Human chondroprogenitors in alginate–collagen hybrid scaffolds produce stable cartilage in vivo

The goal of this study was to evaluate human epiphyseal chondroprogenitor cells (ECPs) as a potential new cell source for cartilage regeneration. ECPs were compared to human bone marrow stromal cells (MSCs) and human adult articular chondrocytes (ACs) for their chondrogenic potential and phenotypic stability in vitro and in vivo. The cells were seeded in Optimaix‐3D scaffolds at 5 × 10⁴ cells/mm³ and gene expression, matrix production and mechanical properties were analysed up to 6 weeks. In vitro, ECPs synthesized consistently high collagen 2 and low collagen 10. AC‐seeded constructs exhibited high donor variability in GAG/DNA values as well as in collagen 2 staining, but showed low collagen 10 production. MSCs, on the other hand, expressed high levels of collagen 2 but also of collagens 1 and 10, and were therefore not considered further. In vivo, there was considerable loss of matrix proteins in ECPs compared to in vitro cultured samples. To overcome this, a second implantation study investigated the effect of mixing cells with alginate prior to seeding in the scaffold. ECPs in alginate maintained their cartilage matrix and resisted mineralization and vessel infiltration better 6 weeks after subcutaneous implantation, whereas ACs lost their chondrogenic matrix completely. This study shows the great potential of ECPs as an off‐the‐shelf, highly chondrogenic cell type that produces stable cartilage in vivo. Copyright © 2016 John Wiley & Sons, Ltd.     

Biomimetic scaffolds and dynamic compression enhance the properties of chondrocyte‐ and MSC‐based tissue‐engineered cartilage

Adult chondrocytes are surrounded by a protein‐ and glycosaminoglycan‐rich extracellular matrix and are subjected to dynamic mechanical compression during daily activities. The extracellular matrix and mechanical stimuli play an important role in chondrocyte biosynthesis and homeostasis. In this study, we aimed to develop scaffold and compressive loading conditions that mimic the native cartilage micro‐environment and enable enhanced chondrogenesis for tissue engineering applications. Towards this aim, we fabricated porous scaffolds based on silk fibroin (SF) and SF with gelatin/chondroitin sulfate/hyaluronate (SF‐GCH), seeded the scaffolds with either human bone marrow mesenchymal stromal cells (BM‐MSCs) or chondrocytes, and evaluated their performance with and without dynamic compression. Human chondrocytes derived from osteoarthritic joints and BM‐MSCs were seeded in scaffolds, precultured for 1 week, and subjected to compression with 10% dynamic strain at 1 Hz, 1 hr/day for 2 weeks. When dynamic compression was applied, chondrocytes significantly increased expression of aggrecan (ACAN) and collagen X (COL10A1) up to fivefold higher than free‐swelling controls. In addition, dynamic compression dramatically improved the chondrogenesis and chondrocyte biosynthesis cultured in both SF and SF‐GCH scaffolds evidenced by glycosaminoglycan (GAG) content, GAG/DNA ratio, and immunostaining of collagen type II and aggrecan. However, both chondrocytes and BM‐MSCs cultured in SF‐GCH scaffolds under dynamic compression showed higher GAG content and compressive modulus than those in SF scaffolds. In conclusion, the micro‐environment provided by SF‐GCH scaffolds and dynamic compression enhances chondrocyte biosynthesis and matrix accumulation, indicating their potential for cartilage tissue engineering applications.     

Cell–cell interaction between vocal fold fibroblasts and bone marrow mesenchymal stromal cells in three‐dimensional hyaluronan hydrogel

Mesenchymal stromal cells (MSCs) are multipotential adult cells present in all tissues. Paracrine effects and differentiating ability make MSCs an ideal cell source for tissue regeneration. However, little is known about how interactions between implanted MSCs and native cells influence cellular growth, proliferation, and behaviour. By using an in vitro three‐dimensional (3D) co‐culture assay of normal or scarred human vocal fold fibroblasts (VFFs) and bone marrow‐derived MSCs (BM‐MSCs) in a uniquely suited hyaluronan hydrogel (HyStem–VF), we investigated cell morphology, survival rate, proliferation and protein and gene expression of VFFs and BM‐MSCs. BM‐MSCs inhibited cell proliferation of both normal and scarred VFFs without changes in VFF morphology or viability. BM‐MSCs demonstrated decreased proliferation and survival rate after 7 days of co‐culture with VFFs. Interactions between BM‐MSCs and VFFs led to a significant increase in protein secretion of collagen I and hepatocyte growth factor (HGF) and a decrease of vascular endothelial growth factor (VEGF), monocyte chemotactic protein‐1 (MCP‐1) and interleukin‐6 (IL‐6). In particular, BM‐MSCs significantly upregulated matrix metalloproteinase 1 (MMP1) and HGF gene expression for scarred VFFs compared to normal VFFs, indicating the potential for increases in extracellular matrix remodelling and tissue regeneration. Application of BM‐MSCs‐hydrogels may play a significant role in tissue regeneration, providing a therapeutic approach for vocal fold scarring. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of chondroitin sulphate C on the in vitro and in vivo chondrogenesis of mesenchymal stem cells in crosslinked type II collagen scaffolds

This study evaluates the crosslinkage effect of chondroitin sulphate C (CSC) and type II collagen (COL II) on chondrogenesis of mesenchymal stem cells (MSCs) in vitro and in vivo. In the in vitro studies, our results show that the weight ratio CSC:COL II that reaches 1.2:100 (CSC_1.2/100–COL II scaffold) can provide an optimal microenvironment for MSC chondrogenesis. When MSCs are cultured in this CSC_1.2/100–COL II scaffold, the chondrogenic gene expression of cultured cells is upregulated, while the osteogenic gene expression of these is downregulated. In addition, MSCs cultivated in the CSC_1.2/100–COL II scaffold are found to express the highest glycosaminoglycans:DNA ratio as compared to those in scaffolds of other CSC:COL II ratios. Histological and immunohistological evidence also supports the result. In the in vivo study, our results show that MSCs cultivated in the CSC_1.2/100–COL II scaffold demonstrate a better repair ability on cartilage lesions than does the COL II scaffold. After 1 month in vivo, the injected MSCs in the CSC_1.2/100–COL II scaffold show lacuna structures and stimulate the formation of type II collagen at the defective sites. Six months after transplantation, the generated cells in the CSC_1.2/100–COL II group show higher gene expressions of type II collagen and aggrecan but lower gene expression of type I collagen at the defective sites than those in the COL II group. The results strongly suggest that CSC_1.2/100–COL II scaffold can serve as a potential candidate for cartilage repair and improve the chondrogenesis of MSCs in general. Copyright © 2012 John Wiley & Sons, Ltd.     

Matrix formation is enhanced in co‐cultures of human meniscus cells with bone marrow stromal cells

The ultimate aim of this study was to assess the feasibility of using human bone marrow stromal cells (BMSCs) to supplement meniscus cells for meniscus tissue engineering and regeneration. Human menisci were harvested from three patients undergoing total knee replacements. Meniscus cells were released from the menisci after collagenase treatment. BMSCs were harvested from the iliac crest of three patients and were expanded in culture until passage 2. Primary meniscus cells and BMSCs were co‐cultured in vitro in three‐dimensional (3D) pellet culture at three different cell–cell ratios for 3 weeks under normal (21% O₂) or low (3% O₂) oxygen tension in the presence of serum‐free chondrogenic medium. Pure BMSCs and pure meniscus cell pellets served as control groups. The tissue generated was assessed biochemically, histochemically and by quantitative RT–PCR. Co‐cultures of primary meniscus cells and BMSCs resulted in tissue with increased (1.3–1.7‐fold) deposition of proteoglycan (GAG) extracellular matrix (ECM) relative to tissues derived from BMSCs or meniscus cells alone under 21% O₂. GAG matrix formation was also enhanced (1.3–1.6‐fold) under 3% O₂ culture conditions. Alcian blue staining of generated tissue confirmed increased deposition of GAG‐rich matrix. mRNA expression of type I collagen (COL1A2), type II collagen (COL2A1) and aggrecan were upregulated in co‐cultured pellets. However, SOX9 and HIF‐1α mRNA expression were not significantly modulated by co‐culture. Co‐culture of primary meniscus cells with BMSCs resulted in increased ECM formation. Co‐delivery of meniscus cells and BMSCs can, in principle, be used in tissue engineering and regenerative medicine strategies to repair meniscus defects. Copyright © 2012 John Wiley & Sons, Ltd.     

Spatial distribution and survival of human and goat mesenchymal stromal cells on hydroxyapatite and β‐tricalcium phosphate

The combination of scaffolds and mesenchymal stromal cells (MSCs) is a promising approach in bone tissue engineering (BTE). Knowledge on the survival, outgrowth and bone‐forming capacity of MSCs in vivo is limited. Bioluminescence imaging (BLI), histomorphometry and immunohistochemistry were combined to study the fate of gene‐marked goat and human MSCs (gMSCs, hMSCs) on scaffolds with different osteoinductive properties. Luciferase–GFP‐labelled MSCs were seeded on hydroxyapatite (HA) or β‐tricalcium phosphate (TCP), cultured for 7 days in vitro in osteogenic medium, implanted subcutaneously in immunodeficient mice and monitored with BLI for 6 weeks. The constructs were retrieved and processed for histomorphometry and detection of luciferase‐positive cells (LPCs). For gMSCs, BLI revealed doubling of signal after 1 week, declining to 60% of input after 3 weeks and remaining constant until week 6. hMSCs showed a constant decrease of BLI signal to 25% of input, indicating no further expansion. Bone formation of gMSCs was two‐fold higher on TCP than HA. hMSCs and gMSCs control samples produced equal amounts of bone on TCP. Upon transduction, there was a four‐fold reduction in bone formation compared with untransduced hMSCs, and no bone was formed on HA. LPCs were detected at day 14, but were much less frequent at day 42. Striking differences were observed in spatial distribution. MSCs in TCP were found to be aligned and interconnected on the surface but were scattered in an unstructured fashion in HA. In conclusion, the spatial distribution of MSCs on the scaffold is critical for cell–scaffold‐based BTE. Copyright © 2012 John Wiley & Sons, Ltd.     

Dynamic cell–cell interactions between cord blood haematopoietic progenitors and the cellular niche are essential for the expansion of CD34+, CD34+CD38− and early lymphoid CD7+ cells

Most clinical applications of haematopoietic stem/progenitor cells (HSCs) would benefit from their ex vivo expansion to obtain a therapeutically significant amount of cells from the available donor samples. We studied the impact of cellular interactions between umbilical cord blood (UCB) haematopoietic cells and bone marrow (BM)‐derived mesenchymal stem cells (MSCs) on the ex vivo expansion and differentiative potential of UCB CD34⁺‐enriched cells. UCB cells were cultured: (a) directly in contact with BM MSC‐derived stromal layers (contact); (b) separated by a microporous membrane (non‐contact); or (c) without stroma (no stroma). Highly dynamic culture events occurred in HSC‐MSC co‐cultures, involving cell–cell interactions, which preceded HSC expansion. Throughout the time in culture [18 days], total cell expansion was significantly higher in contact (fold increase of 280 ± 37 at day 18) compared to non‐contact (85 ± 25). No significant cell expansion was observed in stroma‐free cultures. CD34⁺ cell expansion was also clearly favoured by direct contact with BM MSCs (35 ± 5‐ and 7 ± 3‐fold increases at day 18 for contact and non‐contact, respectively). Moreover, a higher percentage of CD34⁺CD38^? cells was consistently maintained during the time in culture under contact (8.1 ± 1.9% at day 18) compared to non‐contact (5.7 ± 1.6%). Importantly, direct cell interaction with BM MSCs significantly enhanced the expansion of early lymphoid CD7⁺ cells, yielding considerably higher (×3–10) progenitor numbers compared to non‐contact conditions. These results highlight the importance of dynamic cell–cell interactions between UCB HSCs and BM MSCs, towards the maximization of HSC expansion ex vivo to obtain clinically relevant cell numbers for multiple settings, such as BM transplantation or somatic cell gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Mesenchymal stem cells can be recruited to wounded tissue via hepatocyte growth factor‐loaded biomaterials

Mesenchymal stem cells (MSC) are precursor cells of mesodermal tissue and, because of their trophic phenotype, they are known to play beneficial roles in wound healing. In addition, various tissue engineering strategies are based on MSC/biomaterial constructs. As the isolation and expansion of MSCs is a long‐term process, a major goal is to develop an endogenous stem cell recruitment system that circumvents all ex vivo steps generally used for tissue engineering. Therefore collagen and silk fibroin were loaded with hepatocyte growth factor (HGF), a chemoattractant for MSCs. Collagen was mixed with HGF during polymerization, while silk fibroin and HGF were produced as fusion proteins by transgenic silkworms. To demonstrate release of active HGF, enzyme‐linked immunosorbent assay, in vitro migration assays and animal studies were performed to demonstrate MSC migration in vivo, followed by detailed examinations of the immunological effects of the biomaterials. Hepatocyte growth factor was released burst‐like, both from silk fibroin and collagen during the first 8 h and gradually for up to 168 h in vitro. Directed migration in vitro was demonstrated when MSCs were exposed to HGF. In vivo, HGF‐loaded collagen and silk fibroin were tolerated as subcutaneous implants. In addition, it was proved that endogenous MSCs were recruited from the local environment. These results show for the first time recruitment of endogenous MSCs to HGF‐loaded collagen (fast degradable) and silk fibroin scaffolds (long‐term degradable) in vitro and in vivo. This knowledge could be applied to make off‐the‐shelf, readily available constructs for use in patients with chronic wound or burns. Copyright © 2016 John Wiley & Sons, Ltd.     

The differential in vitro and in vivo responses of bone marrow stromal cells on novel porous gelatin–alginate scaffolds

Tissue engineering and stem cell therapy hold great potential of being able to fully restore, repair and replace damaged, diseased or lost tissues in the body. Biocompatible porous scaffolds are used for the delivery of cells to the regeneration sites. Marrow stromal cells (MSCs), also referred to as mesenchymal stem cells, are an attractive cell source for tissue engineering, due to the relative ease of isolation and the ability of in vitro expanded MSCs to generate multiple cell types, including osteoblasts, chondrocytes and adipocytes. This study utilized a novel technique called microwave vacuum drying to fabricate porous gelatin–alginate scaffolds for the delivery of MSCs and investigated the differential in vitro and in vivo responses of MSCs seeded on these scaffolds. Scaffold total porosity was found to decrease with increased cross‐link density but the pore size and pore size distribution were not affected. Although highly porous, the scaffold had relatively small pores and limited interconnectivity. The porous gelatin–alginate scaffold demonstrated excellent biocompatibility with neovascularization on the surfaces and was bioresorbed completely in vivo, depending upon the cross‐link density. MSCs were able to attach and proliferate at the same rate on the scaffolds, and the self‐renewal potential of MSC cultures was similar during both in vitro culture and in vivo implantation. However, the subcutaneous microenvironment was found to suppress MSC differentiation along the osteogenic, chondrogenic and adipogenic lineages compared to in vitro conditions, highlighting the differential responses of MSCs cultured in vitro compared to implantation in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

Ectopic bone formation by aggregated mesenchymal stem cells from bone marrow and adipose tissue: A comparative study

Tissue engineered constructs (TECs) based on spheroids of bone marrow mesenchymal stromal cells (BM‐MSCs) combined with calcium phosphate microparticles and enveloped in a platelet‐rich plasma hydrogel showed that aggregation of MSCs improves their ectopic bone formation potential. The stromal vascular fraction (SVF) and adipose‐derived MSCs (ASCs) have been recognized as an interesting MSC source for bone tissue engineering, but their ectopic bone formation is limited. We investigated whether aggregation of ASCs could similarly improve ectopic bone formation by ASCs and SVF cells. The formation of aggregates with BM‐MSCs, ASCs and SVF cells was carried out and gene expression was analysed for osteogenic, chondrogenic and vasculogenic genes in vitro. Ectopic bone formation was evaluated after implantation of TECs in immunodeficient mice with six conditions: TECs with ASCs, TECs with BM‐MSC, TECs with SVF cells (with and without rhBMP2), no cells and no cells with rhBMP2. BM‐MSCs showed consistent compact spheroid formation, ASCs to a lesser extent and SVF showed poor spheroid formation. Aggregation of ASCs induced a significant upregulation of the expression of osteogenic markers like alkaline phosphatase and collagen type I, as compared with un‐aggregated ASCs. In vivo, ASC and SVF cells both generated ectopic bone in the absence of added morphogenetic proteins. The highest incidence of bone formation was seen with BM‐MSCs (7/9) followed by SVF + rhBMP2 (4/9) and no cells + rhBMP2 (2/9). Aggregation can improve ectopic bone tissue formation by adipose‐derived cells, but is less efficient than rhBMP2. A combination of both factors should now be tested to investigate an additive effect.     

Effect of visco‐elastic silk–chitosan microcomposite scaffolds on matrix deposition and biomechanical functionality for cartilage tissue engineering

Commonly used polymer‐based scaffolds often lack visco‐elastic properties to serve as a replacement for cartilage tissue. This study explores the effect of reinforcement of silk matrix with chitosan microparticles to create a visco‐elastic matrix that could support the redifferentiation of expanded chondrocytes. Goat chondrocytes produced collagen type II and glycosaminoglycan (GAG)‐enriched matrix on all the scaffolds (silk:chitosan 1:1, 1:2 and 2:1). The control group of silk‐only constructs suffered from leaching out of GAG molecules into the medium. Chitosan‐reinforced scaffolds retained a statistically significant (p < 0.02) higher amount of GAG, which in turn significantly increased (p < 0.005) the aggregate modulus (as compared to silk‐only controls) of the construct akin to that of native tissue. Furthermore, the microcomposite constructs demonstrated highly pronounced hysteresis at 4% strain up to 400 cycles, mimicking the visco‐elastic properties of native cartilage tissue. These results demonstrated a step towards optimizing the design of biomaterial scaffolds used for cartilage tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro chondrogenesis with lysozyme susceptible bacterial cellulose as a scaffold

A current focus of tissue engineering is the use of adult human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes for cartilage repair. Several natural and synthetic polymers (including cellulose) have been explored as a biomaterial scaffold for cartilage tissue engineering. While bacterial cellulose (BC) has been used in tissue engineering, its lack of degradability in vivo and high crystallinity restricts widespread applications in the field. Recently we reported the formation of a novel bacterial cellulose that is lysozyme‐susceptible and ‐degradable in vivo from metabolically engineered Gluconacetobacter xylinus. Here we report the use of this modified bacterial cellulose (MBC) for cartilage tissue engineering using hMSCs. MBC's glucosaminoglycan‐like chemistry, combined with in vivo degradability, suggested opportunities to exploit this novel polymer in cartilage tissue engineering. We have observed that, like BC, MBC scaffolds support cell attachment and proliferation. Chondrogenesis of hMSCs in the MBC scaffolds was demonstrated by real‐time RT–PCR analysis for cartilage‐specific extracellular matrix (ECM) markers (collagen type II, aggrecan and SOX9) as well as histological and immunohistochemical evaluations of cartilage‐specific ECM markers. Further, the attachment, proliferation, and differentiation of hMSCs in MBC showed unique characteristics. For example, after 4 weeks of cultivation, the spatial cell arrangement and collagen type‐II and ACAN distribution resembled those in native articular cartilage tissue, suggesting promise for these novel in vivo degradable scaffolds for chondrogenesis. Copyright © 2013 John Wiley & Sons, Ltd.     

In vitro and in vivo co‐culture of chondrocytes and bone marrow stem cells in photocrosslinked PCL–PEG–PCL hydrogels enhances cartilage formation

Chondrocytes (CH) and bone marrow stem cells (BMSCs) are sources that can be used in cartilage tissue engineering. Co‐culture of CHs and BMSCs is a promising strategy for promoting chondrogenic differentiation. In this study, articular CHs and BMSCs were encapsulated in PCL–PEG–PCL photocrosslinked hydrogels for 4 weeks. Various ratios of CH:BMSC co‐cultures were investigated to identify the optimal ratio for cartilage formation. The results thus obtained revealed that co‐culturing CHs and BMSCs in hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. Co‐culture with a 1:4 CH:BMSC ratio significantly increased the synthesis of GAGs and collagen. In vivo cartilage regeneration was evaluated using a co‐culture system in rabbit models. The co‐culture system exhibited a hyaline chondrocyte phenotype with excellent regeneration, resembling the morphology of native cartilage. This finding suggests that the co‐culture of these two cell types promotes cartilage regeneration and that the system, including the hydrogel scaffold, has potential in cartilage tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.     

Modulation of chondrogenic differentiation of human mesenchymal stem cells in jellyfish collagen scaffolds by cell density and culture medium

Studies on tissue‐engineering approaches for the regeneration of traumatized cartilage focus increasingly on multipotent human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes. The present study applied porous scaffolds made of collagen from the jellyfish Rhopilema esculentum for the in vitro chondrogenic differentiation of hMSCs. Culture conditions in those scaffolds differ from conditions in high‐density pellet cultures, making a re‐examination of these data necessary. We systematically investigated the influence of seeding density, basic culture media [Dulbecco's modified Eagle's medium (DMEM), α‐minimum essential medium (α‐MEM)] with varying glucose content and supplementation with fetal calf serum (FCS) or bovine serum albumin (BSA) on the chondrogenic differentiation of hMSCs. Gene expression analyses of selected markers for chondrogenic differentiation and hypertrophic development were conducted. Furthermore, the production of cartilage extracellular matrix (ECM) was analysed by quantification of sulphated glycosaminoglycan and collagen type II contents. The strongest upregulation of chondrogenic markers, along with the highest ECM deposition was observed in scaffolds seeded with 2.4 × 10⁶ cells/cm³ after cultivation in high‐glucose DMEM and 0.125% BSA. Lower seeding densities compared to high‐density pellet cultures were sufficient to induce in vitro chondrogenic differentiation of hMSCs in collagen scaffolds, which reduces the amount of cells required for the seeding of scaffolds and thus the monolayer expansion period. Furthermore, examination of the impact of FCS and α‐MEM on chondrogenic MSC differentiation is an important prerequisite for the development of an osteochondral medium for simultaneous osteogenic and chondrogenic differentiation in biphasic scaffolds for osteochondral tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

A collagen network phase improves cell seeding of open‐pore structure scaffolds under perfusion

Scaffolds with open‐pore morphologies offer several advantages in cell‐based tissue engineering, but their use is limited by a low cell‐seeding efficiency. We hypothesized that inclusion of a collagen network as filling material within the open‐pore architecture of polycaprolactone–tricalcium phosphate (PCL–TCP) scaffolds increases human bone marrow stromal cells (hBMSCs) seeding efficiency under perfusion and in vivo osteogenic capacity of the resulting constructs. PCL–TCP scaffolds, rapid prototyped with a honeycomb‐like architecture, were filled with a collagen gel and subsequently lyophilized, with or without final crosslinking. Collagen‐free scaffolds were used as controls. The seeding efficiency was assessed after overnight perfusion of expanded hBMSCs directly through the scaffold pores using a bioreactor system. By seeding and culturing freshly harvested hBMSCs under perfusion for 3 weeks, the osteogenic capacity of generated constructs was tested by ectopic implantation in nude mice. The presence of the collagen network, independently of the crosslinking process, significantly increased the cell seeding efficiency (2.5‐fold), and reduced the loss of clonogenic cells in the supernatant. Although no implant generated frank bone tissue, possibly due to the mineral distribution within the scaffold polymer phase, the presence of a non‐crosslinked collagen phase led to in vivo formation of scattered structures of dense osteoids. Our findings verify that the inclusion of a collagen network within open morphology porous scaffolds improves cell retention under perfusion seeding. In the context of cell‐based therapies, collagen‐filled porous scaffolds are expected to yield superior cell utilization, and could be combined with perfusion‐based bioreactor devices to streamline graft manufacture. Copyright © 2011 John Wiley & Sons, Ltd.     

Additive and synergistic effects of bFGF and hypoxia on leporine meniscus cell‐seeded PLLA scaffolds

Injuries to avascular regions of menisci do not heal and result in significant discomfort to patients. Current treatments, such as partial meniscectomy, alleviate these symptoms in the short term but lead to premature osteoarthritis as a result of compromised stability and changes in knee biomechanics. Thus, tissue engineering of the meniscus may provide an alternative treatment modality to overcome this problem. In this experiment, a scaffold‐based tissue‐engineering approach was utilized to regenerate the meniscus. Meniscus cells were cultured on poly‐L ‐lactic acid scaffolds in normoxic (～21% oxygen) or hypoxic (～2% oxygen) conditions in the presence or absence of the growth factor, basic fibroblast growth factor (bFGF). At t = 4 weeks, histological sections of constructs showed presence of collagen and glycosaminoglycan (GAG) in all groups. Immunohistochemical staining showed the presence of collagen I in all groups and collagen II in groups cultured under hypoxic conditions. bFGF in the culture medium significantly increased cell number/construct by 25%, regardless of culture conditions. For GAG/construct, synergistic increases were observed in constructs cultured in hypoxic conditions and bFGF (two‐fold) when compared to constructs cultured in normoxic conditions. Compressive tests showed synergistic increases in the relaxation modulus and coefficient of viscosity and additive increases in the instantaneous modulus for constructs cultured under hypoxic conditions and bFGF, when compared to constructs cultured under normoxic conditions. Overall, these results demonstrate that bFGF and hypoxia can significantly enhance the ability of meniscus cells to produce GAGs and improve the compressive properties of tissue‐engineered meniscus constructs in vitro. Copyright © 2009 John Wiley & Sons, Ltd.