A cell leakproof PLGA‐collagen hybrid scaffold for cartilage tissue engineering

A cell leakproof porous poly(DL ‐lactic‐co‐glycolic acid) (PLGA)‐collagen hybrid scaffold was prepared by wrapping the surfaces of a collagen sponge except the top surface for cell seeding with a bi‐layered PLGA mesh. The PLGA‐collagen hybrid scaffold had a structure consisting of a central collagen sponge formed inside a bi‐layered PLGA mesh cup. The hybrid scaffold showed high mechanical strength. The cell seeding efficiency was 90.0% when human mesenchymal stem cells (MSCs) were seeded in the hybrid scaffold. The central collagen sponge provided enough space for cell loading and supported cell adhesion, while the bi‐layered PLGA mesh cup protected against cell leakage and provided high mechanical strength for the collagen sponge to maintain its shape during cell culture. The MSCs in the hybrid scaffolds showed round cell morphology after 4 weeks culture in chondrogenic induction medium. Immunostaining demonstrated that type II collagen and cartilaginous proteoglycan were detected in the extracellular matrices. Gene expression analyses by real‐time PCR showed that the genes encoding type II collagen, aggrecan, and SOX9 were upregulated. These results indicated that the MSCs differentiated and formed cartilage‐like tissue when being cultured in the cell leakproof PLGA‐collagen hybrid scaffold. The cell leakproof PLGA‐collagen hybrid scaffolds should be useful for applications in cartilage tissue engineering. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Chitosan sponges as tissue engineering scaffolds for bone formation

Rat calvarial osteoblasts were grown in porous chitosan sponges fabricated by freeze drying. The prepared chitosan sponges had a porous structure with a 100-200 microm pore diameter, which allowed cell proliferation. Cell density, alkaline phosphatase activity and calcium deposition were monitored for up to 56 d culture. Cell numbers were 4 x 10(6) (day 1), 11 x 10(6) (day 28) and 12 x 10(6) (day 56) per g sponge. Calcium depositions were 9 (day 1), 40 (day 28) and 48 (day 56) microg per sponge. Histological results corroborated that bone formation within the sponges had occurred. These results show that chitosan sponges can be used as effective scaffolding materials for tissue engineered bone formation in vitro.     

Advances in biomimetic collagen mineralisation and future approaches to bone tissue engineering

With an ageing world population and ~20% of adults in Europe being affected by bone diseases, there is an urgent need to develop advanced regenerative approaches and biomaterials capable to facilitate tissue regeneration while providing an adequate microenvironment for cells to thrive. As the main components of bone are collagen and apatite mineral, scientists in the tissue engineering field have attempted in combining these materials by using different biomimetic approaches to favour bone repair. Still, an ideal bone analogue capable of mimicking the distinct properties (i.e., mechanical properties, degradation rate, porosity, etc.) of cancellous bone is to be developed. This review seeks to sum up the current understanding of bone tissue mineralisation and structure while providing a critical outlook on the existing biomimetic strategies of mineralising collagen for bone tissue engineering applications, highlighting where gaps in knowledge exist.     

Nanoparticles for bone tissue engineering

Tissue engineering (TE) envisions the creation of functional substitutes for damaged tissues through integrated solutions, where medical, biological, and engineering principles are combined. Bone regeneration is one of the areas in which designing a model that mimics all tissue properties is still a challenge. The hierarchical structure and high vascularization of bone hampers a TE approach, especially in large bone defects. Nanotechnology can open up a new era for TE, allowing the creation of nanostructures that are comparable in size to those appearing in natural bone. Therefore, nanoengineered systems are now able to more closely mimic the structures observed in naturally occurring systems, and it is also possible to combine several approaches ‐ such as drug delivery and cell labeling ‐ within a single system. This review aims to cover the most recent developments on the use of different nanoparticles for bone TE, with emphasis on their application for scaffolds improvement; drug and gene delivery carriers, and labeling techniques. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:590–611, 2017     

Poly(lactic-co-glycolic acid) hollow fibre membranes for use as a tissue engineering scaffold

Mass transfer limitations of scaffolds are currently hindering the development of 3-dimensional, clinically viable, tissue engineered constructs. We have developed a poly(lactide-co-glycolide) (PLGA) hollow fibre membrane scaffold that will provide support for cell culture, allow psuedovascularisation in vitro and provide channels for angiogenesis in vivo. We produced P(DL)LGA flat sheet membranes using 1, 4-dioxane and 1-methyl-2-pyrrolidinone (NMP) as solvents and water as the nonsolvent, and hollow fibre membranes, using NMP and water, by dry/wet- and wet-spinning. The resulting fibres had an outer diameter of 700 micro m and an inner diameter of 250 micro m with 0.2-1.0 micro m pores on the culture surface. It was shown that varying the air gap and temperature when spinning changed the morphology of the fibres. The introduction of a 50 mm air gap caused a dense skin of 5 micro m thick to form, compared to a skin of 0.5 micro m thick without an air gap. Spinning at 40 degrees C produced fibres with a more open central section in the wall that contained more, larger macrovoids compared to fibres spun at 20 degrees C. Culture of the immortalised osteogenic cell line 560pZIPv.neo (pZIP) was carried out on the P(DL)LGA flat sheets in static culture and in a P(DL)LGA hollow fibre bioreactor under counter-current flow conditions. Attachment and proliferation was statistically similar to tissue culture polystyrene on the flat sheets and was also successful in the hollow fibre bioreactor. The P(DL)LGA hollow fibres are a promising scaffold to address the size limitations currently seen in tissue engineered constructs.     

The utility of human dedifferentiated fat cells in bone tissue engineering in vitro

We compared the osteoblastic differentiation abilities of dedifferentiated fat cells (DFATs) and human bone marrow mesenchymal stem cells (hMSCs) as a cell source for bone regeneration therapies. In addition, the utility of DFATs in bone tissue engineering in vitro was assessed by an alpha-tricalcium phosphate (α-TCP)/collagen sponge (CS). Human DFATs were isolated from the submandibular of a patient by ceiling culture. DFATs and hMSCs at passage 3 were cultured in control medium or osteogenic medium (OM) for 14 days. Runx2 gene expression, alkaline phosphatase (ALP) activity, as well as osteocalcin (OCN) and calcium contents were analyzed to evaluate the osteoblastic differentiation ability of both cell types. DFATs seeded in a α-TCP/CS and cultured in OM for 14 days were analyzed by scanning electron microscopy (SEM) and histologically. Compared with hMSCs, DFATs cultured in OM generally underwent superior osteoblastogenesis by higher Runx2 gene expression at all days tested, as well as higher ALP activity at day 3 and 7, OCN expression at day 14, and calcium content at day 7. In SEM analyses, DFATs seeded in a α-TCP/CS were well spread and covered the α-TCP/CS by day 7. In addition, numerous spherical deposits were found to almost completely cover the α-TCP/CS on day 14. Von Kossa staining showed that DFATs differentiated into osteoblasts in the α-TCP/CS and formed cultured bone by deposition of a mineralized extracellular matrix. The combined use of DFATs and an α-TCP/CS may be an attractive option for bone tissue engineering.     

Biomechanics in bone tissue engineering

Biomechanics may be considered as central in the development of bone tissue engineering. The initial mechanical aspects are essential to the outcome of a functional tissue engineering approach; so are aspects of interface micromotion, bone ingrowths inside the scaffold and finally, the mechanical integrity of the scaffold during its degradation. A proposed view is presented herein on how biomechanical aspects can be synthesised and where future developments are needed. In particular, a distinction is made between the mechanical and the mechanotransductional aspects of bone tissue engineering: the former could be related to osteoconduction, while the latter may be correlated to the osteoinductive properties of the scaffold. This distinction allows biomechanicians to follow a strategy in the development of a scaffold having not only mechanical targets but also incorporating some mechanotransduction principles.     

Optimizing the medium perfusion rate in bone tissue engineering bioreactors

There is a critical need to increase the size of bone grafts that can be cultured in vitro for use in regenerative medicine. Perfusion bioreactors have been used to improve the nutrient and gas transfer capabilities and reduce the size limitations inherent to static culture, as well as to modulate cellular responses by hydrodynamic shear. Our aim was to understand the effects of medium flow velocity on cellular phenotype and the formation of bone‐like tissues in three‐dimensional engineered constructs. We utilized custom‐designed perfusion bioreactors to culture bone constructs for 5 weeks using a wide range of superficial flow velocities (80, 400, 800, 1,200, and 1,800 µm/s), corresponding to estimated initial shear stresses ranging from 0.6 to 20 mPa. Increasing the flow velocity significantly affected cell morphology, cell–cell interactions, matrix production and composition, and the expression of osteogenic genes. Within the range studied, the flow velocities ranging from 400 to 800 µm/s yielded the best overall osteogenic responses. Using mathematical models, we determined that even at the lowest flow velocity (80 µm/s) the oxygen provided was sufficient to maintain viability of the cells within the construct. Yet it was clear that this flow velocity did not adequately support the development of bone‐like tissue. The complexity of the cellular responses found at different flow velocities underscores the need to use a range of evaluation parameters to determine the quality of engineered bone. Bioeng. 2011; 108:1159–1170. © 2010 Wiley Periodicals, Inc.     

基于组织工程研究的可降解支架材料选择策略

目前,器官或组织移植是治疗器官衰竭或大范围组织缺损唯一长期有效的方法,但存在供体短缺、免疫排斥等问题。组织工程技术作为一种潜在的替代治疗方法,支架材料的选择是其中具有决定意义的组成部分。组织工程支架材料按其来源可分为天然及其改性修饰材料、人工合成与复合支架材料3种。组织工程目的就是修复临床上的病损组织或器官,并达到较理想的结构和功能的恢复。因此组织工程支架也必须从基本性质上具有一定的仿生化结构及功能,即"活"支架,这样才能彻底代替病损组织或器官。通过多种支架材料的优化组合(即材料的复合),对材料进行表面改性、制备工艺优化及添加细胞因子缓释微球等技术,模拟病损器官组织的特性及周围环境,有望打开组织工程的新局面。理想的组织工程支架应当以临床需要为根本目的,依靠材料学、分子生物学、工程学等多学科间的交叉研究,取各家之长,优化配比组合,达到仿生的目的。本课题组前期工作已经将骨髓间充质干细胞体外诱导分化为胆管上皮样细胞,并设计出左旋聚乳酸/聚己内酯共聚物(PLCL)胆道支架,内部混有包含生长因子的纳米缓释微球,供细胞因子的远期释放,支架内表面涂有基质胶/胶原混合层,且胶内加入bFGF、EGF,提供诱导因子的早期释放。将诱导细胞与PLCL胆道支架复合,制备组织工程胆管。文中综述了现存各类支架材料的研究状况,简单介绍了制备工艺、表面修饰等影响支架性能的因素,力求探索组织工程支架材料的选择策略。     

Perinatal stem cells: A promising cell resource for tissue engineering of craniofacial bone

In facing the mounting clinical challenge and suboptimal techniques of craniofacial bone defects resulting from various conditions, such as congenital malformations, osteomyelitis, trauma and tumor resection, the ongoing research of regenerative medicine using stem cells and concurrent advancement in biotechnology have shifted the focus from surgical reconstruction to a novel stem cell-based tissue engineering strategy for customized and functional craniofacial bone regeneration. Given the unique ontogenetical and cell biological properties of perinatal stem cells, emerging evidence has suggested these extraembryonic tissue-derived stem cells to be a promising cell source for extensive use in regenerative medicine and tissue engineering. In this review, we summarize the current achievements and obstacles in stem cell-based craniofacial bone regeneration and subsequently we address the characteristics of various types of perinatal stem cells and their novel application in tissue engineering of craniofacial bone. We propose the promising feasibility and scope of perinatal stem cell-based craniofacial bone tissue engineering for future clinical application.     

明胶微球在骨组织工程中的应用

骨组织工程通过联合利用种子细胞、生物活性因子和支架材料等要素来构建骨组织再生微环境,从而促进骨缺损的修复重建来诱导骨再生。明胶微球具有多孔性、生物降解性、生物相容性及生物安全性等优势,是一种极具应用潜能的骨修复材料。明胶微球用于体外培养种子细胞时可实现高效扩增。多官能团结构使其可作为促血管再生因子、促骨再生因子及抗感染因子等多种药物的递送载体,缓释药物的同时也可实现微球的多功能化。在构建明胶微球支架时与其他生物材料复合及血管化性能的赋予可提高支架材料的综合性能,但目前支架的设计还存在如何兼顾材料多孔结构和力学性能的问题。本文主要综述了明胶微球的常见制备技术及其近年来在骨组织工程中的应用,并对未来的发展前景进行展望。     

Numerical and experimental evaluation of TPMS Gyroid scaffolds for bone tissue engineering

The combination of computational methods with 3D printing allows for the control of scaffolds microstructure. Lately, triply periodic minimal surfaces (TPMS) have been used to design porosity-controlled scaffolds for bone tissue engineering (TE). The goal of this work was to assess the mechanical properties of TPMS Gyroid structures with two porosity levels (50 and 70%). The scaffold stiffness function of porosity was determined by the asymptotic homogenisation method and confirmed by mechanical testing. Additionally, microCT analysis confirmed the quality of the printed parts. Thus, the potential of both design and manufacturing processes for bone TE applications is here demonstrated.     

Cell-based approaches to the engineering of vascularized bone tissue

This review summarizes recent efforts to create vascularized bone tissue in vitro and in vivo through the use of cell-based therapy approaches. The treatment of large and recalcitrant bone wounds is a serious clinical problem, and in the United States approximately 10% of all fractures are complicated by delayed union or non-union. Treatment approaches with the use of growth factor and gene delivery have shown some promise, but results are variable and clinical complications have arisen. Cell-based therapies offer the potential to recapitulate key components of the bone-healing cascade, which involves concomitant regeneration of vasculature and new bone tissue. For this reason, osteogenic and vasculogenic cell types have been combined in co-cultures to capitalize on the function of each cell type and to promote heterotypic interactions. Experiments in both two-dimensional and three-dimensional systems have provided insight into the mechanisms by which osteogenic and vasculogenic cells interact to form vascularized bone, and these approaches have been translated to ectopic and orthotopic models in small-animal studies. The knowledge generated by these studies will inform and facilitate the next generation of pre-clinical studies, which are needed to move cell-based orthopaedic repair strategies into the clinic. The science and application of cytotherapy for repair of large and ischemic bone defects is developing rapidly and promises to provide new treatment methods for these challenging clinical problems.     

Proliferation and tenogenic differentiation of bone marrow mesenchymal stem cells in a porous collagen sponge scaffold

BACKGROUND Collagen is one of the most commonly used natural biomaterials for tendon tissue engineering.One of the possible practical ways to further enhance tendon repair is to combine a porous collagen sponge scaffold with a suitable growth factor or cytokine that has an inherent ability to promote the recruitment,proliferation,and tenogenic differentiation of cells.However,there is an incomplete understanding of which growth factors are sufficient and optimal for the tenogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs)in a collagen sponge-based 3D culture system.AIM To identify one or more ideal growth factors that benefit the proliferation and tenogenic differentiation of rat BMSCs in a porous collagen sponge scaffold.METHODS We constructed a 3D culture system based on a type I collagen sponge scaffold.The surface topography of the collagen sponge scaffold was observed by scanning electron microscopy.Primary BMSCs were isolated from Sprague-Dawley rats.Cell survival on the surfaces of the scaffolds with different growth factors was assessed by live/dead assay and CCK-8 assay.The mRNA and protein expression levels were confirmed by quantitative real-time polymerase chain reaction and Western blot,respectively.The deposited collagen was assessed by Sirius Red staining.RESULTS Transforming growth factorβ1(TGF-β1)showed great promise in the tenogenic differentiation of BMSCs compared to growth differentiation factor 7(GDF-7)and insulin-like growth factor 1(IGF-1)in both the 2D and 3D cultures,and the 3D culture enhanced the differentiation of BMSCs into tenocytes well beyond the level of induction in the 2D culture after TGF-β1 treatment.In the 2D culture,the proliferation of the BMSCs showed no significant changes compared to the control group after TGF-β1,IGF-1,or GDF-7 treatment.However,TGF-β1 and GDF-7 could increase the cell proliferation in the 3D culture.Strangely,we also found more dead cells in the BMSC-collagen sponge constructs that were treated with TGF-β1.Moreover,TGF-β1 promoted more collagen deposition in both the 2D and 3D cultures.CONCLUSION Collagen sponge-based 3D culture with TGF-β1 enhances the responsiveness of the proliferation and tenogenic differentiation of rat BMSCs.     

In silico multi‐scale model of transport and dynamic seeding in a bone tissue engineering perfusion bioreactor

Computer simulations can potentially be used to design, predict, and inform properties for tissue engineering perfusion bioreactors. In this work, we investigate the flow properties that result from a particular poly‐L ‐lactide porous scaffold and a particular choice of perfusion bioreactor vessel design used in bone tissue engineering. We also propose a model to investigate the dynamic seeding properties such as the homogeneity (or lack of) of the cellular distribution within the scaffold of the perfusion bioreactor: a pre‐requisite for the subsequent successful uniform growth of a viable bone tissue engineered construct. Flows inside geometrically complex scaffolds have been investigated previously and results shown at these pore scales. Here, it is our aim to show accurately that through the use of modern high performance computers that the bioreactor device scale that encloses a scaffold can affect the flows and stresses within the pores throughout the scaffold which has implications for bioreactor design, control, and use. Central to this work is that the boundary conditions are derived from micro computed tomography scans of both a device chamber and scaffold in order to avoid generalizations and uncertainties. Dynamic seeding methods have also been shown to provide certain advantages over static seeding methods. We propose here a novel coupled model for dynamic seeding accounting for flow, species mass transport and cell advection‐diffusion‐attachment tuned for bone tissue engineering. The model highlights the timescale differences between different species suggesting that traditional homogeneous porous flow models of transport must be applied with caution to perfusion bioreactors. Our in silico data illustrate the extent to which these experiments have the potential to contribute to future design and development of large‐scale bioreactors. Biotechnol. Bioeng. 2013; 110: 1221–1230. © 2012 Wiley Periodicals, Inc.     

Decoration of electrical conductive polyurethane-polyaniline/polyvinyl alcohol matrixes with mussel-inspired polydopamine for bone tissue engineering

Electrospinning is a versatile technology for the fabrication of nanofibrous matrixes to regenerate defects. This study aims to develop a functionalized and electroconductive polymeric matrix to improve rat bone marrow mesenchymal stem cell adhesion, proliferation, and differentiation. Herein, the influence of the chemical composition of the substrate on homogeneous modification of the surface with mussel-inspired polydopamine (PDA) is focused. Accordingly, the deposition of PDA on the surface was proved by Fourier transform infrared spectroscopy. Morphologies of the scaffolds demonstrated homogeneous decoration of the polyvinyl alcohol (PVA)/polyurethane (PU)-polyaniline (PANI) matrixes with PDA, while a lower density of mussel-inspired polymer was observed in bare PU-PANI constructs. Although uniform and dense precipitation of PDA reduced conductivity of scaffolds 1.2 times compared with the samples with a low density of the PDA, 1.1 and 1.2 times enhancement in tensile strength and Young's modulus, respectively, were the strength of the applied process, especially in bone tissue engineering area. Contact angle measurements demonstrated about two times reduction in measured values, which shows improvement in hydrophilicity of PDA-modified PVA/PU-PANI fibers compared with PDA-coated PU-PANI ones. Swelling ratio and mass loss ratio calculations revealed enhancement in measured values as a function of homogeneous and dense coating, which arise from hydrophilicity of the polymeric substrate. The bioactivity test indicated that a dense layer of PDA strongly supports formations of hydroxyapatite-like crystals. Moreover, homogeneous decoration of conductive matrixes with PDA showed suitable cell viability, adhesion, and spreading while cell-scaffolds interactions improved under electrical stimulation. Higher expression of alkaline phosphatase and secretion of Collagen I under the electrical field proved the applicability of modified electroconductive scaffolds for further preclinical and clinical studies to introduce as a reconstructive bone substitute.     

A novel method for quantifying cell migration through tissue engineering scaffolds: evaluation of modified collagen matrices

A novel method to quantify cell migration through potential tissue engineering 3-d scaffolds is described. The migration assay uses a dot-blotting apparatus into which the tissue engineering matrix is placed on top of a nitrocellulose membrane. This assay was used to evaluate human dermal fibroblast migration through four porcine collagen matrices with varying pore diameters and pitch lengths. Fibroblasts were placed on the matrix surface, at between 1 ×10³–3 × 10³ cells mm^–2, and left for 18 h to allow migration. The nitrocellulose membrane was stained with haematoxylin, the membrane digitised and the pixel intensity of the stained cells quantified. We showed that for all matrix variants, migration was more effective with a higher initial seeding density. The application of varying initial cell densities resulted in the greatest extent of cell migration through the matrix variant with pores of 30 m diameter and 400 m pitch length (i.e. 10.3% migration at 1 ×10³ cells mm^–2). This method was coupled with confocal microscopy to evaluate the depth of cell migration within the matrix. At a depth of 20 m cell numbers were similar to those on the matrix surface: at a depth of 100 m only a few cells were observed.     

Analysis of cell growth and diffusion in a scaffold for cartilage tissue engineering

Developments in tissue engineering over the past decade have offered promising future for the repair and reconstruction of damaged tissues. To regenerate three dimensional and weight-bearing implants, advances in biomaterials and manufacturing technologies prompted cell cultivations with natural or artificial scaffolds, in which cells are allowed to proliferate, migrate, and differentiate in vitro. In this article, we develop a mathematical model for cell growth in a porous scaffold. By treating the cell-scaffold construct as a porous medium, a continuum model is set up based on basic principles of mass conservation. In addition to cell growth kinetics, we incorporate cell diffusion in the model to describe the effects of cell random walks. Computational results are compared to experimental data found in the literature. With this model, we are able to investigate cell motility, heterogeneous cell distributions, and non-uniform seeding for tissue engineering applications. Results show that random walks tend to enhance uniform cell spreads in space, which in turn increases the probabilities for cells to acquire nutrients; therefore random walks are likely to be a positive contribution to the overall cell growth on scaffolds.