Hierarchically Inverse Opal Porous Scaffolds from Droplet Microfluidics for Biomimetic 3D Cell Co-Culture

With the advantages of better mimicking the specificity of natural tissues, three-dimensional (3D) cell culture plays a major role in drug development, toxicity testing, and tissue engineering. However, existing scaffolds or microcarriers for 3D cell culture are often limited in size and show suboptimal performance in simulating the vascular complexes of living organisms. Therefore, we present a novel hierarchically inverse opal porous scaffold made via a simple microfluidic approach for promoting 3D cell co-culture techniques. The designed scaffold is constructed using a combined concept involving an emulsion droplet template and inert polymer polymerization. This work demonstrates that the resultant scaffolds ensure a sufficient supply of nutrients during cell culture, so as to achieve large-volume cell culture. In addition, by serially planting different cells in the scaffold, a 3D co-culture system of endothelial-cell-encapsulated hepatocytes can be developed for constructing certain functional tissues. It is also demonstrated that the use of the proposed scaffold for a co-culture system helps hepatocytes to maintain specific in vivo functions. These hierarchically inverse opal scaffolds lay the foundation for 3D cell culture and even the construction of biomimetic tissues.     

3-D Nanofibrous electrospun multilayered construct is an alternative ECM mimicking scaffold

Extra cellular matrix (ECM) is a natural cell environment, possesses complicated nano- and macro- architecture. Mimicking this three-dimensional (3-D) web is a challenge in the modern tissue engineering. This study examined the application of a novel 3-D construct, produced by multilayered organization of electrospun nanofiber membranes, for human bone marrow-derived mesenchymal stem cells (hMSCs) support. The hMSCs were seeded on an electrospun scaffold composed of poly ε-caproloactone (PCL) and collagen (COL) (1:1), and cultured in a dynamic flow bioreactor prior to in vivo implantation. Cell viability after seeding was analyzed by AlamarBlue™ Assay. At the various stages of experiment, cell morphology was examined by histology, scanning electron microscopy (SEM) and confocal microscopy. Results: A porous 3-D network of randomly oriented nanofibers appeared to support cell attachment in a way similar to traditionally used tissue culture polysterene plate. The following 6 week culture process of the tested construct in the dynamic flow system led to massive cell proliferation with even distribution inside the scaffold. Subcutaneous implantation of the cultured construct into nude mice demonstrated good integration with the surrounding tissues and neovascularization. Conclusion: The combination of electrospinning technology with multilayer technique resulted in the novel 3-D nanofiber multilayered construct, able to contain efficient cell mass necessary for a successful in vivo grafting. The success of this approach with undifferentiated cells implies the possibility of its application as a platform for development of constructs with cells directed into various tissue types.     

开环乳糖交联壳聚糖微载体的制备及肝细胞亲和性研究

以液体石蜡作分散介质,开环乳糖作交联剂,反相悬浮法制备了性能优良的壳聚糖微栽体。用其进行原代大鼠肝细胞培养。利用相差显微镜和扫描电镜对培养结果进行观察,并测定了细胞的代谢活性。肝细胞在开环乳糖交联壳聚糖微载体上保持良好的球形状态,白蛋白的分泌量达到36.4μg／24h／mL,明显高于未修饰壳聚糖和Cytodex3微载体上肝细胞的白蛋白分泌量(分别为26．0和26.7μg／24h／mL),表明开环乳糖交联壳聚糖微载体是一种优良的肝细胞培养支架。     

Design of novel 3D gene activated PEG scaffolds with ordered pore structure

The ability to genetically modify cells seeded inside synthetic hydrogel scaffolds offers a suitable approach to induce and control tissue repair and regeneration guiding cell fate. In fact the transfected cells can act as local in vivo bioreactor, secreting plasmid encoded proteins that augment tissue regeneration processes. We have realized a DNA bioactivated high porous poly(ethylene glycol) (PEG) matrix by polyethyleneimine (PEI)/DNA complexes adsorption. As the design of the microarchitectural features of a scaffold also contributes to promote and influence cell fate, we appropriately designed the inner structure of gene activated PEG hydrogels by gelatine microparticles templating. Microarchitectural properties of the scaffold were analysed by scanning electron microscopy. 3D cell migration and transfection were monitored through time-lapse videomicroscopy and confocal laser scanning microscopy.     

3D-Cultivation of bone marrow stromal cells on hydroxyapatite scaffolds fabricated by dispense-plotting and negative mould technique

The main principle of a bone tissue engineering (BTE) strategy is to cultivate osteogenic cells in an osteoconductive porous scaffold. Ceramic implants for osteogenesis are based mainly on hydroxyapatite (HA), since this is the inorganic component of bone. Rapid Prototyping (RP) is a new technology in research for producing ceramic scaffolds. This technology is particularly suitable for the fabrication of individually and specially tailored single implants. For tissue engineering these scaffolds are seeded with osteoblast or osteoblast precursor cells. To supply the cultured osteoblastic cells efficiently with nutrition in these 3D-geometries a bioreactor system can be used. The aim of this study was to analyse the influence of differently fabricated HA-scaffolds on bone marrow stromal cells. For this, two RP-techniques, dispense-plotting and a negative mould method, were used to produce porous ceramics. The manufactured HA-scaffolds were then cultivated in a dynamic system (bioreactor) with an osteoblastic precursor cell line. In our study, the applied RP-techniques give the opportunity to design and process HA-scaffolds with defined porosity, interconnectivity and 3D pore distribution. A higher differentiation of bone marrow stromal cells could be detected on the negative mould fabricated scaffolds, while cell proliferation was higher on the dispense-plotted scaffolds. Nevertheless, both scaffold types can be used in tissue engineering applications.     

Simultaneous expansion and harvest of hematopoietic stem cells and mesenchymal stem cells derived from umbilical cord blood

The simultaneous expansion and harvest of hematopoietic stem cells and mesenchymal stem cells derived from umbilical cord blood were carried out using bioreactors. The co-culture of umbilical cord blood (UCB)-derived hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) was performed within spinner flasks and a rotating wall vessel (RWV) bioreactor using glass-coated styrene copolymer (GCSC) microcarriers. The medium used was composed of serum-free IMDM containing a cocktail of SCF 15 ng·mL^?1, FL 5 ng·mL^?1, TPO 6 ng·mL^?1, IL-3 15 ng·mL^?1, G-CSF 1 ng·mL^?1 and GM-CSF 5 ng·mL^?1. Accessory stromal cells derived from normal allogeneic adipose tissue were encapsulated in alginate-chitosan (AC) beads and used as feeding cells. The quality of the harvested UCB-HSCs and MSCs was assessed by immunophenotype analysis, methylcellulose colony and multi-lineage differentiation assays. After 12 days of culture, the fold-expansion of total cell numbers, colony-forming units (CFU-C), CD34⁺/CD45⁺/CD105^? (HSCs) cells and CD34^?/CD45^?/CD105⁺ (MSCs) cells using the RWV bioreactor were (3.7 ± 0.3)- , (5.1 ± 1.2)- , (5.2 ± 0.4)- , and (13.9 ± 1.2)-fold respectively, significantly better than those obtained using spinner flasks. Moreover, UCB-HSCs and UCB-MSCs could be easily separated by gravity sedimentation after the co-culture period as only UCB-MSCs adhered on to the microcarriers. Simultaneously, we found that the fibroblast-like cells growing on the surface of the GCSC microcarriers could be induced and differentiated towards the osteoblastic, chondrocytic and adipocytic lineages. Phenotypically, these cells were very similarly to the MSCs derived from bone marrow positively expressing the MSCs-related markers CD13, CD44, CD73 and CD105, while negatively expressing the HSCs-related markers CD34, CD45 and HLA-DR. It was thus demonstrated that the simultaneous expansion and harvest of UCB-HSCs and UCB-MSCs is possible to be accomplished using a feasible bioreactor culture system such as the RWV bioreactor with the support of GCSC microcarriers.     

A comparative study of the chondrogenic potential between synthetic and natural scaffolds in an in vivo bioreactor

Abstract

The clinical demand for cartilage tissue engineering is potentially large for reconstruction defects resulting from congenital deformities or degenerative disease due to limited donor sites for autologous tissue and donor site morbidities. Cartilage tissue engineering has been successfully applied to the medical field: a scaffold pre-cultured with chondrocytes was used prior to implantation in an animal model. We have developed a surgical approach in which tissues are engineered by implantation with a vascular pedicle as an in vivo bioreactor in bone and adipose tissue engineering. Collagen type II, chitosan, poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) were four commonly applied scaffolds in cartilage tissue engineering. To expand the application of the same animal model in cartilage tissue engineering, these four scaffolds were selected and compared for their ability to generate cartilage with chondrocytes in the same model with an in vivo bioreactor. Gene expression and immunohistochemistry staining methods were used to evaluate the chondrogenesis and osteogenesis of specimens. The result showed that the PLGA and PCL scaffolds exhibited better chondrogenesis than chitosan and type II collagen in the in vivo bioreactor. Among these four scaffolds, the PCL scaffold presented the most significant result of chondrogenesis embedded around the vascular pedicle in the long-term culture incubation phase.     

Biocompatible biodegradable polycaprolactone/basil seed mucilage scaffold for cell culture

In this study, a polymer obtained from the basil seed mucilage (BSM) in combination with polycaprolactone (PCL), was used in the 2D scaffold production process for cell culture. First, combinations of two polymers with different ratios and concentrations were prepared and electrospun. Among these samples, a sample with a BSM/PCL ratio of 2/3 was used to perform different tests due to its fibre uniformity and appropriate diameter. The Fourier transform infrared spectrometer test was carried out to chemically analyse the scaffold, the X‐ray diffraction test to determine the crystallinity of the scaffold, and the contact angle test to determine the hydrophilicity of the scaffold. The strength, porosity, and degradation percentage of the scaffold were also studied. With appropriate conditions of the scaffold for cell culture determined, Vero epithelial cells were cultured on the scaffold. Results obtained from cell culture indicated that the adhesion of the scaffold was suitable for the appropriate growth cells.Inspec keywords: adhesion, porosity, hydrophilicity, tissue engineering, X‐ray diffraction, electrospinning, cellular biophysics, contact angle, biodegradable materials, biomedical materials, polymer blends, Fourier transform infrared spectraOther keywords: X‐ray diffraction test, contact angle test, cell culture, biocompatible biodegradable scaffold, polycaprolactone‐basil seed mucilage scaffold, Fourier transform infrared spectrometer test, scaffold hydrophilicity, Vero epithelial cells, scaffold crystallinity, 2D scaffold production process     

Bioreactors in Tissue Engineering

Tissue engineering is a promising interdisciplinary scientific field of regenerative medicine. Aiming at the structural and functional restoration of damaged tissues and organs, it possesses a role of significant socioeconomical impact. In the course towards the ultimate goal of artificially constructed natural organs, our knowledge of the elementary constitutive components of living organisms and the intrinsic mechanisms that drive their interactions is greatly enhanced. Bioreactors are valuable tools providing the technological means to investigate fundamental issues for basic research and to improve tissue‐engineering products for clinical applications. They are devices enabling the in vitro simulation of the in vivo biological, physical and mechanical environment of growing tissues. In this review paper, we discuss the general demands defining the design considerations for modern bioreactor systems. These criteria originate from physiological characteristics of the cells and biochemical and mechanical properties of the extracellular matrix (ECM). In this context, we present an overview of the various bioreactor systems dedicated to the study of specific functional tissues developed by numerous research groups.     

Development of three-dimensional tissue engineered bone-oral mucosal composite models

Tissue engineering of bone and oral mucosa have been extensively studied independently. The aim of this study was to develop and investigate a novel combination of bone and oral mucosa in a single 3D in vitro composite tissue mimicking the natural structure of alveolar bone with an overlying oral mucosa. Rat osteosarcoma (ROS) cells were seeded into a hydroxyapatite/tri-calcium phosphate scaffold and bone constructs were cultured in a spinner bioreactor for 3 months. An engineered oral mucosa was fabricated by air/liquid interface culture of immortalized OKF6/TERET-2 oral keratinocytes on collagen gel-embedded fibroblasts. EOM was incorporated into the engineered bone using a tissue adhesive and further cultured prior to qualitative and quantitative assessments. Presto Blue assay revealed that ROS cells remained vital throughout the experiment. The histological and scanning electron microscope examinations showed that the cells proliferated and densely populated the scaffold construct. Micro computed tomography (micro-CT) scanning revealed an increase in closed porosity and a decrease in open and total porosity at the end of the culture period. Histological examination of bone-oral mucosa model showed a relatively differentiated parakeratinized epithelium, evenly distributed fibroblasts in the connective tissue layer and widely spread ROS cells within the bone scaffold. The feasibility of fabricating a novel bone-oral mucosa model using cell lines is demonstrated. Generating human ‘normal’ cell-based models with further characterization is required to optimize the model for in vitro and in vivo applications.     

Design and 3D Printing of Scaffolds and Tissues

A growing number of three-dimensional(3D)-print- ing processes have been applied to tissue engineering. This paper presents a state-of-the-art study of 3D-printing technologies for tissue-engineering applications, with particular focus on the development of a computer-aided scaffold design system; the direct 3D printing of functionally graded scaffolds; the modeling of selective laser sintering(SLS) and fused deposition modeling(FDM) processes; the indirect additive manufacturing of scaffolds, with both micro and macro features; the development of a bioreactor; and 3D/4D bioprinting. Technological limitations will be discussed so as to highlight the possibility of future improvements for new 3D-printing methodologies for tissue engineering.     

Expansion and preservation of multipotentiality of rabbit bone-marrow derived mesenchymal stem cells in dextran-based microcarrier spin culture

The use of mesenchymal stem cells (MSCs) in tissue repair and regeneration despite their multipotentiality has been limited by their cell source quantity and decelerating proliferative yield efficiency. A study was thus undertaken to determine the feasibility of using microcarrier beads in spinner flask cultures for MSCs expansion and compared to that of conventional monolayer cultures and static microcarrier cultures. Isolation and characterization of bone marrow derived MSCs were conducted from six adult New Zealand white rabbits. Analysis of cell morphology on microcarriers and culture plates at different time points (D0, D3, D10, D14) during cell culture were performed using scanning electron microscopy and bright field microscopy. Cell proliferation rates and cell number were measured over a period of 14 days, respectively followed by post-expansion characterization. MTT proliferation assay demonstrated a 3.20 fold increase in cell proliferation rates in MSCs cultured on microcarriers in spinner flask as compared to monolayer cultures (p < 0.05). Cell counts at day 14 were higher in those seeded on stirred microcarrier cultures (6.24 ± 0.0420 cells/ml) × 10⁵ as compared to monolayer cultures (0.22 ± 0.004 cells/ml) × 10⁵ and static microcarrier cultures (0.20 ± 0.002 cells/ml) × 10⁵. Scanning electron microscopy demonstrated an increase in cell colonization of the cells on the microcarriers in stirred cultures. Bead-expanded MSCs were successfully differentiated into osteogenic and chondrogenic lineages. This system offers an improved and efficient alternative for culturing MSCs with preservation to their phenotype and multipotentiality.     

PAN hollow fiber membranes elicit functional hippocampal neuronal network

This study focuses on the development of an advanced in vitro biohybrid culture model system based on the use of hollow fibre membranes (HFMs) and hippocampal neurons in order to promote the formation of a high density neuronal network. Polyacrylonitrile (PAN) and modified polyetheretherketone (PEEK-WC) membranes were prepared in hollow fibre configuration. The morphological and metabolic behaviour of hippocampal neurons cultured on PAN HF membranes were compared with those cultured on PEEK-WC HF. The differences of cell behaviour between HFMs were evidenced by the morphometric analysis in terms of axon length and also by the investigation of metabolic activity in terms of neurotrophin secretion. These findings suggested that PAN HFMs induced the in vitro reconstruction of very highly functional and complex neuronal networks. Thus, these biomaterials could potentially be used for the in vitro realization of a functional hippocampal tissue analogue for the study of neurobiological functions and/or neurodegenerative diseases.     

Fabrication of nanocrystalline hydroxyapatite doped degradable composite hollow fiber for guided and biomimetic bone tissue engineering

Natural bone tissue possesses a nanocomposite structure interwoven in a three-dimensional (3-D) matrix, which plays critical roles in conferring appropriate physical and biological properties to the bone tissue. Single type of material may not be sufficient to mimic the composition, structure and properties of native bone, therefore, composite materials consisting of both polymers, bioceramics, and other inorganic materials have to be designed. Among a variety of candidate materials, polymer-nanoparticle composites appear most promising for bone tissue engineering applications because of superior mechanical properties, improved durability, and surface bioactivity when compared with conventional polymers or composites. The long term objective of this project is to use highly aligned, bioactive, biodegradable scaffold mimicking natural histological structure of human long bone, and to engineer and regenerate human long bone both in vitro and in vivo. In this study, bioactive, degradable, and highly permeable composite hollow fiber membranes (HFMs) were fabricated using a wet phase phase-inversion approach. The structure of the hollow fiber membranes was examined using scanning electron microscopy (SEM); degradation behavior was examined using weigh loss assay, gel permeation chromatography (GPC), and differential scanning calorimetry (DSC); and bioactivity was evaluated with the amount of calcium deposition from the culture media onto HFM surface. Doping PLGA HFMs with nanoHA results in a more bioactive and slower degrading HFM than pure PLGA HFMs.     

Fabrication of hierarchical polycaprolactone/gel scaffolds via combined 3D bioprinting and electrospinning for tissue engineering

It is a severe challenge to construct 3D scaffolds which hold controllable pore structure and similar morphology of the natural extracellular matrix(ECM).In this study,a compound technology is proposed by combining the 3D bioprinting and electrospinning process to fabricate 3D scaffolds,which are composed by orthogonal array gel microfibers in a grid-like arrangement and intercalated by a nonwoven structure with randomly distributed polycaprolactone(PCL) nanofibers.Human adiposederived stem cells(hASCs) are seeded on the hierarchical scaffold and cultured 21 d for in vitro study.The results of cells culturing show that the microfibers structure with controlled pores can allow the easy entrance of cells and the efficient diffusion of nutrients,and the nanofiber webs layered in the scaffold can significantly improve initial cell attachment and proliferation.The present work demonstrates that the hierarchical PCL/gel scaffolds consisting of controllable 3D architecture with interconnected pores and biomimetic nanofiber structures resembling the ECM can be designed and fabricated by the combination of 3D bioprinting and electrospinning to improve biological performance in tissue engineering applications.     

Biodegradable GdPO4·H2O/PLGA microcarriers for stem cell delivery and non-invasive MRI translocation tracing

Within brain tissue engineering, stem cell implantation assisted by biodegradable injectable scaffolds is a crucial method. However, implanted scaffolds, especially microcarriers, may translocate due to the fluidity of the materials. Therefore, the development of an MRI contrast enhancement microcarrier, which could noninvasively trace the location of the implant in vivo, is necessary to guide the evaluation of treatment prognosis. In this study, GdPO₄·H₂O nanoparticles were utilized as the dispersed phase to endow PLGA microcarriers with T1-weighted MRI contrast ability. By adjusting the dispersed phase of Gd compound nanoparticle in PLGA Matrix, a cross-comparison experiment between MRI contrast enhancement imaging and biocompatibility was conducted, and 0.8% Gd was found to be the most suitable rate for preparing nano-composites. Moreover, 0.8% Gd/PLGA microcarriers were suspension-cultured with stem cells in vitro and implanted into traumatic brain injury rats in vivo. Excellent cytocompatibility and enhancement of the T1 phase of MRI were confirmed. This work created a T1-weighted MRI contrast-enhanced microcarrier, which provides a clinical noninvasive tracing cell scaffold for brain tissue engineering.

     

陶瓷中空纤维透氧膜的制备与性能

应用相转化法制备了La_0.6Sr_0.4Co_0.2Fe_0.8O_3-α(LSCF)氧离子-电子混合传导陶瓷中空纤维膜, 该陶瓷中空纤维膜具有由多孔层和致密层组成的非对称结构. 经 1300℃的4h烧结后, 可得到致密的LSCF陶瓷中空纤维膜. 烧结后, LSCF粒度变大而其钙钛矿型晶相结构没有发生变化. LSCF中空纤维膜的透氧速率大大高于一般管式膜的氧透量.     

Preparation of polyurethane/poly(vinylidene fluoride) blend hollow fibre membrane using melt spinning and stretching

Melt spinning and stretching process was used to prepare polyurethane/poly(vinylidene difluoride) (PU/PVDF) blend hollow fibre membrane in this study. Rheological properties of melts of PU, PVDF and their blends were studied by observing their melting viscosity changing with shear rate and temperature. Morphologies of PU/PVDF blend hollow fibre membranes with different mass ratios were also observed by SEM and results showed that PU/PVDF mass ratio of 3:1 was relatively better condition for membrane preparation. The forming mechanism of the interfacial microvoids in PU/PVDF blends was also investigated and PU/PVDF blend hollow fibre membrane with water flux of ～2174 L m^?2 h^?1 was finally obtained under the pressure of 0·1 MPa.     

Drop-on-demand printing of cells and materials for designer tissue constructs

Adapting bottom-up approaches to tissue engineering is a real challenge. Since the first application of fused deposition modeling for tissue engineering scaffolds, considerable effort has been focused on printing synthetic biodegradable scaffolds. Concurrently a variety of rapid prototyping techniques have been developed to define macroscopically the shapes of deposited biomaterials, including photolithography, syringe-based gel deposition, and solid freeform fabrication. These designed scaffolds have shown promise in regenerating tissues at least equivalent to other scaffolding methods.An exciting advance in scaffold aided tissue regeneration is presented here, that of cell and organ printing, which allows direct printing of cells and proteins within 3D hydrogel structures. Cell printing opens the possibility to programmed deposition of scaffold structure and cell type, thus controlling the type of tissue that can be regenerated within the scaffold. Several examples of printed tissues will be presented including contractile cardiac hybrids. The hybrid materials have properties that can be tailored in 3D to achieve desired porosities, mechanical and chemical properties. The materials include alginate hydrogels with controlled microshell structures that can be built by spraying cross-linkers onto ungelled alginic acid.Endothelial cells were seen to attach to the inside of these microshells. The cells remained viable in constructs as thick as 1 cm due to the programmed porosity. Finite element modeling was used to predict the mechanical properties and to generate CAD models with properties matching cardiac tissue. These results suggest that the printing method could be used for hierarchical design of functional cardiac patches, balanced with porosity for mass transport and structural support.     

Performance of evacuated calcium phosphate microcarriers loaded with mesenchymal stem cells within a rat calvarium defect

Tissue engineering of stem cells in concert with 3-dimensional (3D) scaffolds is a promising approach for regeneration of bone tissues. Bioactive ceramic microspheres are considered effective 3D stem cell carriers for bone tissue engineering. Here we used evacuated calcium phosphate (CaP) microspheres as the carrier of mesenchymal stem cells (MSCs) derived from rat bone marrow. The performance of the CaP-MSCs construct in bone formation within a rat calvarium defect was evaluated. MSCs were first cultured in combination with the evacuated microcarriers for 7?days in an osteogenic medium, which was then implanted in the 6?mm-diameter calvarium defect for 12?weeks. For comparison purposes, a control defect and cell-free CaP microspheres were also evaluated. The osteogenic differentiation of MSCs cultivated in the evacuated CaP microcarriers was confirmed by alkaline phosphatase staining and real time polymerase chain reaction. The in vivo results confirmed the highest bone formation was attained in the CaP microcarriers combined with MSCs, based on microcomputed tomography and histological assays. The results suggest that evacuated CaP microspheres have the potential to be useful as stem cell carriers for bone tissue engineering.