A bioreactor test system to mimic the biological and mechanical environment of oral soft tissues and to evaluate substitutes for connective tissue grafts

Gingival cells of the oral connective tissue are exposed to complex mechanical forces during mastication, speech, tooth movement and orthodontic treatments. Especially during wound healing following surgical procedures, internal and external forces may occur, creating pressure upon the newly formed tissue. This clinical situation has to be considered when developing biomaterials to augment soft tissue in the oral cavity. In order to pre‐evaluate a collagen sponge intended to serve as a substitute for autogenous connective tissue grafts (CTGs), a dynamic bioreactor system was developed. Pressure and shear forces can be applied in this bioreactor in addition to a constant medium perfusion to cell‐material constructs. Three‐dimensional volume changes and stiffness of the matrices were analyzed. In addition, cell responses such as cell vitality and extracellular matrix (ECM) production were investigated. The number of metabolic active cells constantly increased under fully dynamic culture conditions. The sponges remained elastic even after mechanical forces were applied for 14 days. Analysis of collagen type I and fibronectin revealed a statistically significant accumulation of these ECM molecules (P < 0.05–0.001) when compared to static cultures. An increased expression of tenascin‐c, indicating tissue remodeling processes, was observed under dynamic conditions only. The results indicate that the tested in vitro cell culture system was able to mimic both the biological and mechanical environments of the clinical situation in a healing wound. Biotechnol. Bioeng. 2010;107: 1029–1039. © 2010 Wiley Periodicals, Inc.     

Differential effect of ECM molecules on re-expression of cartilaginous markers in near quiescent human chondrocytes

The limited source of healthy primary chondrocytes restricts the clinical application of tissue engineering for cartilage repair. Therefore, method to maintain or restore the chondrocyte phenotype during in vitro expansion is essential. The objective of this study is to establish the beneficial effect of ECM molecules on restoring the re‐expression of cartilaginous markers in primary human chondrocytes after extensive monolayer expansion. During the course of chondrocyte serial expansion, COL2A1, SOX9, and AGN mRNA expression levels, and GAG accumulation level were reduced significantly in serially passaged cells. Exogenous type II collagen dose‐dependently elevated GAG level and induced the re‐expression of cartilaginous marker mRNAs in P7 chondrocytes. Chondroitin sulfate did not show significant effect on P7 chondrocytes, while hyaluronic acid inhibited the expression of SOX9 and AGN mRNAs. Upon treatment with type II collagen, FAK, ERK1/2, and JNK were activated via phosphorylation in P7 chondrocytes within 15 min. Furthermore, GFOGER integrin blocking peptide, MEK inhibitor and JNK inhibitor, not p38 inhibitor, significantly reduced the type II collagen‐induced GAG deposition level. Finally, in the presence of TGF‐β1 and IGF‐I, P7 chondrocytes cultured in 3D type II collagen matrix exhibited better cartilaginous features than those cells cultured in the type I collagen matrix. In conclusion, type II collagen alone can effectively restore cartilaginous features of expanded P7 human chondrocytes. It is probably mediated via the activation of FAK‐ERK1/2 and FAK‐JNK signaling pathways. The potential application of type II collagen in expanding a scarcity of healthy chondrocytes in vitro for further tissue engineering is implicated. J. Cell. Physiol. 226: 1981–1988, 2011. © 2010 Wiley‐Liss, Inc.     

Tissue engineering of cartilage using a mechanobioreactor exerting simultaneous mechanical shear and compression to simulate the rolling action of articular joints

The effect of dynamic mechanical shear and compression on the synthesis of human tissue‐engineered cartilage was investigated using a mechanobioreactor capable of simulating the rolling action of articular joints in a mixed fluid environment. Human chondrocytes seeded into polyglycolic acid (PGA) mesh or PGA–alginate scaffolds were precultured in shaking T‐flasks or recirculation perfusion bioreactors for 2.5 or 4 weeks prior to mechanical stimulation in the mechanobioreactor. Constructs were subjected to intermittent unconfined shear and compressive loading at a frequency of 0.05 Hz using a peak‐to‐peak compressive strain amplitude of 2.2% superimposed on a static axial compressive strain of 6.5%. The mechanical treatment was carried out for up to 2.5 weeks using a loading regime of 10 min duration each day with the direction of the shear forces reversed after 5 min and release of all loading at the end of the daily treatment period. Compared with shaking T‐flasks and mechanobioreactor control cultures without loading, mechanical treatment improved the amount and quality of cartilage produced. On a per cell basis, synthesis of both major structural components of cartilage, glycosaminoglycan (GAG) and collagen type II, was enhanced substantially by up to 5.3‐ and 10‐fold, respectively, depending on the scaffold type and seeding cell density. Levels of collagen type II as a percentage of total collagen were also increased after mechanical treatment by up to 3.4‐fold in PGA constructs. Mechanical treatment had a less pronounced effect on the composition of constructs precultured in perfusion bioreactors compared with perfusion culture controls. This work demonstrates that the quality of tissue‐engineered cartilage can be enhanced significantly by application of simultaneous dynamic mechanical shear and compression, with the greatest benefits evident for synthesis of collagen type II. Biotechnol. Bioeng. 2012; 109:1060–1073. © 2011 Wiley Periodicals, Inc.     

Effect of reduced oxygen tension and long-term mechanical stimulation on chondrocyte-polymer constructs

We have investigated the influence of long-term confined dynamic compression and surface motion under low oxygen tension on tissue-engineered cell-scaffold constructs. Porous polyurethane scaffolds (8 mm × 4 mm) were seeded with bovine articular chondrocytes and cultured under normoxic (21% O₂) or hypoxic (5% O₂) conditions for up to 4 weeks. By means of our joint-simulating bioreactor, cyclic axial compression (10–20%; 0.5 Hz) was applied for 1 h daily with a ceramic ball, which simultaneously oscillated over the construct surface (±25°; 0.5 Hz). Culture under reduced oxygen tension resulted in an increase in mRNA levels of type II collagen and aggrecan, whereas the expression of type I collagen was down-regulated at early time points. A higher glycosaminoglycan content was found in hypoxic than in normoxic constructs. Immunohistochemical analysis showed more intense type II and weaker type I collagen staining in hypoxic than in normoxic cultures. Type II collagen gene expression was slightly elevated after short-term loading, whereas aggrecan mRNA levels were not influenced by the applied mechanical stimuli. Of importance, the combination of loading and low oxygen tension resulted in a further down-regulation of collagen type I mRNA expression, contributing to the stabilization of the chondrocytic phenotype. Histological results confirmed the beneficial effect of mechanical loading on chondrocyte matrix synthesis. Thus, mechanical stimulation combined with low oxygen tension is an effective tool for modulating the chondrocytic phenotype and should be considered when chondrocytes or mesenchymal stem cells are cultured and differentiated with the aim of generating cartilage-like tissue in vitro. This work was supported by the Swiss National Science Foundation (grant no. 3200B0-104083).     

Improved cartilage integration and interfacial strength after enzymatic treatment in a cartilage transplantation model

The objective of the present study was to investigate whether treatment of articular cartilage with hyaluronidase and collagenase enhances histological and mechanical integration of a cartilage graft into a defect. Discs of 3 mm diameter were taken from 8-mm diameter bovine cartilage explants. Both discs and annulus were either treated for 24 hours with 0.1% hyaluronidase followed by 24 hours with 10 U/ml collagenase or left untreated (controls). Discs and annulus were reassembled and implanted subcutaneously in nude mice for 5 weeks. Integration of disc with surrounding cartilage was assessed histologically and tested biomechanically by performing a push-out test. After 5 weeks a significant increase in viable cell counts was seen in wound edges of the enzyme-treated group as compared with controls. Furthermore, matrix integration (expressed as a percentage of the total interface length that was connected; mean ± standard error) was 83 ± 15% in the treated samples versus 44 ± 40% in the untreated controls. In the enzyme-treated group only, picro-Sirius Red staining revealed collagen crossing the interface perpendicular to the wound surface. Immunohistochemical analyses demonstrated that the interface tissue contained cartilage-specific collagen type II. Collagen type I was found only in a small region of fibrous tissue at the level of the superficial layer, and collagen type III was completely absent in both groups. A significant difference in interfacial strength was found using the push-out test: 1.32 ± 0.15 MPa in the enzyme-treated group versus 0.84 ± 0.14 MPa in the untreated controls. The study shows that enzyme treatment of cartilage wounds increases histological integration and improves biomechanical bonding strength. Enzymatic treatment may represent a promising addition to current techniques for articular cartilage repair.     

Three hours of perfusion culture prior to 28 days of static culture, enhances osteogenesis by human cells in a collagen GAG scaffold

In tissue engineering, bioreactors can be used to aid in the in vitro development of new tissue by providing biochemical and physical regulatory signals to cells and encouraging them to undergo differentiation and/or to produce extracellular matrix prior to in vivo implantation. This study examined the effect of short term flow perfusion bioreactor culture, prior to long‐term static culture, on human osteoblast cell distribution and osteogenesis within a collagen glycosaminoglycan (CG) scaffold for bone tissue engineering. Human fetal osteoblasts (hFOB 1.19) were seeded onto CG scaffolds and pre‐cultured for 6 days. Constructs were then placed into the bioreactor and exposed to 3 × 1 h bouts of steady flow (1 mL/min) separated by 7 h of no flow over a 24‐h period. The constructs were then cultured under static osteogenic conditions for up to 28 days. Results show that the bioreactor and static culture control groups displayed similar cell numbers and metabolic activity. Histologically, however, peripheral cell‐encapsulation was observed in the static controls, whereas, improved migration and homogenous cell distribution was seen in the bioreactor groups. Gene expression analysis showed that all osteogenic markers investigated displayed greater levels of expression in the bioreactor groups compared to static controls. While static groups showed increased mineral deposition; mechanical testing revealed that there was no difference in the compressive modulus between bioreactor and static groups. In conclusion, a flow perfusion bioreactor improved construct homogeneity by preventing peripheral encapsulation whilst also providing an enhanced osteogenic phenotype over static controls. Bioeng. 2011; 108:1203–1210. © 2010 Wiley Periodicals, Inc.     

Engineered cartilage constructs subject to very low regimens of interstitial perfusion

We have studied an in vitro engineered cartilage model, consisting of bovine articular chondrocytes seeded on micro-porous scaffolds and perfused with very low regimens of interstitial flow. Our previous findings suggested that synthesis of sulphated glycosaminoglycans (sGAG) was promoted in this model, if the level of shear generated on cells was maintained below 10 mPa (0.1 dyn/cm2). Constructs were stimulated with a median shear stress of 1.2 and 6.7 mPa using two independent culture chambers. Quantification of the applied stresses and of oxygen consumption rates was obtained from computational modelling. Experimentally, we set a time zero reference at 24 hours after cell seeding and total culture time at two weeks. The cell metabolic activity, measured by MTT, was significantly lower in all constructs at two weeks (-73% in static controls, -66% in the 1.2 mPa group and -60% in the 6.7 mPa group) vs. the time zero group, and significantly higher (+33%) in the 7 mPa group vs. static controls. The ratio between synthesis of collagen type II/type I, measured by Western Blot, was significantly higher in the 1.2 mPa constructs (+109% vs. the 6.7 mPa group, +120% vs. the time zero group and +286% vs. static controls). A trend of decreased alpha-actin expression was observed with increased ratio of type II to type I collagen, in all groups. These results reinforce the notion that, at early time points in culture, hydrodynamic shear below 10 mPa may promote formation of extra-cellular matrix specific to hyaline cartilage in chondrocyte-seeded constructs.     

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

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

Cartilage production by rabbit articular chondrocytes on polyglycolic acid scaffolds in a closed bioreactor system

Rabbit articular chondrocytes were seeded onto three-dimensional polyglycolic acid (PGA) scaffolds and placed into a closed bioreactor system. After 4 weeks of growth, meshes were examined for cartilage formation. Gross examination revealed solid, glistening material that had the appearance of cartilaginous tissue. Histologic examination revealed cell growth and deposition of extracellular matrix throughout the mesh with a less dense central core. Alcian blue and Safranin 0 staining showed deposition of glycosaminoglycans (GAGs). Immunostaining showed positive reactivity for type II collagen and chondroitin sulfate and no reactivity for type I collagen. Biochemical analysis showed collagen and GAG values to be 15% and 25% dry weight, respectively. Our results indicate that this type of system compares well with those previously described and should be useful for producing cartilage for evaluation in a clinical setting. (c) 1995 John Wiley & Sons, Inc.     

Mechanobiology of engineered cartilage cultured under a quantified fluid-dynamic environment

Natural cartilage remodels both in vivo and in vitro in response to mechanical forces and hence mechanical stimulation is believed to have a potential as a tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage. Fluid-induced shear is known to enhance chondrogenesis on animal cells. A well-defined hydrodynamic environment is required to study the biochemical response to shear of three-dimensional engineered cell systems. We have developed a perfused-column bioreactor in which the culture medium flows through chondrocyte-seeded porous scaffolds, together with a computational fluid-dynamic model of the flow through the constructs' microstructure. A preliminary experiment of human chondrocyte growth under static versus dynamic conditions is described. The median shear stress imposed on the cells in the bioreactor culture, as predicted by the CFD model, is 3 × 10⁻³ Pa (0.03 dyn/cm²) at a flow rate of 0.5 ml/min corresponding to an inlet fluid velocity of 44.2 μm/s. Providing a fluid-dynamic environment to the cells yielded significant differences in cell morphology and in construct structure. Received: 22 December 2001 / Accepted: 18 February 2002     

Effects of extracellular matrix proteins in chondrocyte‐derived matrices on chondrocyte functions

Loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier to the application of chondrocytes for tissue engineering. In previous study, we showed that dedifferentiation of chondrocytes during the passage culture was delayed by matrices formed by primary chondrocytes (P0‐ECM). In this study, we investigated bovine chondrocyte functions when being cultured on isolated extracellular matrix (ECM) protein‐coated substrata and P0‐ECM. Low chondrocyte attachment was observed on aggrecan‐coated substratum and P0‐ECM. Cell proliferation on aggrecan‐ and type II collagen/aggrecan‐coated substrata and P0‐ECM was lower than that on the other ECM protein (type I collagen and type II collagen)‐coated substrata. When chondrocytes were subcultured on aggrecan‐coated substratum, decline of cartilaginous gene expression was delayed, which was similar to the cells subcultured on P0‐ECM. These results indicate that aggrecan plays an important role in the regulation of chondrocyte functions and P0‐ECM may be a good experimental control for investigating the role of each ECM protein in cartilage ECM. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1331–1336, 2013     

Effects of co‐cultures of meniscus cells and articular chondrocytes on PLLA scaffolds

The knee meniscus, a fibrocartilaginous tissue located in the knee joint, is characterized by heterogeneity in extracellular matrix and biomechanical properties. To recreate these properties using a tissue engineering approach, co‐cultures of meniscus cells (MCs) and articular chondrocytes (ACs) were seeded in varying ratios (100:0, 75:25, 50:50, 25:75, and 0:100) on poly‐L ‐lactic acid (PLLA) scaffolds and cultured in serum‐free medium for 4 weeks. Histological, biochemical, and biomechanical tests were used to assess constructs at the end time point. Strong staining for collagen and glycosaminoglycan (GAG) was observed in all groups. Constructs with 100% MCs were positive for collagen I and constructs cultured with 100% ACs were positive for collagen II, while a mixture of collagen I and II was observed in other co‐culture groups. Total collagen and GAG per construct increased as the percentage of ACs increased (27 ± 8 µg, 0% AC to 45 ± 8 µg, 100% ACs for collagen and 12 ± 4 µg, 0% ACs to 40 ± 5 µg, 100% ACs for GAG). Compressive modulus (instantaneous and relaxation modulus) of the constructs was significantly higher in the 100% ACs group (63 ± 12 and 22 ± 9 kPa, respectively) when compared to groups with higher percentage of MCs. No differences in tensile properties were noted among groups. Specific co‐culture ratios were identified mimicking the GAG/DW of the inner (0:100, 25:75, and 50:50) and outer regions (100:0) of the meniscus. Overall, it was demonstrated that co‐culturing MCs and ACs on PLLA scaffolds results in functional tissue engineered meniscus constructs with a spectrum of biochemical and biomechanical properties. Biotechnol. Bioeng. 2009;103: 808–816. © 2009 Wiley Periodicals, Inc.     

Tissue engineering strategies for cartilage generation--micromass and three dimensional cultures using human chondrocytes and a continuous cell line

Utilizing ATDC5 murine chondrogenic cells and human articular chondrocytes, this study sought to develop facile, reproducible three-dimensional models of cartilage generation with the application of tissue engineering strategies, involving biodegradable poly(glycolic acid) scaffolds and rotating wall bioreactors, and micromass pellet cultures. Chondrogenic differentiation, assessed by histology, immunohistochemistry, and gene expression analysis, in ATDC5 and articular chondrocyte pellets was evident by the presence of distinct chondrocytes, expressing Sox-9, aggrecan, and type II collagen, in lacunae embedded in a cartilaginous matrix of type II collagen and proteoglycans. Tissue engineered explants of ATDC5 cells were reminiscent of cartilaginous structures composed of numerous chondrocytes, staining for typical chondrocytic proteins, in lacunae embedded in a matrix of type II collagen and proteoglycans. In comparison, articular chondrocyte explants exhibited areas of Sox-9, aggrecan, and type II collagen-expressing cells growing on fleece, and discrete islands of chondrocytic cells embedded in a cartilaginous matrix.     

The expression of the fibrillar collagen genes during fracture healing: heterogeneity of the matrices and differentiation of the osteoprogenitor cells

The cells that express the genes for the fibrillar collagens, types I, II, III and V, during callus development in rabbit tibial fractures healing under stable and unstable mechanical conditions were localized. The fibroblast-like cells in the initial fibrous matrix express types I, III and V collagen mRNAs. Osteoblasts, and osteocytes in the newly formed membranous bone under the periosteum, express the mRNAs for types I, III and V collagens, but osteocytes in the mature trabeculae express none of these mRNAs. Cartilage formation starts at 7 days in calluses forming under unstable mechanical conditions. The differentiating chondrocytes express both types I and II collagen mRNAs, but later they cease expression of type I collagen mRNA. Both types I and II collagens were located in the cartilaginous areas. The hypertrophic chondrocytes express neither type I, nor type II, collagen mRNA. Osteocalcin protein was located in the bone and in some cartilaginous regions. At 21 days, irrespective of the mechanical conditions, the callus consists of a layer of bone; only a few osteoblasts lining the cavities now express type I collagen mRNA.We suggest that osteoprogenitor cells in the periosteal tissue can differentiate into either osteoblasts or chondrocytes and that some cells may exhibit an intermediate phenotype between osteoblasts and chondrocytes for a short period. The finding that hypertrophic chondrocytes do not express type I collagen mRNA suggests that they do not transdifferentiate into osteoblasts during endochondral ossification in fracture callus.     

Adaptation of cellular mechanical behavior to mechanical loading for osteoblastic cells

Numerous cellular biochemical responses to mechanical loading are transient, indicating a cell's ability to adapt its behavior to a new mechanical environment. Since load-induced cellular deformation can initiate these biochemical responses, the overall goal of this study was to investigate the adaptation of global, or whole-cell, mechanical behavior, i.e., cellular deformability, in response to mechanical loading for osteoblastic cells. Confluent cell cultures were subjected to 1 or 2 Pa flow-induced shear stress for 2 h. Whole-cell mechanical behavior was then measured for individual cells using an atomic force microscope. Compared to cells maintained under static conditions, whole-cell stiffness was 1.36-fold (p=0.006) and 1.70-fold (p<0.001) greater for cells exposed to 1 and 2 Pa shear loading, respectively. The increase in shear stress magnitude from 1 to 2 Pa also caused a statistically significant, 1.25-fold increase in cell stiffness (p=0.02). Increases in cell stiffness were not altered in either flow group for 70 min after flow was terminated (p=0.15). Flow-induced rearrangement of the actin cytoskeleton was also maintained for at least 90 min after flow was terminated. Taken together, these findings support the hypothesis that cells become mechanically adapted to their mechanical environment via cytoskeletal modifications. Accordingly, cellular mechanical adaptation may play a key role in regulation of cellular mechanosensitivity and the related effects on tissue structure and function.     

周期性机械应力对髓核细胞增殖和细胞外基质表达的影响

目的：探讨周期性机械应力对髓核细胞增殖和细胞外基质表达的影响。方法：对兔髓核细胞进行体外细胞培养,对细胞施加周期性机械应力（0.25Mpa,0.1Hz）。实验分为2组,不加压组和加压组,不加压组置于单纯旋转式生物反应器内,加压组每天置于周期性机械应力场内2小时。分别于3天,7天检测细胞数目以及聚集蛋白聚糖（aggrecan）和Ⅱ型胶原的基因表达。结果：髓核细胞的增殖和聚集蛋白聚糖、Ⅱ型胶原基因的表达水平与周期性压力密切相关,在周期性机械应力刺激下髓核细胞增殖明显,细胞外基质的分泌增加,组织工程髓核细胞的活性显著提高。结论：周期性机械应力能够显著促进髓核细胞增殖,同时上调聚集蛋白聚糖、Ⅱ型胶原的基因的表达。     

Recellularization of Decellularized Lung Scaffolds Is Enhanced by Dynamic Suspension Culture

Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.     

Regenerative medicine bioprocessing: Concentration and behavior of adherent cell suspensions and pastes

Regenerative medicines based on human cells demand their harvesting, culture, and processing. Manufacturing processes are likely to include cell concentration and subsequent controlled dosing of concentrates, for example, to the patient or tissue construct. The integrity and functionality of the cells must be maintained during these processing stages. In this study the performance of two different cell concentration protocols (involving centrifugation and resuspension) are compared and consideration given to possible causes of cell loss. Further studies examine cell size and rheological behavior of anchorage‐dependent mammalian cell suspensions, and the effect of capillary flow stress (0.5–15 Pa, laminar flow regime) on cell number and membrane integrity as quantified by flow cytometry. The cell concentration protocols achieved maximum cell volume fraction of around 0.3 and the improved protocol exhibited intact cell yield of 80 ± 13%, demonstrating proof‐of principle for achieving tissue‐like cell concentrations by a process of centrifugation and orbital shaking. Volume mean cell diameter (cell diameter at the mean cell volume) for the rat aortic smooth muscle cells (CRL‐1444) used in this study was 22.4 µm. Concentrated cell suspension rheology approximated to power law behavior and exhibited similar trends to reports for plant and yeast cells. Capillary transfer at 2–15 Pa (wall shear stress) did not significantly affect cell number or membrane integrity while losses observed at low shear (0.5, 1.0 Pa) were probably due to surface attachment of cells in the apparatus. Biotechnol. Bioeng. 2009;103: 1236–1247. © 2009 Wiley Periodicals, Inc.     

Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development

A concentric cylinder bioreactor has been developed to culture tissue engineered cartilage constructs under hydrodynamic loading. This bioreactor operates in a low shear stress environment, has a large growth area for construct production, allows for dynamic seeding of constructs, and provides for a uniform loading environment. Porous poly-lactic acid constructs, seeded dynamically in the bioreactor using isolated bovine chondrocytes, were cultured for 4 weeks at three seeding densities (60, 80, 100 x 10(6) cells per bioreactor) and three different shear stresses (imposed at 19, 38, and 76 rpm) to characterize the effect of chondrocyte density and hydrodynamic loading on construct growth. Construct seeding efficiency with chondrocytes is greater than 95% within 24 h. Extensive chondrocyte proliferation and matrix deposition are achieved so that after 28 days in culture, constructs from bioreactors seeded at the highest cell densities contain up to 15 x 10(6) cells, 2 mg GAG, and 3.5 mg collagen per construct and exhibit morphology similar to that of native cartilage. Bioreactors seeded with 60 million chondrocytes do not exhibit robust proliferation or matrix deposition and do not achieve morphology similar to that of native cartilage. In cultures under different steady hydrodynamic loading, the data demonstrate that higher shear stress suppresses matrix GAG deposition and encourages collagen incorporation. In contrast, under dynamic hydrodynamic loading conditions, cartilage constructs exhibit robust matrix collagen and GAG deposition. The data demonstrate that the concentric cylinder bioreactor provides a favorable hydrodynamic environment for cartilage construct growth and differentiation. Notably, construct matrix accumulation can be manipulated by hydrodynamic loading. This bioreactor is useful for fundamental studies of construct growth and to assess the significance of cell density, nutrients, and hydrodynamic loading on cartilage development. In addition, studies of cartilage tissue engineering in the well-characterized, uniform environment of the concentric cylinder bioreactor will develop important knowledge of bioprocessing parameters critical for large-scale production of engineered tissues.