The prepared hydroxyapatite scaffold was coated with a nanocrystalline bioactive glass layer to be subsequently sintered at different temperatures. The bioactivity and degradability of the coated scaffolds were investigated by standard procedures. The ability to induce Ca-P formation in SBF (simulated body fluid) was also investigated semi-quantitatively. BS1 scaffolds (scaffolds sintered at 800 °C with a holding time of 2 h) showed remarkable bioactivity and degradability simultaneously. Formation of a nanocrystalline phase (Si₂PO₇) during the sintering considerably decreased the capability of BS1 scaffolds for Ca-P formation and the rate of degradation but enhanced their mechanical properties. The BS1 scaffolds showed not only significant bioactivity but also good degradability and suitable mechanical property.