Rotating Wall Vessel Bioreactor Research Articles

Organotypic 3D cell culture models: using the rotating wall vessel to study host–pathogen interactions

Appropriately simulating the three-dimensional (3D) environment in which tissues normally develop and function is crucial for engineering in vitro models that can be used for the meaningful dissection of host-pathogen interactions. This Review highlights how the rotating wall vessel bioreactor has been used to establish 3D hierarchical models that range in complexity from a single cell type to multicellular co-culture models that recapitulate the 3D architecture of tissues observed in vivo. The application of these models to the study of infectious diseases is discussed.

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 15ng·mL(-1), FL 5ng·mL(-1), TPO 6ng·mL(-1), IL-3 15ng·mL(-1), G-CSF 1ng·mL(-1) and GM-CSF 5ng·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 12days 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.

The Effect of Simulated Microgravity on Human Mesenchymal Stem Cells Cultured in an Osteogenic Differentiation System: A Bioinformatics Study

One proposed strategy for bone regeneration involves ex vivo tissue engineering, accomplished using bone-forming cells, biodegradable scaffolds, and dynamic culture systems, with the goal of three-dimensional tissue formation. Rotating wall vessel bioreactors generate simulated microgravity conditions ex vivo, which lead to cell aggregation. Human mesenchymal stem cells (hMSCs) have been extensively investigated and shown to possess the potential to differentiate into several cell lineages. The goal of the present study was to evaluate the effect of simulated microgravity on all genes expressed in hMSCs, with the underlying hypothesis that many important pathways are affected during culture within a rotating wall vessel system. Gene expression was analyzed using a whole genome microarray and clustering with the aid of the National Institutes of Health's Database for Annotation, Visualization and Integrated Discovery database and gene ontology analysis. Our analysis showed 882 genes that were downregulated and 505 genes that were upregulated after exposure to simulated microgravity. Gene ontology clustering revealed a wide variety of affected genes with respect to cell compartment, biological process, and signaling pathway clusters. The data sets showed significant decreases in osteogenic and chondrogenic gene expression and an increase in adipogenic gene expression, indicating that ex vivo adipose tissue engineering may benefit from simulated microgravity. This finding was supported by an adipogenic differentiation assay. These data are essential for further understanding of ex vivo tissue engineering using hMSCs.

The generation of 3-D tissue models based on hyaluronan hydrogel-coated microcarriers within a rotating wall vessel bioreactor

With the increasing necessity for functional tissue- and organ equivalents in the clinic, the optimization of techniques for the in vitro generation of organotypic structures that closely resemble the native tissue is of paramount importance. The engineering of a variety of highly differentiated tissues has been achieved using the rotating wall vessel (RWV) bioreactor technology, which is an optimized suspension culture allowing cells to grow in three-dimensions (3-D). However, certain cell types require the use of scaffolds, such as collagen-coated microcarrier beads, for optimal growth and differentiation in the RWV. Removal of the 3-D structures from the microcarriers involves enzymatic treatment, which disrupts the delicate 3-D architecture and makes it inapplicable for potential implantation. Therefore, we designed a microcarrier bead coated with a synthetic extracellular matrix (ECM) composed of a disulfide-crosslinked hyaluronan and gelatin hydrogel for 3-D tissue engineering, that allows for enzyme-free cell detachment under mild reductive conditions (i.e. by a thiol-disulfide exchange reaction). The ECM-coated beads (ECB) served as scaffold to culture human intestinal epithelial cells (Int-407) in the RWV, which formed viable multi-layered cell aggregates and expressed epithelial differentiation markers. The cell aggregates remained viable following dissociation from the microcarriers, and could be returned to the RWV bioreactor for further culturing into bead-free tissue assemblies. The developed ECBs thus offer the potential to generate scaffold-free 3-D tissue assemblies, which could further be explored for tissue replacement and remodeling.

Dynamic Three-Dimensional Culture Methods Enhance Mesenchymal Stem Cell Properties and Increase Therapeutic Potential

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation along the osteogenic, chondrogenic, and adipogenic lineages and have potential applications in a range of therapies. MSCs can be cultured as monolayers on tissue culture plastic, but there are indications that they lose cell-specific properties with time in vitro and so poorly reflect in vivo MSC behavior. We developed dynamic three-dimensional (3D) techniques for in vitro MSC culture using spinner flasks and a rotating wall vessel bioreactor. We characterized the two methods for dynamic 3D MSC culture and compared the properties of these cultures with monolayer MSCs. Our results showed that under optimal conditions, MSCs form compact cellular spheroids and remain viable in dynamic 3D culture. We demonstrated altered cell size and surface antigen expression together with enhanced osteogenic and adipogenic differentiation potential in MSCs from dynamic 3D conditions. By microarray analysis of monolayer and spinner flask MSCs, we identified many differences in gene expression, including those confirming widespread changes to the cellular architecture and extracellular matrix. The upregulation of interleukin 24 in dynamic 3D cultures was shown to selectively impair the viability of prostate cancer cells cultured in medium conditioned by dynamic 3D MSCs. Overall, this work suggests a novel therapeutic application for dynamic 3D MSCs and demonstrates that these methods are a viable alternative to monolayer techniques and may prove beneficial for retaining MSC properties in vitro.

Impact of scaffold micro and macro architecture on Schwann cell proliferation under dynamic conditions in a rotating wall vessel bioreactor

Over the last decade tissue engineering has emerged as a powerful alternative to regenerate lost tissues owing to trauma or tumor. Evidence shows that Schwann cell containing scaffolds have improved performance in vivo as compared to scaffolds that depend on cellularization post implantation. However, owing to limited supply of cells from the patients themselves, several approaches have been taken to enhance cell proliferation rates to produce complete and uniform cellularization of scaffolds. The most common approach is the application of a bioreactor to enhance cell proliferation rate and therefore reduce the time needed to obtain sufficiently significant number of glial cells, prior to implantation. In this study, we show the application of a rotating wall bioreactor system for studying Schwann cell proliferation on nanofibrous spiral shaped scaffolds, prepared by solvent casting and salt leaching techniques. The scaffolds were fabricated from polycaprolactone (PCL), which has ideal mechanical properties and upon degradation does not produce acidic byproducts. The spiral scaffolds were coated with aligned or random nanofibers, produced by electrospinning, to provide a substrate that mimics the native extracellular matrix and the essential contact guidance cues. At the 4 day time point, an enhanced rate of cell proliferation was observed on the open structured nanofibrous spiral scaffolds in a rotating wall bioreactor, as compared to static culture conditions. However, the cell proliferation rate on the other contemporary scaffolds architectures such as the tubular and cylindrical scaffolds show reduced cell proliferation in the bioreactor as compared to static conditions, at the same time point. Moreover, the rotating wall bioreactor does not alter the orientation or the phenotype of the Schwann cells on the aligned nanofiber containing scaffolds, wherein, the cells remain aligned along the length of the scaffolds. Therefore, these open structured spiral scaffolds pre-cultured with Schwann cells, in bioreactors could potentially shorten the time needed for grafts for peripheral nerve regeneration.

A Novel Buoyancy Technique Optimizes Simulated Microgravity Conditions for Whole Sensory Organ Culture in Rotating Bioreactors

Whole-organ culture of a sensory organ in a rotating wall vessel bioreactor provides a powerful in vitro model for physiological and pathophysiological investigation as previously demonstrated for the postnatal inner ear. The model is of specific relevance as a tool for regeneration research. In the immature inner ear explant, the density was only 1.29 g/cm(3). The high density of 1.68 g/cm(3) of the functionally mature organ resulted in enhanced settling velocity and deviation from its ideal circular orbital path causing enhanced shear stress. The morphometric and physical properties, as well as the dynamic motion patterns of explants, were analyzed and numerically evaluated by an orbital path index. Application of a novel buoyancy bead technique resulted in a 6.5- to 14.8-fold reduction of the settling velocity. The deviation of the explant from its ideal circular orbital path was adjusted as indicated by an optimum value for the orbital path index (-1.0). Shear stress exerted on the inner ear explant was consequently reduced 6.4- to 15.0-fold. The culture conditions for postnatal stages were optimized, and the preconditions for transferring this in vitro model toward mature high-density stages established. This buoyancy technique may also be useful in tissue engineering of other high-density structures.

0304 軟骨再生医療のためのGMP対応自動回転培養システムの構築(OS11:軟骨の形態と機能)

An RWV(rotating wall vessel) bioreactor is known as a useful tool for three dimensional culture. Our group reported the rapid regeneration of three dimensional large cartilaginous tissue from rabbit and human bone marrow cells using the bioreactor. This system is quite useful, however, the present system needs manual operation of controlling rotation speed changing with the mass increase of the growing tissue. The medium exchange procedure is quite complex. Thus we developed a GMP grade automatic RWV culture system including functions of automatic rotation control and medium exchange, consisting an RWV bioreactor, two illumination systems, a CCD camera, and an automatic medium exchange unit. The detailed results using the automatic system will be reported.

The influence of leptin on the activity of lung lymphocytes under simulated microgravity

Exposure to microgravity has been implicated in the compromised immune function in space travellers, resulting in opportunistic infections, poor wound healing, and cancer. Since recent studies have suggested that leptin was capable of modulating immune responses, the purpose of this study was to examine effects of microgravity on the activation and proliferation of rat lung lymphocytes and then to examine the effects of leptin-mediated signal transduction mechanisms of lymphocyte activation in these same conditions. In control conditions (T-flasks cultured cells) leptin was not able by itself to increase lymphocytes proliferation, or induce significant increase of either IL-2 production or expression of lymphocytes activation markers, such as CD25 and CD71, while it markedly enhanced the positive effects induced on these parameters by concanavalin A (ConA). Using clinostatic rotating wall vessel (RWV) bioreactors to simulate a microgravity environment, we found that ConA responsiveness was inhibited. Moreover, under these conditions, leptin was not able to reverse these impaired functions. Accordingly with the above cited inhibitory effects exerted by the simulated microgravity environment, evidence was also obtained of defects in lymphocyte intracellular signal transduction induced by the incubation in RWV bioreactors, namely concerning decreased ConA-mediated PKC activity, and reduced expression of NF-kappaB, c-fos, and ERK1/2. Again, leptin appeared to be unable in restoring a physiologic increase of these parameters, different from what could be observed after complementation of the ConA-mediated signalling with phorbol myristate acetate, which instead demonstrated to overcome the inhibition of lymphocytes activating functions, in the presence of simulated microgravity conditions.

Differential Translocation of Nuclear Factor-KappaB in a Cardiac Muscle Cell Line Under Gravitational Changes

Microgravity (micro-g) environments have been shown to elicit dysregulation of specific genes in a wide assay of cell types. It is known that the activation of transcription factors and molecular signaling pathways influence various physiological outcomes associated with stress and adaptive responses. Nuclear factor-kappa B (NF-kappaB) is one of the most prevailing oxidation-sensitive transcription factors. It is hypothesized that simulated microgravity would activate NF-kappaB and its downstream transcriptional networks, thus suggesting a role for NF-kappaB in microgravity induced muscle atrophy. To investigate the activation of NF-kappaB in a rat cardiac cell line (H9c2) under micro-g, rotating wall vessel bioreactors were used to simulate micro-g conditions. Western blotting revealed that mean nuclear translocation of NF-kappaB p65 subunit was 69% for micro-g and 46% for unit-g dynamic control as compared with a 30 min TNF-alpha positive control (p<0.05, n=3). The results from western blots were confirmed by enzyme-linked immunosorbent assay, which showed 66% for micro-g and 45% for dynamic control as compared with positive control (p<0.05, n=3). These results show significant differential translocation of NF-kappaB p65 under simulated micro-g. These results may be expanded upon to explain physiological changes such as muscle atrophy and further identify the regulatory pathways and effector molecules activated under exposure to micro-g.

Transplantation of Sertoli-Islet Cell Aggregates Formed by Microgravity: Prolonged Survival in Diabetic Rats

Transplantation of pancreatic islets is a potentially attractive treatment for type I diabetes. We generated the transplantable, tissue-like aggregates composed of Sertoli cells and islets in rotating wall vessel bioreactors, SICA (Sertoli-islet cell aggregates), to improve their biological function in vitro and in vivo. The isolated islet equivalent and Sertoli cells were purified from Wistar rats and cocultured for 5 days in bioreactor to generate SICA. The SICA, islets aggregates, and fresh isolated islets were transplanted under the kidney capsule of diabetic Sprague-Dawley (SD) rats, respectively. The functions of different grafts were ascertained by blood glucose level measurements and an in vivo glucose tolerance test. In response to elevated glucose, insulin secretion from SICA was 1.4-fold higher (P<0.05, n=5) than islet aggregates cultured alone. Of the rats that received SICA, 90% (9/10) remained normoglycemic at 60 days post-transplantation, and the survival significantly increased compared with recipients bearing homotypic islets aggregates or freshly isolated islets. The former responded similarly with healthy rats to the glucose tolerance test. Our results support the usefulness of SICA for the treatment of type 1 diabetes without any immunosuppressive agents.

Rotating three‐dimensional dynamic culture of adult human bone marrow‐derived cells for tissue engineering of hyaline cartilage

The method of constructing cartilage tissue from bone marrow-derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow-derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per microg DNA of the tissues were 10.01 +/- 3.49 microg/microg DNA in the case of the RWV bioreactor and 6.27 +/- 3.41 microg/microg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three-dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow-derived cells.

Bioreactor Transient Exposure Activates Specific Neurotrophic Pathway in Cortical Neurons

Altered gravity forces might influence neu- roplasticity and can provoke changes in biochemical mechanisms. In this contest, neurotrophins have a piv- otal role, particularly nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF). A suspen- sion of dissociated cortical cells from rat embryos was exposed to 24 h of microgravity before plating in nor- mal adherent culture system. Expression and transduc- tional signalling pathways of NGF and BDNF were assessed at the end of maturational process (8-10 days in vitro). Rotating wall vessel bioreactor (RWV) pre- exposition did not induce changes in NGF expression and its high affinity receptor TrkA. On the contrary both BDNF expression and its high affinity recep- tor TrkB were strongly up-regulated, inducing Erk- 5, but not Erk-1/2 activation and, in turn, MEF2C over-expression and activation. According to our pre- vious and present results, we postulate that relatively short microgravitational stimuli, applied to neural cells

Cartilage tissue regeneration from bone marrow cells by RWV bioreactor using collagen sponge scaffold

Three dimensional culture techniques have attracted much attention in the field of tissue engineering. Among them, the RWV (rotating wall vessel) bioreactor, which simulates a microgravity environment for cells, is becoming recognized as a useful tool for several kinds of the cells for tissue regeneration such as chondrocytes, osteoblasts etc. In this paper, we report successful control of the cartilage tissue shape from rabbit bone marrow cells by RWV bioreactor using a collagen sponge scaffold. Moreover, the used scaffold was proved to enhance the glycosaminoglycan (GAG) content and strengthened the compression strength of the product, which reached 0.9 MPa in only one-week RWV culture. The usage of a collagen sponge scaffold shortened the necessary culture period for tissue transplantation.

Cell carrier function of hollow-fiber membrane in rotating wall vessel bioreactor

Large-scale expansion of the osteoblasts of a Sprague-Dawley (SD) rat was studied in a rotating wall hollow-fiber membrane bioreactor (RWHMB) by using hollow-fiber membrane as the carrier. For the sake of contrast, cells were also expanded in a T-flask using a hollow-fiber membrane as carrier and in a rotating wall vessel bioreactor (RWVB) using a microcarrier. During the culture period, the cells were sampled every 12 h, and after 5 days, the cells were harvested and evaluated with scanning electron microscopy (SEM), hematoxylin-eosin (HE) staining and alkaline phosphatase (ALP) staining. Moreover, von-Kossa staining and Alizarin Red S staining were carried out for mineralized nodules formation. The results show that in RWHMB, the cells present better morphology and vitality and secrete much more extracellular matrix. It is concluded that the RWHMB combines the advantages of the rotating wall vessel and hollow-fiber membrane bioreactors. The hydrodynamic stimulation within it accelerates the metabolism of the osteoblast and mass transfer, which is propitious to cell differentiation and proliferation.

Three‐dimensional fabrication of engineered bone with human bio‐derived bone scaffolds in a rotating wall vessel bioreactor

Bone tissue engineering has emerged as a promising strategy in the effort to regenerate and repair diseased or damaged bone. The bioreactor, within which engineered bone tissue is cultured, plays a key role in the development of engineered bone graphs. In this work, the potentials of the rotating wall vessel bioreactor (RWVB) and the human bio-derived bone scaffolds (BDBS) for 3D bone culture are evaluated. The osteoblasts isolated from the cranium of neonatal Sprague-Dawley (SD) rat of 3 days old were expanded firstly with microcarrier suspension culture in a RWVB. After the assessment of the biological functions of the expanded cells by histomorphometry, the cells were seeded at 2 x 10(6) and 1 x 10(6) cells/mL, respectively, onto the 3D human BDBS and cultured for 3 weeks in the RWVB. The cells metabolism and nutrient concentration were monitored in the whole culture processes. The structure of the harvested bone tissues was observed with optical microscope and scanning electron microscope (SEM). The biological properties of the engineered bone were detected by alkaline phosphatase (ALP) expression and alizarin red staining to visualize the newly formed bone. Acridine orange/ethidium bromide (AO/EB) double fluorescence staining was used to analyze the cell activity. For a comparative study, cell seeded constructs were also cultured in static conditions. The results indicate that the bone grafts cultured in RWVB with two different seeded cell densities grew well, and the cell number expanded in RWVB was five times as that in T-flask and spinner flask. There were significantly more collagen fibers mineralized nodules and new osteoid tissue formed than those in T-flask and spinner flask. It also demonstrated that with the stress stimulation inside the fluid in the RWVB, the ALP expression could be increased; the formation of mineralized nodules can be accelerated.

Repair of large osteochondral defects with allogeneic cartilaginous aggregates formed from bone marrow‐derived cells using RWV bioreactor

Our objective was to examine the technique of regenerating cartilage tissue from bone marrow-derived cells by three-dimensional (3D) culture using the rotating wall vessel (RWV) bioreactor. Three-dimensional and cylindrical aggregates of allogeneic cartilage with dimensions of 10 x 5 mm (height x diameter) formed by the RWV bioreactor were transplanted into osteochondral defects of Japanese white rabbits (Group T, n = 15). For the control, some osteochondral defects were left empty (Group C, n = 18). At 4, 8, and 12 weeks postimplantation, the reparative tissues were evaluated macroscopically, histologically, and biochemically. In Group T at as early as 4 weeks, histological observation, especially via safranin-O staining, suggested that the reparative tissues resembled hyaline cartilage. And we observed no fibrous tissues between reparative tissue and adjacent normal tissues. In the deeper portion of the bony compartment, the osseous tissues were well remodeled. At 4 and 8 weeks postimplantation, the mean histological score of Group T was significantly better than that of Group C (p < 0.05). The glycosaminoglycans (GAG)/DNA ratio in both groups increased gradually from 4 to 8 weeks and then decreased from 8 to 12 weeks. We herein report the first successful regeneration of cartilage in osteochondral defects in vivo using allogeneic cartilaginous aggregates derived from bone marrow-derived cells by 3D culture using the RWV bioreactor.

Characterization of Primary Breast Carcinomas Grown in Three-Dimensional Cultures

The process of progression and spread of cancer is not easily replicated in animal models and is difficult to examine in vitro. This is particularly true for human primary breast carcinoma cells, whose in vitro growth is shown to be limited to one or two passages in monolayer culture. Three-dimensional (3D) growth of breast cancer cells suggests that cell aggregates grown in this manner have many similarities to in vivo behavior. Primary tumors obtained from five breast cancer patients were grown in 3D cultures using the rotating-wall vessel bioreactor. Tumor aggregates were assessed for DNA ploidy, cell cycle kinetics, and expression of tumor markers and cytokines. Comparisons between fresh tumor cells and 3D aggregates were performed. All five breast cancers were found to be aneuploid after 3D culture, with elevated S-phase fractions. Reverse transcription-polymerase chain reaction analyses revealed mRNA expression of HER2/neu, H-ras, K-ras, p53, transforming growth factor-alpha, transforming growth factor-beta, interleukin-1, and interleukin-6 in 3D-grown tumor cells; in most cases, expression appeared increased when compared with mRNA obtained from freshly isolated primary tumor cells. After prolonged 3D growth in the rotating wall bioreactor, complex tissue-like constructs of primary breast tumor cells exhibited significantly increased proliferative activity in conjunction with oncogene activation and developed into aggressive aneuploid populations.

Oxygen transport and consumption by suspended cells in microgravity: A multiphase analysis

A rotating bioreactor for the cell/tissue culture should be operated to obtain sufficient nutrient transfer and avoid damage to the culture materials. Thus, the objective of the present study is to determine the appropriate suspension conditions for the bead/cell distribution and evaluate oxygen transport in the rotating wall vessel (RWV) bioreactor. A numerical analysis of the RWV bioreactor is conducted by incorporating the Eulerian-Eulerian multiphase and oxygen transport equations. The bead size and rotating speed are the control variables in the calculations. The present results show that the rotating speed for appropriate suspensions needs to be increased as the size of the bead/cell increases: 10 rpm for 200 microm; 12 rpm for 300 microm; 14 rpm for 400 microm; 18 rpm for 600 microm. As the rotating speed and the bead size increase from 10 rpm/200 microm to 18 rpm/600 microm, the mean oxygen concentration in the 80% midzone of the vessel is increased by approximately 85% after 1-h rotation due to the high convective flow for 18 rpm/600 microm case as compared to 10 rpm/200 microm case. The present results may serve as criteria to set the operating parameters for a RWV bioreactor, such as the size of beads and the rotating speed, according to the growth of cell aggregates. In addition, it might provide a design parameter for an advanced suspension bioreactor for 3-D engineered cell and tissue cultures.

Encapsulation and osteoinduction of human periodontal ligament fibroblasts in chitosan–hydroxyapatite microspheres

Periodontal ligament cells play a crucial role in the regeneration of periodontal tissues and an undifferentiated mesenchymal cell subset is thought to exist within this population. The aim of this study was to assess the osteogenic differentiation potential of human periodontal ligament fibroblasts (hPDLFs) in three dimensional (3D)-osteogenic culture environment following encapsulation in chitosan-hydroxyapatite (C/HA) microspheres with the size range of 350-450 microm. Human PDLF cultures were established and three experimental groups were formed: (i) two-dimensional (2D)-culture as single cell monolayer, (ii) 3D-static culture of C/HA encapsulated hPDLFs, and (iii) 3D-dynamic culture of C/HA encapsulated hPDLFs in a rotating wall vessel bioreactor. The cells were cultured in standard culture medium supplemented with beta-glycerophosphate, dexamethasone, and ascorbic acid. After 21 days, immunohistochemistry was performed using antibodies against osteonectin, osteopontin, bone-sialoprotein, and osteocalcin as osteogenic differentiation markers. Phase-contrast and scanning electron microscopy observations were used for histological and morphological evaluation. The combined effects of osteoinductive medium and HA-containing composite microsphere material on encapsulated hPDLFs resulted in the transformation of a considerable portion of the cells into osteoblastic lineage at the end of the experiments. Results demonstrate the ability of hPDLFs to undergo osteogenic differentiation upon induction in vitro, both under 2D and 3D culture conditions. C/HA microspheres in microgravity bioreactor may serve as a suitable 3D environment to support the osteogenic differentiation of human PDLFs, in vitro.