Tissue Culture Plastic Controls Research Articles

Quasi-Spherical Cell Clusters Induced by a Polyelectrolyte Multilayer.

Fibroblasts cultured on polyelectrolyte multilayers, PEMUs, made from poly(diallyldimethylammonium), PDADMA, and poly(styrene sulfonate), PSS, showed a variety of attachment modes, depending on the charge of the last layer and deposition conditions. PEMUs terminated with PDADMA (cationic) were cytotoxic when built in 1.0 M NaCl but cytophilic when built in 0.15 M NaCl. Cells adhered poorly to all PSS-capped (anionic) films. PEMUs built in 0.15 M NaCl but terminated with a layer of PSS in 1.0 M NaCl induced most cells to form spherical clusters after about 48 h of culture. These clusters still interrogated the surface, and when they were replated on control tissue culture plastic, cells emerged with close to 100% viability. Differences between the various surfaces were probed in an effort to identify the mechanism responsible for this unusual behavior, which did not follow accepted correlations between substrate stiffness and cell adhesion. No significant differences in roughness or wetting were observed between cluster-inducing PSS-capped multilayers and those that did not produce clusters. When the surface charge was assayed with radiolabeled ions a strong increase in negative surface charge was revealed. Viewing the multilayer as a zwitterionic solid and comparing its surface charge density to that of a cell membrane yields similarities that suggest a mechanism for preventing protein adhesion to the surface, a necessary step in the integrin-mediated mechanotransduction properties of a cell.

Tissue Engineering for Intervertebral Disc Repair and Regeneration

Introduction Tissue engineering aims to combine isolated cells, engineered materials and small molecule biochemical factors to generate neotissues with specific form and function to replace or repair damaged or degenerate native tissue. Intervertebral disc (IVD) degeneration directly causes back pain, and understanding how to regenerate this tissue may help alleviate this costly global problem. Several studies have combined isolated nucleus pulposus (NP) disc cells with novel scaffold materials to produce promising therapeutics toward IVD repair. We have taken three avenues toward IVD tissue engineering: culturing isolated IVD cells on continuous expansion surfaces to enhance cell phenotypes, utilizing novel injectable hydrogels for cell delivery to intact discs, and production of 3D printed scaffold geometries enhancing disc matrix production by seeded NP cells. Material and Methods Bovine NP cells were isolated from caudal discs of freshly slaughtered steer (∼ 24 months) obtained from a local slaughterhouse. Bovine NP cells are expanded on either tissue culture plastic with two passages, or on highly elastic silicone rubber dishes which slowly expands the surface from 12 to 78 cm2 and marker gene expression is compared. Isolated cells are labeled with fluorescent membrane binding dye, and cells are mixed with an injectable, chitosan/hyaluronic acid hydrogel. Approximately 100 µL of cell/gel (106 cells/µL of gel) mixtures is injected into isolated whole bovine discs (obtained as described earlier, with vertebral bone drilled down to the cartilage end plate). Discs are then placed into a custom bioreactor, and dynamic diurnal loading is applied to the injected discs. After 14 days, localization and viability of injected cells is assessed by confocal microscopy. For 3D printed constructs, 10 × 10 × 0.3 mm plastic scaffolds were printed using a desktop 3D printer, with pore size of approximately 700 µm. Overall, 5 × 105 bovine NP cells were seeded on scaffolds, and cell viability, DNA content, proteoglycan content, and collagen type II content was assessed after 21 days in culture. Results When cultured on elastic silicone dishes, continuous expansion of bovine NP cells preserve cell phenotype and promotes higher aggrecan and collagen type II expression compared with tissue culture plastic controls. NP cells embedded in a hydrogel and injected into whole discs were localized throughout the discs. Cells also showed high viability after 14 days in culture. Bovine NP cells seeded on 3D printed orthogonal plastic scaffolds grow extremely well and fill in the large 700 µm pores over 21 days culture. The cultured constructs have high viability and produce ample aggrecan and collagen type II protein. Conclusion By combining three different approaches we show the following: (1) individual NP cells can be engineered to maintain superior phenotypic qualities compared with standard culture procedures, (2) combination of expanded cells with injectable hydrogels can be delivered to intact discs for potential repair, and (3) simple 3D printed scaffolds can promote NP matrix production by seeded cells. Combination of these avenues may further enhance IVD regenerative capacities.

Enhanced osteogenic gene expression in microcarrier-bound scalably expanded fetal bone marrow MSCS

Mesenchymal stem cell (MSC) therapy is a promising strategy to address the unmet clinical need for improved bone regeneration. However, challenges to the scalable stem cell production hinder the clinical translation of MSC therapy. Furthermore, the effects of scalable microcarrier-based culture and enzymatic cell harvest on the phenotype and molecular identity of MSCs upon directed osteogenic differentiation are not well understood. In order to address these issues, we expanded fetal bone marrow MSCs on either commercially-available microcarriers in spinner flask culture or on control tissue culture plastic (TCP) monolayer surfaces. At the end of this expansion, we characterized the phenotype of microcarrier- or TCP monolayer-expanded MSCs in terms of cytokine, gene and surface marker expression. We then differentiated microcarrier-bound, microcarrier-harvested or plastic-harvested MSCs which were seeded onto collagen-coated plates or polycaprolactone-tricalcium phosphate scaffolds. In both cases, microcarrier-bound MSCs displayed increased expression of osteogenic genes such as RUNX2 and ALPL compared to microcarrier-harvested and plastic-harvested cells upon osteogenic induction. These results indicate that microcarrier-bound MSCs undergo enhanced osteogenic differentiation. Our findings also suggest that compared to conventional methods of delivering MSC cell suspensions that were harvested either from microcarriers cultured in agitated bioreactors or from TCP surfaces, in vivo delivery of MSC-bound microcarriers may present a promising method for bone regeneration.

Effect of bone graft density on in vitro cell behavior with enamel matrix derivative.

Bone replacement grafting materials play an important role in regenerative dentistry. Despite a large array of tested bone-grafting materials, little information is available comparing the effects of bone graft density on in vitro cell behavior. Therefore, the aim of the present study is to compare the effects of cells seeded on bone grafts at low and high density in vitro for osteoblast adhesion, proliferation, and differentiation. The response of osteoblasts to the presence of a growth factor (enamel matrix derivative, (EMD)) in combination with low (8mg per well) or high (100mg per well) bone grafts (BG; natural bone mineral, Bio-Oss®) density, was studied and compared for osteoblast cell adhesion, proliferation, and differentiation as assessed by real-time PCR. Standard tissue culture plastic was used as a control with and without EMD. The present study demonstrates that in vitro testing of bone-grafting materials is largely influenced by bone graft seeding density. Osteoblast adhesion was up to 50% lower when cells were seeded on high-density BG when compared to low-density BG and control tissue culture plastic. Furthermore, proliferation was affected in a similar manner whereby cell proliferation on high-density BG (100mg/well) was significantly increased when compared to that on low-density BG (8mg/well). In contrast, cell differentiation was significantly increased on high-density BG as assessed by real-time PCR for markers collagen 1 (Col 1), alkaline phosphatase (ALP), and osteocalcin (OC) as well as alizarin red staining. The effects of EMD on osteoblast adhesion, proliferation, and differentiation further demonstrated that the bone graft seeding density largely controls in vitro results. EMD significantly increased cell attachment only on high-density BG, whereas EMD was able to further stimulate cell proliferation and differentiation of osteoblasts on control culture plastic and low-density BG when compared to high-density BG. The results from the present study demonstrate that the in vitro conditions largely influence cell behavior of osteoblasts seeded on bone grafts and in vitro testing. These results also illustrate the necessity for careful selection of bone graft seeding density to optimize in vitro testing and provide the clinician with a more accurate description of the osteopromotive potential of bone grafts.

Cell-Secreted Matrices Enhance Osteogenesis in Adipose-Derived Stem Cells Undergoing Lineage Specification

Osteogenic differentiation of mesenchymal stem cells (MSC) is important in the field of bone tissue engineering. The identification of biological factors that influence osteogenesis is vital for developing a broader understanding of how complex microenvironments play a role in differentiation. The aim of this study was to demonstrate that adipose-derived stem cell (ADSC) osteogenesis is enhanced through interaction with extracellular matrices (ECM) secreted by ADSC undergoing osteogenesis. ADSC were obtained from human patients following elective abdominoplasty. Cells were selected for plastic adherence, characterized, and induced to differentiate using osteogenic supplements (OS; dexamethasone, ascorbic acid, and beta-glycerol phosphate). Cells were removed at several time points during osteogenesis and the secreted ECM was isolated. Undifferentiated cells were re-seeded onto the cell secreted ECMs and induced to differentiate with OS. At several time points, cells cultured on ECMs or tissue culture plastic controls (i.e. uncoated surface) were collected and RNA isolated. QPCR and gene array analysis revealed enrichment of osteogenic markers and more rapid progression through osteogenic maturational phases in cells seeded onto ECM secreted at the midpoint in differentiation (ca. 15 days). Our results demonstrate that the cumulative deposition of ECM reaches a critical point at approximately 15 days, before which there appear to be no definitive osteogenic cues from the matrix, and after which, strong drivers of osteogenesis are present. The creation of microenvironments that contain essential morphogenic matrix signals is an important step towards methods of growing and differentiating MSC in a rapid effective manner, particularly for bone-related clinical applications.

Novel porous scaffolds of poly(lactic acid) produced by phase-separation using room temperature ionic liquid and the assessments of biocompatibility

Here we prepared three-dimensional (3D) porous-structured biodegradable polymer scaffolds for tissue regeneration using room temperature ionic liquids (RTILs) as a novel porogen, and addressed their biological properties, including in vitro cell growth and differentiation and in vivo tissue compatibility. RTIL based on 1-butyl-3-methylimidazolium ([bmim]) bearing hydrophilic anion Cl was introduced within the polymer structure to provide a pore network. A mixture of poly(lactic acid) (PLA) with RTIL dissolved in an organic solvent formed a bi-continuous network during the drying process. Selective dissolution of the RTIL phase was facilitated in ethanol, which resulted in a porous network of the polymer phase with complete removal of the RTIL. The RTILs-assisted porous scaffolds showed a typical open-channeled network with pore sizes over 100 μm and porosities of about 86-94%. For the biocompatibility assessments of the scaffolds, mesenchymal stem cells (MSCs) derived from rat bone marrow were seeded onto the PLA scaffold, and the cell proliferation and osteoblastic differentiation behaviors were examined. Results showed a typical on-going increase in the cell population with a level comparable to that observed on the tissue culture plastic control, indicating good cell compatibility. When cultured in an osteogenic medium, the alkaline phosphatase (ALP) activity of the cells on the PLA scaffolds was stimulated to increase with time from 7 to 14 days, in a similar manner to that on the control. Moreover, the expression of genes related to osteoblasts, including collagen type I, osteocalcin and bone sialoprotein, was stimulated on the 3D PLA scaffold during culture for up to 14 days, with levels higher than those on the control, suggesting the developed scaffold provided a 3D matrix condition for osteogenesis. An in vivo pilot study conducted subcutaneously in rat for 4 weeks revealed good tissue compatibility of the scaffold, with the ingrowth of cells and formation of collageneous tissue around and deep within the pores of the scaffold and no significant inflammatory reaction. Taken together, this novel method of using RTILs as a pore generator is considered to be useful in the development of biocompatible porous polymer scaffolds for tissue regeneration.

In vitro studies on the influence of surface modification of Ni–Ti alloy on human bone cells

The in vitro cell behavior on Nitinol after different surface treatments was investigated. As references samples, commercially pure titanium (cpTi) and bioactive titanium were used. The surface treatments influenced the topography, surface energy, crystallographic structure, ion release, chemistry, and ability to form apatite layer from simulated body fluids. Regardless of the surface treatment, the bioactivity study showed that the kinetics of apatite film formation was similar for all tested samples. No clear indication of the surface characteristics influence on the ability for calcium-phosphate precipitation was evident. Cell activity studies showed that ground nickel titanium, spark oxidized and thermally oxidized (at 400 degrees C and below) had higher cellular activity and caused increased alkaline phosphatase (ALP) and osteocalcin (OC) expression which was comparable to control tissue culture plastic and titanium reference samples. Regardless of surface modifications, preimmersion of the samples in media for 72 h resulted in cell proliferation at the same level for all samples. Therefore, it can be concluded that preconditioning of samples alters surface properties and modulates the cell response regardless of the initial surface treatment and its properties. Moreover, a detrimental effect on cell response was observed after 7 and 14 days in culture for alkali treated samples. This was attributed to a high surface nickel concentration and a high nickel ion release rate from these surfaces.

Engineering Biomimetic Culture Systems: Impact On Human Bone Marrow-Derived Stem Cells.

Abstract 3628Poster Board III-564The bone marrow (BM) harbours haematopoietic stem/progenitor cells (HSCs) in anatomically distinct sites (niches) where HSCs are subjected to regulatory cues such as cytokines, cell-cell contacts and extra-cellular matrix (ECM) all of which control stem cell fate. In particular mesenchymal stromal cells (MSCs) are an integral part of the bone marrow and are known to be key regulators of the HSC niche. We have previously shown that bio-artificial scaffolds can have a significant impact on the in vitro behaviour of MSCs. Here, we are therefore focussing on the role of (native) ECM within the MSC-HSC microenvironment by building on our previous findings and published data (Seib et al.,Tissue Eng Part A., 2009 in press). Thus the aim of the current study is (a) to identify niche-specific ECM components and (b) the use of such ECMs for in vitro culture of BM-derived stem cells. To mimic the natural ECM composition of the BM, different ECM types were generated from BM-derived cells using (a) Dexter cultures, (b) standard MSC cultures, (c) MSCs subjected to osteogenic differentiation. After 10 days of culture those MSC-derived ECMs were decellularised using 0.5% Triton-X and 20mM NH4OH leaving only the ECM behind (verified by scanning electron microscopy). Those ECMs were used as a substrate for a second culture of MSCs, which were analysed for their proliferation and differentiation potential. Cell-free ECM from standard MSC cultures improved MSC proliferation compared to cells grown on regular tissue culture plastic (TCP) over the period of 8 days. Most notably, all cell-free ECM preparations lead to a significant difference in the cytoskeletal arrangement of MSCs during the first 2 days of culture compared to TCP controls. Cultivation of MSCs on native ECM provided a guiding structure for those cells to grow into, and helped to maintain an elongated cell shape compared to substantial cell spreading on TCP (roundness 0.2 versus 0.5 and cell area of 2.2 versus 8.2mm2, respectively, p<0.001, n=60. A factor of 1 was set to equate to a perfect circle). Next, we investigate if native ECM could either directly improve HSC cultures or maximise MSC feeder characteristics. For the latter set of studies MSCs were initially cultured for 7 days on cell-free ECM (from standard MSC cultures) and subsequently co-cultured with human peripheral blood CD34+ HSCs in serum free medium supplemented with cytokines (Tpo, Flt3, and SCF at 10ng/ml). Following a 14 day culture period up to 3.5-fold more CD34+ cells were present in ECM co-cultures compared to TCP co-cultures that was accompanied with an overall expansion of CD45+ cells of 109-fold versus 35-fold, respectively. Our data suggest that ECM preparations derived from MSCs might be useful to accomplish better expansion of HSCs under defined culture conditions. In addition, this system permits the identification of bimolecular key components that can be utilized in the future design of simple and robust carrier systems for improved HSC maintenance in vitro. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Polarization of hydroxyapatite: Influence on osteoblast cell proliferation

Hydroxyapatite (HA) has been used clinically to treat bone defects. However, modifications of the surface properties of HA could improve and control bone matrix deposition and localized host tissue integration. The aim of this study was to investigate the effect of developing a surface charge on HA discs with respect to osteoblast activity in vitro. HA discs (12 mm × 2 mm) were sintered in either air or water vapour. The HA discs were then electrically polarized (positive and negative surfaces) or non-polarized (controls) and seeded with MC3T3-E1 cells. Polarized HA sintered in water vapour was shown to retain six times more charge than polarized HA sintered in air. Picogreen analysis demonstrated that at 4 h cell number was significantly higher on the negatively and positively charged HA surface (water sintered) in comparison to the non-charged water and air-sintered HA controls. At 7 days there was a significant increase in cell number on the negatively charged HA (air sintered) sample in comparison to the negatively charged water vapour sintered HA sample and the non-charged water vapour sintered control sample. Also at 7 days, the picogreen data showed a significant increase in cell number on the positively charged water-treated HA sample in comparison to both the air- and water-treated HA non-charged control HA samples. An alamarBlue assay at 7 days demonstrated significant cell metabolic activity on the charged surfaces (both positive and negative) in comparison to the non-charged HA and the tissue culture plastic controls. This study demonstrated that all of the HA discs tested supported cell viability/attachment. However, cell attachment/proliferation/metabolic activity was significantly increased as a result of developing a charge on the HA surface.

Biocompatibility of polyhydroxyalkanoate as a potential material for ligament and tendon scaffold material

There is a strong need for new biodegradable materials that are suitable for scaffolds in tissue engineering of tendons and ligaments. In many cases, quick degradation rates are favorable, however, with respect to ligament and tendon replacement, slowly degrading polymers are clearly favored. Prime candidates are members of the large class of polyhydroxyalkanoates (PHAs), which are thermoplastic/elastomeric biopolyesters that are slowly degraded by surface erosion. Moreover, their physico-mechanical properties can be tailored during biosynthesis in bacteria or by chemical modifications. They may be spun into fibers, coated on surfaces or be part of composites. This study has investigated the biocompatability of seven different thermoplastic or elastomeric PHAs using L929 murine fibroblast cells. Cell viability and proliferation over 7 days was analyzed with live/dead staining and a picogreen assay. In addition, extracellular matrix production was measured with the hydroxyproline assay after 14 days. It was found that cell attachment to the PHA film ranged from 85-99% after 7 days. Three PHA films (PHBV (92/8), PHOUE-POSS and PHUE-O3) supported similar cell viability in comparison to the controls performed on tissue culture plastic (polystyrene), whereas the biomaterials (PHUA, PHUE, PHB and PHOUE) showed fewer viable cells than in controls. PHB, PHUE-O3, and PHBV with a water contact angle below 85 degrees supported a similar amount of collagen production in comparison to the tissue culture plastic controls. PHUA, PHUE, PHOUE, and PHOUE-POSS showed a decrease in collagen production in comparison to the controls after 14 days. Overall, PHB, PHBV, and PHUE-O3 demonstrated good performance with regards to potential use as a tissue-engineering scaffold.

Effects of Immobilized Glycosaminoglycans on the Proliferation and Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are adult stem cells with potential for multilineage differentiation. They represent an attractive cell source alternative to embryonic stem cells for therapeutic applications. Optimal utilization of MSCs for tissue engineering requires improved biomaterials that can enhance their growth and direct differentiation. The biological activity of glycosaminoglycans (GAGs) has been previously exploited for use in tissue engineering applications. In this study, MSC proliferation and differentiation was studied on GAG-derivatized chitosan membranes. The GAGs included heparin, heparan sulfate, dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, and hyaluronic acid. The covalent GAG immobilization method and amount of immobilized GAG were varied. It was found that MSC growth increased as much as fivefold on GAG-immobilized surfaces compared to tissue culture plastic and chitosan-only controls. The MSC growth rates increased significantly with increasing GAG density on the culture surfaces. The MSC proliferation rates on heparin, heparan sulfate, dermatan sulfate, and chondroitin 6-sulfate exhibited nonlinear increases with the level of fibronectin binding on these surfaces. In contrast, MSC proliferation on hyaluronic acid and chondroitin 4-sulfate was found to be independent of fibronectin or vitronectin binding on the surfaces, suggesting that these GAGs influenced MSC proliferation through different mechanisms. In conclusion, the results indicate that GAG immobilization on chitosan scaffolds provides an effective means of manipulating MSC proliferation and has promising potential for directing MSC differentiation in tissue engineering applications employing chitosan.

Osteoblast expression of cytokines is altered on MTA surfaces

Mineral trioxide aggregate (MTA) is used within areas of periradicular inflammation. Therefore, the purpose of this study was to determine if osteoblasts that attach to MTA surfaces alter their expression of inflammatory cytokines. Mouse MC3T3-E1 preosteoblasts were seeded onto ProRoot (Pr-MTA) and Tooth-Colored (Tc-MTA) MTA surfaces, as well as commercially pure titanium (cpTi) and tissue culture plastic (TCP) controls. Cell-surface interactions were visualized by inverted fluorescence microscopy. After 24 hours of attachment, the cells were stimulated with bacterial lipopolysaccharide (LPS) and analyzed by reverse-transcription polymerase chain reaction. Numerous cells attached and spread out onto Pr-MTA and Tc-MTA, as they did on cpTi and TCP. They formed plentiful extensions and a spindle-shaped morphology on the granular MTA surfaces. Cells grown on TCP and cpTi expressed an abundance of interleukin (IL-6) and IL-1alpha upon LPS exposure. Cells on Pr-MTA and Tc-MTA produced IL-6 but failed to express IL-1alpha despite LPS stimulation. Osteoblast expression of inflammatory cytokines is altered on endodontic MTA surfaces compared with titanium implants and plastic controls.

Cytocompatibility and Effect of Increasing MgO Content in a Range of Quaternary Invert Phosphate-based Glasses

Recently, phosphate-based glass (PBG) fibers have been used to reinforce the biodegradable polymers polycaprolactone and polylactic acid, in order to fabricate materials suitable for use as resorbable bone fracture fixation devices. However, the PBG fibers investigated tended to degrade too quickly for application. Therefore, more durable PBG formulations were sought with emphasis remaining firmly placed on their biocompatibility. In this study, four invert PBG formulations (in the system P2O5-CaO-MgO-Na2O) were produced with fixed phosphate and calcium content at 40 and 25 mol%, respectively. MgO was added at 10-30 mol% in place of Na2O and the maximum divalent cation to phosphate ratio obtained was 1.375. Thermal analyses showed a linear increase in T(g) with increasing MgO content. This was proposed to be due to an increase in the cross-link density of the glass network, which also improved the chemical durability of the glass. EDX analyses were also conducted to verify the final composition of the glass. XRD analyses confirmed the amorphous nature of the glasses investigated. Rapid quenching of the Mg30 glass revealed a degree of surface crystallization, which was shown to be a CaMgP2O7 phase. The degradation rates of the glasses investigated decreased with increasing MgO content. The decrease in rate seen was almost two orders of magnitude (a x 50 difference was seen between glass Mg0 and Mg30). The cytocompatibility studies of the formulations investigated showed good cellular response over time for up to 14 days. Statistical analysis revealed that the formulations investigated gave a response comparable to the tissue culture plastic control. It is suggested that invert PBG provide degradation profiles and the cytocompatibility response desired to make these glasses useful for bone repair applications.

Osteoblasts interact with MTA surfaces and express Runx2

Periradicular healing involves osteoblasts that are dependent on the Runt-related transcription factor 2 (Runx2). The purpose of this study was to determine if mineral trioxide aggregate (MTA) root-end filling materials support Runx2 expression in osteoblasts. Human alveolar bone cells were grown on alternative formulations of MTA. Cell-surface interactions were visualized by scanning electron microscopy. Gene expression was examined by reverse-transcription polymerase chain reaction and Western blot analysis. Cells attached to and spread out on MTA surfaces within 24 hours and formed a collagenous matrix overlay within 1 week of growth. Runx2 expression increased from low levels in the 24-hour cultures to an abundance during 2 weeks of growth and differentiation on MTA surfaces and on tissue culture plastic controls. The cells responded similarly to ProRoot, Tooth-Colored MTA, and MTA mixed with local anesthetic solution. Mineral trioxide aggregate materials support cell attachment and Runx2 expression in osteoblasts.

Electrospun Silk Fibroin Mats for Tissue Engineering

AbstractProcessing Silk Fibroin (SF) with electrospinning (ES) offers a very attractive opportunity for producing a variety of 2D and 3D matrices with great potential for tissue regeneration and repair due to the superior biocompatibility and mechanical properties of SF. Different combinations of ES parameters were explored to investigate the best experimental set‐up related to the dimension and uniformity of the fibers in the electrospun silk fibroin (ES‐SF) mats. Using SEM it was found that the ES‐SF mats contain uniform fibers with a diameter in the nanometric range obtained by electrospinning a 7.5 % w/v SF solution in formic acid, with an electric field of 2.4 kV/cm and a spinneret‐collector distance of 10 cm. FT‐IR and DSC analyses were performed to investigate the structure of the ES‐SF mats before and after immersion in methanol for different times (5, 10, and 15 min). The methanol treatment was able to promote the crystallization of SF by conformational transition of random coil and other poorly ordered conformations (turns and bends) to the β‐sheet structure. The degree of crystallinity was enhanced as shown by the trend of both the FT‐IR crystallinity index and the melting/decomposition peak temperature (from DSC). To study the cytocompatibility of ES‐SF mats, tests with L929 murine fibroblasts were carried out. Samples were seeded with the cells and incubated for 1, 3, and 7 days at 37 °C. At each time point, SEM investigations and Alamar blue tests were performed. The SEM images showed cell adhesion and proliferation just after 1 day and cell confluence at 7 days. Alamar blue test demonstrated that there were very low differences between cell viability on ES‐SF mats and the tissue culture plastic control.

Bombyx moriSilk Fibroin Membranes as Potential Substrata for Epithelial Constructs Used in the Management of Ocular Surface Disorders

Membranes were prepared from fibroin, a protein isolated from the domesticated silkworm (Bombyx mori) silk, and evaluated as a potential substratum for corneal limbal epithelial cells. These membranes (i.e., B. mori silk fibroin [BMSF] membranes) were cast from dialyzed solutions of fibroin protein (4% w/v) dispensed into 35-mm-diameter culture dishes and dried at room temperature (23-24 degrees C). The resulting material was transparent, easy to handle, and supported levels of human limbal epithelial (HLE) cell growth comparable to that observed on tissue culture plastic. Remarkably, these results were obtained utilizing a commercial serum-free medium (CnT-20) designed for the ex vivo expansion of corneal epithelial progenitor cells. The potential benefits of serum proteins on this culture system were examined through addition of fetal bovine serum (FBS) either to fibroin stocks prior to membrane casting or by supplementation of the CnT-20 medium. Membranes cast in the presence of FBS displayed increasing opacity and induced little change in HLE growth. Supplementation of CnT-20 medium with FBS deterred cell growth on all substrata, including tissue culture plastic control substrata. The remarkable properties of BMSF membranes demonstrated under serum-free conditions warrant investigation of this material as a substratum in the creation of tissue-engineered constructs for the restoration of diseased or damaged ocular surface.

Cell viability, proliferation and extracellular matrix production of human annulus fibrosus cells cultured within PDLLA/Bioglass ® composite foam scaffolds in vitro

The objective of this study was to assess cell viability, attachment, morphology, proliferation, and collagen and sulphated glycosaminoglycan (s-GAG) production by human annulus fibrosus (HAF) cells cultured in vitro in poly( d, l-lactide) (PDLLA)/Bioglass ® composite foams. PDLLA foams with different percentages (0, 5 and 30 wt.%) of Bioglass ® particles were prepared by thermally induced phase separation (TIPS) and characterized by scanning electron microscopy (SEM). HAF cell viability in the PDLLA/Bioglass ® foam was analysed using Live/Dead staining. HAF cell attachment was observed using SEM. An assessment of cell proliferation was conducted using the WST-1 assay. The level of s-GAG and collagen produced by HAF cells was quantified using the 1,9-dimethylmethylene blue (DMMB) assay and Sircol™ assay after 4 weeks of culture. The presence of collagen types I and II within the PDLLA/Bioglass ® composite foams was analysed using immunohistochemistry. Live/dead staining showed that many viable HAF cells were present on the top surface of the foams as well as penetrating into the internal pore structure, suggesting that the PDLLA/Bioglass ® composite materials are non-toxic and that the presence of Bioglass ® particles within PDLLA scaffolds does not inhibit HAF cell growth. The SEM observations revealed that more clusters of HAF cells were attached to the pore walls of both the PDLLA/5BG foam and the PDLLA/30BG foam when compared with the PDLLA/0BG foam. WST-1 assay performed over a period of 4 weeks showed an increased tendency of HAF cells to proliferate within both the PDLLA/5BG foam and the PDLLA/30BG foam when compared with both the tissue culture plastic control and the PDLLA/0BG foam, indicating the presence of Bioglass ® in the foam has a positive effect on HAF cell proliferation. Sircol™ and DMMB assays showed that HAF cells cultured within the PDLLA/30BG foam had a greater ability to deposit collagen and proteoglycan when compared with the control and the PDLLA/0BG foam after 4 weeks in culture, suggesting that the increase of Bioglass ® content may induce microenvironmental changes which promote the production of extracellular matrix containing abundant collagen and s-GAG. The immunohistochemical analysis of collagen production demonstrated that collagen produced in all cultures was predominantly of type I. These findings provide preliminary evidence for the use of PDLLA/Bioglass ® composite as cell-carrier materials for future treatments of the intervertebral disc with damaged AF region.

Intact endothelial cell sheet harvesting from thermoresponsive surfaces coated with cell adhesion promoters

Recently, with the development of smart polymers, research has looked to using thermoresponsive polymers as cell culture substrates. These novel surfaces allow the cultivation of cells without enzymes using the thermoresponsive phase transition property of poly(N-isopropylacrylamide) (PNIPAAm). However, this requires expensive techniques to generate a sufficiently thin film that allows cell adhesion. In this study, we looked at simple solvent cast films which normally show poor cell adhesion, but here the films are coated with cell adhesion promoters (CAPs) to improve cell growth without altering the copolymer thermoresponsive behaviour.A copolymer of PNIPAAm and N-tert-butylacrylamide (NtBAm) with a ratio of 85:15, respectively, was synthesized and solvent cast. The copolymer films were coated with CAPs, such as collagen, fibronectin and laminin, to increase their cell adhesion and growth properties. Cell activity measured by the alamarBlue assay showed similar results for coated copolymer films and standard tissue culture plastic controls. Deposition of CAPs on to the copolymer films was characterized by scanning electron microscopy and atomic force microscopy. Cell detachment from the copolymer films is not affected by the surface coatings of CAPs, and endothelial cells are recovered as an intact sheet, which has great potential for uses in tissue engineering applications. The results demonstrate a versatile method for the cultivation of cells while eliminating the need for the use of digestive enzymes such as trypsin. This study shows that cultivation on physically bonded PNIPAAm copolymers is viable and achievable by relatively simple methods.

Cell growth and detachment from protein‐coated PNIPAAm‐based copolymers

The cultivation of cells requires the use of unfavorable proteolytic enzymes, which cause cell-surface modification and also need considerable optimization. Recently, with the development of smart polymers, research has looked to using thermoresponsive polymers as cell culture substrates. These novel surfaces allow the cultivation of cells without using enzymes by utilizing the thermoresponsive phase transition property of poly(N-isopropylacrylamide) (PNIPAAm). Copolymers of PNIPAAm and N-tert-butyl-acrylamide (NtBAm) with varying ratios were synthesized and solvent cast. The copolymer films are coated with cell adhesion promoters such as collagen, poly-L-lysine, and laminin to increase their cell adhesion and growth properties. Cell activity measured by the alamarBlue and PicoGreen assays is similar for coated copolymer films and standard tissue culture plastic controls. Deposition of cell adhesion promoters onto the copolymer films was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Cell detachment from the copolymer films is dependent on copolymer composition and is not affected by the surface coatings of extracellular matrix (ECM) proteins. The results demonstrate a versatile method for the cultivation of cells while eliminating the need for the use of digestive enzymes such as trypsin.

Bone cell attachment and growth on well‐characterized chitosan films

AbstractChitosan has been widely researched for bone tissue and implant applications. While initial results are promising, there are inconsistent reports regarding the biological responses. This may be due to inadequate evaluation of chitosan material properties. This study evaluated normal human osteoblast precursor cell attachment and proliferation on a series of well‐characterized chitosan films. The chitosan films exhibited a range of properties: 76–96% degree of de‐acetylation (DDA), 2400–8200 kDa viscosity‐average molecular weight, 62–90° contact angle, 0.24–2.46% residual ash, 5.3–287 µg cm−2 residual protein and 23–40% crystallinity. There was no trend or correlation between DDA, crystallinity, contact angle, molecular weight, residual ash or protein content and the attachment or growth of bone cells on chitosan films. All films supported higher levels of bone cell proliferation than tissue culture plastic, which supports the general hypothesis that chitosans are osteocompatible. The 78 and 92% DDA chitosan films supported the most cell proliferation, approximately 16 times that of tissue culture plastic controls, but no chitosan physiochemical property correlated with the increased cell growth. The lack of correlation is hindered since more than one physiochemical property changed for each chitosan material. Data do indicate that there may be much variability in chitosan materials, and this variability may make understanding and comparing biological performance of chitosan materials difficult. These results highlight the need for systematic characterizations of chitosan materials for predictable biomedical applications. Copyright © 2006 Society of Chemical Industry