Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices

The cell seeding density and spatial distribution in a 3-D scaffold are critical to the morphogenetic development of an engineered tissue. A dynamic depth-filtration seeding method was developed to improve the initial cell seeding density and spatial distribution in 3-D nonwoven fibrous matrices commonly used as tissue scaffolds. In this work, trophoblast-like ED27 cells were seeded in poly(ethylene terephthalate) (PET) matrices with various porosities (0.85-0.93). The effects of the initial concentration of cells in the suspension used to seed the PET matrix and the pore size of the matrix on the resulting seeding density and subsequent cell proliferation and tissue development were studied. Compared to the conventional static seeding method, the dynamic depth-filtration seeding method gave a significantly higher initial seeding density (2-4 x 10(7) vs 4 x 10(6) cells/cm3), more uniform cell distribution, and a higher final cell density in the tissue scaffold. The more uniform initial cell spatial distribution from the filtration seeding method also led to more cells in S phase and a prolonged proliferation period. However, both uniform spatial cell distribution and the pore size of the matrices are important to cell proliferation and morphological development in the seeded tissue scaffold. Large-pore matrices led to the formation of cell aggregates and thus might reduce cell proliferation. The dynamic depth-filtration seeding method is better in providing a higher initial seeding density and more uniform cell distribution and is easier to apply to large tissue scaffolds. A depth-filtration model was also developed and can be used to simulate the seeding process and to predict the maximum initial seeding densities in matrices with different porosities.     

Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: dynamic cell seeding and construct development

Human mesenchymal stem cells (hMSCs) have great potential for therapeutic applications. A bioreactor system that supports long-term hMSCs growth and three-dimensional (3-D) tissue formation is an important technology for hMSC tissue engineering. A 3-D perfusion bioreactor system was designed using non-woven poly (ethylene terepthalate) (PET) fibrous matrices as scaffolds. The main features of the perfusion bioreactor system are its modular design and integrated seeding operation. Modular design of the bioreactor system allows the growth of multiple engineered tissue constructs and provides flexibility in harvesting the constructs at different time points. In this study, four chambers with three matrices in each were utilized for hMSC construct development. The dynamic depth filtration seeding operation is incorporated in the system by perfusing cell suspensions perpendicularly through the PET matrices, achieving a maximum seeding efficiency of 68%, and the operation effectively reduced the complexity of operation and the risk of contamination. Statistical analyses suggest that the cells are uniformly distributed in the matrices. After seeding, long-term construct cultivation was conducted by perfusing the media around the constructs from both sides of the matrices. Compared to the static cultures, a significantly higher cell density of 4.22 x 10(7) cell/mL was reached over a 40-day culture period. Cellular constructs at different positions in the flow chamber have statistically identical cell densities over the culture period. After expansion, the cells in the construct maintained the potential to differentiate into osteoblastic and adipogenic lineages at high cell density. The perfusion bioreactor system is amenable to multiple tissue engineered construct production, uniform tissue development, and yet is simple to operate and can be scaled up for potential clinical use. The results also demonstrate that the multi-lineage differentiation potential of hMSCs are preserved even after extensive expansion, thus indicating the potential of hMSCs for functional tissue construct development. The system has important applications in stem cell tissue engineering.     

Human mesenchymal stem cells tissue development in 3D PET matrices

Human mesenchymal stem cells (hMSCs) are attractive cell sources for engineered tissue constructs with broad therapeutic potential. Three-dimensional (3D) hMSC tissue development in nonwoven poly(ethylene terephthalate) (PET) fibrous matrices was investigated. HMSCs were seeded onto 3D PET scaffolds and were cultured for over 1 month. Their proliferation rates were affected by seeding density but remained much lower than those of 2D controls. Compared to 2D surfaces, hMSCs grown in 3D scaffolds secreted and embedded themselves in an extensive ECM network composed of collagen I, collagen IV, fibronectin, and laminin. HMSCs were influenced by the orientation of adjacent PET fibers to organize the ECM proteins into highly aligned fibrils. We observed the increased expressions of alpha(2)beta(1) integrin but a slight decrease in the expression of alpha(5)beta(1) integrin in 3D compared to 2D culture and found that alpha(V)beta(3) was expressed only in 2D. Paxillin expression was down-regulated in 3D culture with a concomitant change in its localization patterns. We demonstrated the multi-lineage potentials of the 3D tissue constructs by differentiating the cells grown in the scaffolds into osteoblasts and adipocytes. Taken together, these results showed that hMSCs grown in 3D scaffolds display tissue development patterns distinct from their 2D counterparts and provide important clues for designing 3D scaffolds for developing tissue engineered constructs.     

Optimization of cardiac cell seeding and distribution in 3D porous alginate scaffolds

Cardiac tissue engineering has evolved as a potential therapeutic approach to assist in cardiac regeneration. We have recently shown that tissue-engineered cardiac graft, constructed from cardiomyocytes seeded within an alginate scaffold, is capable of preventing the deterioration in cardiac function after myocardial infarction in rats. The present article addresses cell seeding within porous alginate scaffolds in an attempt to achieve 3D high-density cardiac constructs with a uniform cell distribution. Due to the hydrophilic nature of the alginate scaffold, its >90% porosity and interconnected pore structure, cell seeding onto the scaffold was efficient and short, up to 30 min. Application of a moderate centrifugal force during cell seeding resulted in a uniform cell distribution throughout the alginate scaffolds, consequently enabling the loading of a large number of cells onto the 3D scaffolds. The percent cell yield in the alginate scaffolds ranged between 60-90%, depending on cell density at seeding; it was 90% at seeding densities of up to 1 x 10(8) cells/cm(3) scaffold and decreased to 60% at higher densities. The highly dense cardiac constructs maintained high metabolic activity in culture. Scanning electron microscopy revealed that the cells aggregated within the scaffold pores. Some of the aggregates were contracting spontaneously within the matrix pores. Throughout the culture there was no indication of cardiomyocyte proliferation within the scaffolds, nor was it found in 3D cultures of cardiofibroblasts. This may enable the development of cardiac cocultures, without domination of cardiofibroblasts with time.     

Modifications of nonwoven polyethylene terephthalate fibrous matrices via NaOH hydrolysis: Effects on pore size, fiber diameter, cell seeding and proliferation

A simple NaOH treatment method was developed for fabricating nonwoven fibrous matrices of polyethylene terephthalate (PET) with predictable porosity, pore size, and fiber diameter. Matrices with various porosities (90–97%), fiber diameters (13.5–25 μm), and pore sizes (54–65 μm) were prepared by treating with 1N NaOH at 70 °C for up to 120 h, resulting in up to 70% hydrolysis of the PET polymer. The hydrolysis of PET polymer by NaOH was found to follow a second-order kinetics with respect to the fiber surface area. Accordingly, mathematical models were developed to predict matrix porosity, fiber diameter, and apparent pore size of the PET matrices. The exponential decay coefficient of PET polymer was found to be 0.0147 h⁻¹. The matrices were used to study the effects of pore size and fiber diameter on cell seeding and proliferation. The seeding study demonstrated that cell adhesion on PET fibers can be enhanced, largely due to the increased surface roughness of the PET fibers. Decreasing the fiber diameter increases the surface curvature of the fibers and decreases available surface area for cell attachment, which, however, only resulted in a small decrease in the cell growth rate.     

Oxygen mapping: Probing a novel seeding strategy for bone tissue engineering

Bone tissue engineering (BTE) utilizing biomaterial scaffolds and human mesenchymal stem cells (hMSCs) is a promising approach for the treatment of bone defects. The quality of engineered tissue is crucially affected by numerous parameters including cell density and the oxygen supply. In this study, a novel oxygen‐imaging sensor was introduced to monitor the oxygen distribution in three dimensional (3D) scaffolds in order to analyze a new cell‐seeding strategy. Immortalized hMSCs, pre‐cultured in a monolayer for 30–40% or 70–80% confluence, were used to seed demineralized bone matrix (DBM) scaffolds. Real‐time measurements of oxygen consumption in vitro were simultaneously performed by the novel planar sensor and a conventional needle‐type sensor over 24 h. Recorded oxygen maps of the novel planar sensor revealed that scaffolds, seeded with hMSCs harvested at lower densities (30–40% confluence), exhibited rapid exponential oxygen consumption profile. In contrast, harvesting cells at higher densities (70–80% confluence) resulted in a very slow, almost linear, oxygen decrease due to gradual achieving the stationary growth phase. In conclusion, it could be shown that not only the seeding density on a scaffold, but also the cell density at the time point of harvest is of major importance for BTE. The new cell seeding strategy of harvested MSCs at low density during its log phase could be a useful strategy for an early in vivo implantation of cell‐seeded scaffolds after a shorter in vitro culture period. Furthermore, the novel oxygen imaging sensor enables a continuous, two‐dimensional, quick and convenient to handle oxygen mapping for the development and optimization of tissue engineered scaffolds. Biotechnol. Bioeng. 2017;114: 894–902. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Effects of mixing intensity on cell seeding and proliferation in three-dimensional fibrous matrices

Nonwoven fibrous matrices have been widely used in cell and tissue cultures because their three-dimensional (3-D) structures with large surface areas and pore spaces can support high-density cell growth. Although cell adherence and growth on 2-D surfaces have been thoroughly investigated, very little is known for cells cultured in 3-D matrices. The effects of mixing intensity on cell seeding, adherence, and growth in fibrous matrices were thus investigated. Chinese Hamster Ovary and osteosarcoma cells were inoculated into nonwoven polyethylene terephthalate matrices by dynamic and static seeding methods, of which the former was found to be superior in seeding efficiency and cell distribution in the matrices. Dynamic seeding increased seeding efficiency from approximately 40% to more than 90%. When higher mixing intensities were applied, both cell attachment and detachment rates increased. Cell attachment was transport limited, as indicated by the increased attachment rate with increasing the mass transfer coefficient of the cells. Meanwhile, cell detachment from the 3-D matrix can be described by the Bell model. The effects of matrix pore size on cell adherence and proliferation were also investigated. In general, the smaller pore size is favorable to cell attachment and proliferation. Further analysis revealed that the interaction between mixing intensity and pore size played a vital role in hydrodynamic damage to cells, which was found to be significant when the Kolomogorov eddy size was smaller than the matrix pores. Increasing mixing intensity also increased oxygen transfer, decreased the lactate yield from glucose, and improved cell growth.     

Improved seeding of chondrocytes into polyglycolic acid scaffolds using semi-static and alginate loading methods

Cell seeding and attachment in three-dimensional scaffolds is a key step in tissue engineering with implications for cell differentiation and tissue development. In this work, two new seeding methods were investigated using human chondrocytes and polyglycolic acid (PGA) fibrous mesh scaffolds. A simple semi-static seeding method using culture plates and tissue flasks was developed as an easy-to-perform modification of static seeding. An alginate-loading method was also studied, using alginate hydrogel as an adjuvant for entrapping cells within PGA scaffolds. Both the semi-static and PGA-alginate methods produced more homogeneous cell distributions than conventional static and dynamic seeding. Using 20 × 10(6) cells, whereas the seeding efficiency for static seeding was only 52%, all other techniques produced seeding efficiencies of ≥ 90%. With 40 × 10(6) cells, the efficiency of semi-static seeding declined to 74% while the dynamic and PGA-alginate methods retained their ability to accommodate high cell numbers. The seeded scaffolds were cultured in recirculation bioreactors to determine the effect of seeding method on cartilage production. Statically seeded scaffolds did not survive the 5-week cultivation period. Deposition of extracellular matrix in scaffolds seeded using the semi-static and PGA-alginate methods was more uniform compared with scaffolds seeded using the dynamic method. The new semi-static and PGA-alginate seeding methods developed in this work are recommended for tissue engineering because they provide substantial benefits compared with static seeding in terms of seeding efficiency, cell distribution, and cartilage deposition while remaining simple and easy to execute.     

Microplate‐reader compatible perfusion microbioreactor array for modular tissue culture and cytotoxicity assays

One important application of tissue engineering is to provide novel in vitro models for cell‐based assays. Perfusion microbioreactor array provides a useful tool for microscale tissue culture in parallel. However, high‐throughput data generation has been a challenge. In this study, a 4 × 4 array of perfusion microbioreactors was developed for plate‐reader compatible, time‐series quantification of cell proliferation, and cytotoxicity assays. The device was built through multilayer soft lithography. Low‐cost nonwoven polyethylene terephthalate fibrous matrices were integrated as modular tissue culture scaffolds. Human colon cancer HT‐29 cells with stable expression of enhanced green fluorescent protein were cultured in the device with continuous perfusion and reached a cell density over 5 × 10⁷ cells/mL. The microbioreactor array was used to test a chemotherapeutic drug 5‐FU for its effect on HT‐29 cells in continuous perfusion 3D culture. Compared with conventional 2D cytotoxicity assay, significant drug resistance was observed in the 3D perfusion culture. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Perfusion affects the tissue developmental patterns of human mesenchymal stem cells in 3D scaffolds

Human mesenchymal stem cells (hMSCs) developed in three‐dimensional (3D) scaffolds are significantly affected by culture conditions. We hypothesized that the hydrodynamic forces generated in perfusion bioreactors significantly affected hMSC functionality in 3D scaffolds by shaping the extracellular matrix (ECM) proteins. In this study, hMSCs were grown in 3D poly(ethylene terephthalate) (PET) scaffolds in static and a parallel perfusion system under similar initial conditions for up to 35 days. Results demonstrated that even at very low media velocities (O [10^?4 cm/sec]), perfusion cultures affected the ability of hMSCs to form an organized ECM network as illustrated by the immunostaining of collagen I and laminin fibrous structure. The change in the ECM microenvironment consequently influenced the nuclear shape. The hMSCs grown at the lower surface of static culture displayed a 15.2 times higher nuclear elongation than those at the upper surface, whereas cells grown in the perfusion bioreactor displayed uniform spherical nuclei on both surfaces. The difference in ECM organization and nuclear morphology associated with gene expression and differentiation characteristics of hMSCs. The cells exhibited lower CFU‐F colony forming ability and decreased expressions of stem‐cell genes of Rex‐1 and Oct‐4, implying a less primitive stem‐cell phenotype was maintained in the perfusion culture relative to the static culture conditions. The significantly higher expression level of osteonectin gene in the perfusion culture at day 28 indicated an upregulation of osteogenic ability of hMSCs. The study highlights the critical role of dynamic culture conditions on 3D hMSC construct development and properties. J. Cell. Physiol. 219: 421–429, 2009. © 2009 Wiley‐Liss, Inc.     

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.     

接种方式对细胞在三维材料中接种率和分布的影响

考察了静态和动态接种方式对成纤维细胞在胶原壳聚糖支架材料中接种率和分布的影响。将人成纤维细胞制成细胞悬液,分别采用静态接种、转瓶接种和灌注接种方式将细胞接入三维胶原壳聚糖海绵。通过MTT法和切片HE染色分别考察细胞接种率及细胞在三维材料中的分布。实验结果表明：在低的接种密度下静态接种有较高的接种率(889%),但随着接种密度的增加接种率下降显著,细胞结团且分布不均匀;转瓶接种的接种率约为60%,细胞分布也不均匀;灌注接种的接种率始终维持在77%以上,能得到高的起始细胞密度,且细胞分布均匀,是一种理想的接种方式。细胞接种方式的优化为改善工程化组织的结构和功能、缩短体外构建时间奠定了基础。     

Flow cytometric cell cycle analysis of muscle precursor cells cultured within 3D scaffolds in a perfusion bioreactor

It has been widely demonstrated that perfusion bioreactors improve in vitro three‐dimensional (3D) cultures in terms of high cell density and uniformity of cell distribution; however, the studies reported in literature were primarily based on qualitative analysis (histology, immunofluorescent staining) or on quantitative data averaged on the whole population (DNA assay, PCR). Studies on the behavior, in terms of cell cycle, of a cell population growing in 3D scaffolds in static or dynamic conditions are still absent. In this work, a perfusion bioreactor suitable to culture C₂C₁₂ muscle precursor cells within 3D porous collagen scaffolds was designed and developed and a method based on flowcytometric analyses for analyzing the cell cycle in the cell population was established. Cells were extracted by enzymatic digestion of the collagen scaffolds after 4, 7, and 10 days of culture, and flow cytometric live/dead and cell cycle analyses were performed with Propidium Iodide. A live/dead assay was used for validating the method for cell extraction and staining. Moreover, to investigate spatial heterogeneity of the cell population under perfusion conditions, two stacked scaffolds in the 3D domain, of which only the upstream layer was seeded, were analyzed separately. All results were compared with those obtained from static 3D cultures. The live/dead assay revealed the presence of less than 20% of dead cells, which did not affect the cell cycle analysis. Cell cycle analyses highlighted the increment of cell fractions in proliferating phases (S/G₂/M) owing to medium perfusion in long‐term cultures. After 7–10 days, the percentage of proliferating cells was 8–12% for dynamic cultures and 3–5% for the static controls. A higher fraction of proliferating cells was detected in the downstream scaffold. From a general perspective, this method provided data with a small standard deviation and detected the differences between static and dynamic cultures and between upper and lower scaffolds. Our methodology can be extended to other cell types to investigate the influence of 3D culture conditions on the expression of other relevant cell markers. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Primary rat hepatocyte culture on 3D nanofibrous polymer scaffolds for toxicology and pharmaceutical research

Primary rat hepatocytes are a widely used experimental model to estimate drug metabolism and toxicity. In currently used two‐dimensional (2D) cell culture systems, typical problems like morphological changes and the loss of liver cell‐specific functions occur. We hypothesize that the use of polymer scaffolds could overcome these problems and support the establishment of three‐dimensional (3D) culture systems in pharmaceutical research. Isolated primary rat hepatocytes were cultured on collagen‐coated nanofibrous scaffolds for 7 days. Cell loading efficiency was quantified via DNA content measurement. Cell viability and presence of liver‐cell‐specific functions (albumin secretion, glycogen storage capacity) were evaluated. The activity of liver‐specific factors was analyzed by immunofluorescent staining. RNA was isolated to establish quantitative real‐time PCR. Our results indicate that primary rat hepatocytes cultured on nanofibrous scaffolds revealed high viability and well‐preserved glycogen storage. Albumin secretion was existent during the entire culture period. Hepatocytes remain HNF‐4 positive, indicating highly preserved cell differentiation. Aggregated hepatocytes re‐established positive signaling for Connexin 32, a marker for differentiated hepatocyte interaction. ZO‐1‐positive hepatocytes were detected indicating formation of tight junctions. Expression of cytochrome isoenzymes was inducible. Altogether the data suggest that nanofibrous scaffolds provide a good in vitro microenvironment for neo tissue regeneration of primary rat hepatocytes. Biotechnol. Bioeng. 2011; 108:141–150. © 2010 Wiley Periodicals, Inc.     

Various seeding methods for tissue development of human umbilical-cord-derived mesenchymal stem cells in 3-dimensional PET matrix

Mesenchymal stem cells derived from human umbilical cords (hUCMSCs) are attractive as a new cell source for tissue engineering. It is essential to investigate and optimize the seeding process of these cells for the success of cell culture and tissue regeneration in vitro. In this study, a static seeding method (SSM), a centrifugal seeding method (CSM), and a novel method-cycling filtration seeding method (CFSM) are evaluated in terms of seeding efficiency, cell damage, and distribution inside the scaffolds, cell proliferation, and osteogenic differentiation. Cells were seeded on three-dimensional (3-D) nonwoven PET discs at a density of 1×10⁴ cells/disc, followed by 21 days of cell culture and 20 days of osteogenic differentiation. Cells grown in 3-D conditions exhibited higher metabolic activity than those grown on a 2-D control surface. The CSM and CFSM groups showed higher seeding efficiency, proliferation capacity, and differentiation potential. H&E staining indicated a more uniform spatial distribution of cells in CFSM groups. LDH level measurements suggested that more cell damage was caused by the CFSM process. Above all, the results showed that the cells maintained their proliferation ability and differentiation potential ex vivo during approximately 7 weeks of culture. The CSM and CFSM are recommended for hUCMSC tissue engineering, although the seeding parameters still require further investigation and optimization.     

Thermo-responsive non-woven scaffolds for "smart" 3D cell culture

The thermo‐responsive polymer poly(N‐isopropylacrylamide) has received widespread attention for its in vitro application in the non‐invasive, non‐destructive release of adherent cells on two dimensional surfaces. In this study, 3D non‐woven scaffolds fabricated from poly(propylene) (PP), poly(ethylene terephthalate) (PET), and nylon that had been grafted with PNIPAAm were tested for their ability to support the proliferation and subsequent thermal release of HC04 and HepG2 hepatocytes. Hepatocyte viability and proliferation were estimated using the Alamar Blue assay and Hoechst 33258 total DNA quantification. The assays revealed that the pure and grafted non‐woven scaffolds maintained the hepatocytes within the matrix and promoted 3D proliferation comparable to that of the commercially available Algimatrix? alginate scaffold. Albumin production and selected cytochrome P450 genes expression was found to be superior in cells growing on pure and grafted non‐woven PP scaffolds as compared to cells grown as a 2D monolayer. Two scaffolds, namely, PP‐g‐PNIPAAm‐A and PP‐g‐PNIPAAm‐B were identified as having far superior thermal release capabilities; releasing the majority of the cells from the matrices within 2 h. This is the first report for the development of 3D non‐woven, thermo‐responsive scaffolds able to release cells from the matrix without the use of any enzymatic assistance or scaffold degradation. Biotechnol. Bioeng. 2012; 109:2147–2158. © 2012 Wiley Periodicals, Inc.     

Perfusion seeding of channeled elastomeric scaffolds with myocytes and endothelial cells for cardiac tissue engineering

The requirements for engineering clinically sized cardiac constructs include medium perfusion (to maintain cell viability throughout the construct volume) and the protection of cardiac myocytes from hydrodynamic shear. To reconcile these conflicting requirements, we proposed the use of porous elastomeric scaffolds with an array of channels providing conduits for medium perfusion, and sized to provide efficient transport of oxygen to the cells, by a combination of convective flow and molecular diffusion over short distances between the channels. In this study, we investigate the conditions for perfusion seeding of channeled constructs with myocytes and endothelial cells without the gel carrier we previously used to lock the cells within the scaffold pores. We first established the flow parameters for perfusion seeding of porous elastomer scaffolds using the C2C12 myoblast line, and determined that a linear perfusion velocity of 1.0 mm/s resulted in seeding efficiency of 87% ± 26% within 2 hours. When applied to seeding of channeled scaffolds with neonatal rat cardiac myocytes, these conditions also resulted in high efficiency (77.2% ± 23.7%) of cell seeding. Uniform spatial cell distributions were obtained when scaffolds were stacked on top of one another in perfusion cartridges, effectively closing off the channels during perfusion seeding. Perfusion seeding of single scaffolds resulted in preferential cell attachment at the channel surfaces, and was employed for seeding scaffolds with rat aortic endothelial cells. We thus propose that these techniques can be utilized to engineer thick and compact cardiac constructs with parallel channels lined with endothelial cells. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

3D Non-Woven Polyvinylidene Fluoride Scaffolds: Fibre Cross Section and Texturizing Patterns Have Impact on Growth of Mesenchymal Stromal Cells

Several applications in tissue engineering require transplantation of cells embedded in appropriate biomaterial scaffolds. Such structures may consist of 3D non-woven fibrous materials whereas little is known about the impact of mesh size, pore architecture and fibre morphology on cellular behavior. In this study, we have developed polyvinylidene fluoride (PVDF) non-woven scaffolds with round, trilobal, or snowflake fibre cross section and different fibre crimp patterns (10, 16, or 28 needles per inch). Human mesenchymal stromal cells (MSCs) from adipose tissue were seeded in parallel on these scaffolds and their growth was compared. Initial cell adhesion during the seeding procedure was higher on non-wovens with round fibres than on those with snowflake or trilobal cross sections. All PVDF non-woven fabrics facilitated cell growth over a time course of 15 days. Interestingly, proliferation was significantly higher on non-wovens with round or trilobal fibres as compared to those with snowflake profile. Furthermore, proliferation increased in a wider, less dense network. Scanning electron microscopy (SEM) revealed that the MSCs aligned along the fibres and formed cellular layers spanning over the pores. 3D PVDF non-woven scaffolds support growth of MSCs, however fibre morphology and mesh size are relevant: proliferation is enhanced by round fibre cross sections and in rather wide-meshed scaffolds.     

Imaging stem cell distribution,growth, migration,and differentiation in 3‐D scaffolds for bone tissue engineering using mesoscopic fluorescence tomography

Regenerative medicine has emerged as an important discipline that aims to repair injury or replace damaged tissues or organs by introducing living cells or functioning tissues. Successful regenerative medicine strategies will likely depend upon a simultaneous optimization strategy for the design of biomaterials, cell‐seeding methods, cell‐biomaterial interactions, and molecular signaling within the engineered tissues. It remains a challenge to image three‐dimensional (3‐D) structures and functions of the cell‐seeded scaffold in mesoscopic scale (>2 ～ 3 mm). In this study, we utilized angled fluorescence laminar optical tomography (aFLOT), which allows depth‐resolved molecular characterization of engineered tissues in 3‐D to investigate cell viability, migration, and bone mineralization within bone tissue engineering scaffolds in situ.     

Exploring cellular adhesion and differentiation in a micro‐/nano‐hybrid polymer scaffold

Polymer scaffolds play an important role in three dimensional (3‐D) cell culture and tissue engineering. To best mimic the archiecture of natural extracellular matrix (ECM), a nano‐fibrous and micro‐porous combined (NFMP) scaffold was fabricated by combining phase separation and particulate leaching techniques. The NFMP scaffold possesses architectural features at two levels, including the micro‐scale pores and nano‐scale fibers. To evaluate the advantages of micro/nano combination, control scaffolds with only micro‐pores or nano‐fibers were fabricated. Cell grown in NFMP and control scaffolds were characterized with respect to morphology, proliferation rate, diffentiation and adhesion. The NFMP scaffold combined the advantages of micro‐ and nano‐scale structures. The NFMP scaffold nano‐fibers promoted neural differentiation and induced “3‐D matrix adhesion”, while the NFMP scaffold micro‐pores facilitated cell infiltration. This study represents a systematic comparison of cellular activities on micro‐only, nano‐only and micro/nano combined scaffolds, and demonstrates the unique advantages of the later. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010