Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Loss of chondrogenic potential in dedifferentiated chondrocytes correlates with deficient Shc-Erk interaction and apoptosis

OBJECTIVE: If dedifferentiated chondrocytes could be induced to redifferentiate in vitro, then we might thereby be furnished with a population of phenotypically stable cells for autologous implantation in reconstructive surgery. We therefore investigated the redifferentiation capabilities of chondrocytes which, having migrated from alginate beads to form a monolayer, were subsequently passaged. We also characterized the molecular traits of irreversibly dedifferentiated cells. METHODS: Human chondrocytes that had migrated from alginate beads to form a monolayer (passage 1) were passaged seven times (passages 2-8). Cells from each passage were then recultivated in alginate beads. We assessed the synthesis of type-II collagen, cartilage-specific proteoglycans, adhesion molecules (integrins), signaling proteins (Src-homology collagen [Shc] and extracellular-signal-regulated kinase [Erk]) and the apoptosis marker 'activated' caspase-3 in monolayer or secondary alginate cultures. RESULTS: The synthesis of cartilage-specific type-II collagen, alpha 3-integrin, Shc and activated Erk1/2 decreased rapidly after four passages in monolayer culture. Up to passage 4, cells redifferentiated in alginate culture. However, between passages 5 and 8, cells began to produce activated caspase-3; these cells not only failed to redifferentiate when recultivated in alginate, but underwent apoptosis. CONCLUSION: We conclude that the loss of chondrogenic potential by chondrocytes maintained in monolayer culture is associated with a decrease in the synthesis of cartilage markers and with a suppressed activation of key signaling proteins in the Ras-mitogen-activated protein kinase pathway (Shc and Erk1/2). These events lead to apoptosis. A decrease in Shc/Erk expression/interaction could serve as a recognition marker for irreversibly dedifferentiated chondrocytes in tissue engineering.     

Response of human chondrocytes prepared for autologous implantation to growth factors

This study investigated metabolism of autologous chondrocytes after initial expansion immediately before implantation. Chondrocytes cultured in either monolayers or alginate beads were treated with insulin-like growth factor-1 (IGF-1), osteogenic protein-1 (OP-1), or a combination. Proteoglycan synthesis and DNA content were tested in both cultures. Alginate beads also were analyzed with live/dead cell assay, safranin O/fast green stain for histology, and immunohistochemistry with antibodies against collagen type II and VI, aggrecan, decorin, and fibronectin. In monolayers, autologous chondrocytes changed their morphologic appearance. In alginate, they maintained chondrocytic phenotype. Growth factors, especially combined, promoted cell survival and induced chondrocyte proliferation. OP-1 stimulated the largest cartilage-specific matrix and the most accumulation of collagen type II and fibronectin, although the overall matrix synthesized by autologous chondrocyte implantation cells was smaller than that produced by normal chondrocytes. The clinical implications of this study suggest a significant promise for anabolic growth factors in cartilage repair as a potential modifying therapy for the enhancement of chondrocytic phenotype of autologous chondrocytes.     

Bone morphogenetic protein (BMP)-2 enhances the expression of type II collagen and aggrecan in chondrocytes embedded in alginate beads

OBJECTIVE: For autologous chondrocyte transplantation (ACT) chondrocytes are expanded in vitro. During expansion these cells may dedifferentiate. This change in phenotype is characterized by a raised expression of type I collagen and a decrease in type II collagen expression. Since high expression of type II collagen is of central importance for the properties of hyaline cartilage, we investigated if the growth factor bone morphogenetic protein-2 (BMP-2) may modulate the chondrogenic phenotype in monolayer cell cultures and in three-dimensional culture systems. DESIGN: Chondrocytes from articular knee cartilage of 11 individuals (average age: 39.8 years) with no history of joint disease were isolated and seeded either in monolayer cultures or embedded in alginate beads in presence or absence of human recombinant BMP-2 (hr-BMP-2). Then, cells were harvested and analysis of the chondrogenic phenotype was performed using quantitative RT-PCR, immunocytochemistry and ELISA. RESULTS: Addition of BMP-2 to chondrocytes expanded in two-dimensional (2D) cultures during the first subculture (P1) had no effect on mRNA amounts encoding type II collagen and interleukin-1beta (IL-1beta). In contrast, seeding chondrocytes in three-dimensional (3D) alginate cultures raised type II collagen expression significantly and addition of BMP-2 enhanced this effect. CONCLUSIONS: We conclude that chondrocytes during expansion for ACT may benefit from BMP-2 activation only when seeded in an appropriate 3D culture system.     

Rapid phenotypic changes in passaged articular chondrocyte subpopulations.

Articular chondrocytes are often expanded in vitro and then used to assist in healing articular cartilage defects. We investigated the extent of dedifferentiation in monolayer-passaged, zonal articular chondrocytes by using quantitative, real-time PCR. The relative gene expressions for collagen type I and II, aggrecan, and superficial zone protein were analyzed for relevant passage numbers (P0-P4) to determine how the expansion of chondrocytes affects the expression of cartilage extracellular matrix proteins. Results reveal that dramatic changes occur as early as first passage. Furthermore, these changes are shown to persist even when the expanded cells are encapsulated in 3D, alginate beads. Successful tissue engineering and autologous cell transplantation procedures rely heavily on having a cell source that expresses the chondrocytic phenotype. The results of this study suggest that major problems exist at the front-end of cartilage regeneration efforts.     

Characterization of human nasal septal chondrocytes cultured in alginate

BACKGROUND: After serial passages in monolayer, chondrocytes dedifferentiate into a fibroblast-like phenotype. Our objective was to determine if culture in alginate affects the phenotype of dedifferentiated human nasal septal chondrocytes. STUDY DESIGN: Human nasal septal chondrocytes were seeded at low density and passaged in monolayer culture. At passages (P) 1, 2, and 3 a portion of cells were cultured in alginate. Collagen, glycosaminoglycan (GAG), and DNA production were assessed. RESULTS: Chondrocytes in alginate proliferated less yet produced higher levels of GAG and collagen than those in monolayer culture. Alginate encapsulated P1 chondrocytes stained strongly for GAG and collagen type II, and minimally for collagen type I. Monolayer cells at P0 and P1 stained positively for collagen type II. All monolayer passages stained positive for collagen type I with minimal GAG staining. CONCLUSIONS: Compared with monolayer culture, alginate stimulates deposition of GAG and collagen type II, and supports the chondrocyte phenotype through P1, but does not promote redifferentiation.     

Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro

Chondrocytes that were isolated from adult human articular cartilage changed phenotype during monolayer tissue culture, as characterized by a fibroblastic morphology and cellular proliferation. Increased proliferation was accompanied by downregulation of the cartilage-specific extracellular matrix proteoglycan, aggrecan, by cessation of type-II collagen expression, and by upregulation of type-I collagen and versican. This phenomenon observed in monolayer was reversible after the transfer of cells to a suspension culture system. The transfer of chondrocytes to suspension culture in alginate beads resulted in the rapid upregulation of aggrecan and type-II collagen and the downregulation of expression of versican and type-I collagen. Type-X collagen and osteopontin, markers of chondrocyte hypertrophy and commitment to endochondral ossification, were not expressed by adult articular chondrocytes cultured in alginate, even after 5 months. In contrast, type-X collagen was expressed within 2 weeks in a population of cells derived from a fetal growth plate. The inability of adult articular chondrocytes to express markers of chondrocyte hypertrophy has underscored the fundamental distinction between the differentiation pathways that lead to articular cartilage or to bone. Adult articular chondrocytes expressed only hyaline articular cartilage markers without evidence of hypertrophy.     

Smooth muscle actin expression by human articular chondrocytes and their contraction of a collagen-glycosaminoglycan matrix in vitro.

Recent studies have demonstrated that human articular chondrocytes can express the gene for a contractile muscle actin, alpha-smooth muscle actin (SMA), in situ. One objective of this work was to evaluate the SMA-content of isolated human articular chondrocytes using Western blot analysis and to correlate the amount of SMA in the cells with passage number and the number of days in culture. A second objective was to determine if articular cartilage-derived cells expressing the gene for SMA in vitro also continue to express type II collagen. A final aim of the current study was to determine if SMA-containing cartilage-derived cells were capable of contracting a collagen glycosaminoglycan analog of extracellular matrix in vitro. Articular chondrocytes were isolated from 13 patients undergoing total joint arthroplasty. Cells were serially passaged through passage 7. Samples were allocated for Western blot analysis of SMA. Cells in monolayer culture were also stained immunohistochemically for SMA and type II collagen. Cells from passage 3 and 7 were seeded into a porous type I collagen-glycosaminoglycan matrix and the diameter of the scaffolds measured every other day for 21 days. Immunohistochemistry of the articular cartilage samples revealed SMA in the articular chondrocytes in situ with a greater percentage of cells staining positive in the superficial half (60 +/- 1.2%; mean +/- SEM) of the cartilage than in the basal half (28 +/- 1.3%). There was an increasing amount of SMA in the cells in monolayer culture with passage number and a meaningful correlation of the SMA content with the days in culture (linear regression analysis; R2 = 0.72). Double staining for SMA and type II collagen showed that type II collagen-expressing cells in monolayer could also express SMA. SMA-containing cells were found to contract the collagen glycosaminoglycan matrix, with the cells containing more SMA (passage 7 cells) displaying more matrix contraction than those with a lesser amount of SMA (passage 3 cells). The results indicate that control of the expression of SMA may be important when employing articular chondrocytes, expanded in monolayer culture, for implantation alone or in a cell-seeded matrix for cartilage repair procedures.     

骨髓间质干细胞体外定向诱导分化为软骨细胞的实验研究

目的：探讨分离骨髓间充质干细胞（MSCs）并诱导其向软骨细胞转化的体外培养方法,为软骨组织工程的种子细胞来源提供实验依据。方法：抽取兔髂骨骨髓液,经梯度离心法和贴壁法进行体外培养,贴壁细胞传代,取第3代细胞在培养基中添加软骨分化诱导剂[含转化生长因子（TGF-β2）10ng／ml、地塞米松10^7mol／L、维生素C50μmol／L,经7、14、21d诱导培养后,倒置显微镜观察细胞形态,免疫组织化学染色检测软骨特异性Ⅱ型胶原表达。将诱导细胞与软骨支架材料-聚磷酸钙纤维／左旋聚乳酸（CPP／PLLA）复合,1周后终止培养,扫描电镜观察细胞黏附情况。结果：诱导后细胞体外扩增能力显著降低,细胞形态由成纤维样梭形向多角形、多边形或类圆形转变,诱导21d后细胞形态变化最为显著,Ⅱ型胶原免疫组化染色深而均匀。诱导后的MSCs可在支架材料内良好黏附生长。结论：体外培养的MSCs可定向诱导分化为软骨细胞,分泌软骨细胞特异性基质,可用作软骨组织工程的种子细胞。     

Bone cell culture in a three-dimensional polymer bead stabilizes the differentiated phenotype and provides evidence that osteoblastic cells synthesize type III collagen and fibronectin.

We report a novel method to culture chick embryo osteoblasts in vitro. Primary cells were grown from explants of calvaria and then cultured within alginate polymer beads. Enriched cultures of primary osteoblasts were obtained because these cells grow readily within alginate beads but other cell types present in the initial outgrowth from calvarial fragments, such as fibroblasts, do not. A reproducible bone cell phenotype was observed in calvarial cells cultured in the alginate polymer for as long as 8 months. Alginate is a uronic acid monomer that reversibly polymerizes based on the presence or absence of divalent cations. Osteoblasts derived from the alginate beads elaborated and mineralized an extracellular matrix in vitro that contained fibronectin, type III collagen, and type I collagen. The synthesis and deposition of these matrix molecules was also demonstrated in the chick embryo calvaria in vivo. Together, these in vitro and in vivo observations provide the first evidence that type III collagen and fibronectin colocalize with type I collagen during the development of avian membranous bone. They also indicate that the phenotype of chick embryo osteoblasts can be expanded to include the synthesis of fibronectin and type III collagen.     

Culture of human septal chondrocytes in a rotary bioreactor

Objectives (1) To show that extracellular matrix deposition in 3-dimensional culture of human septal chondrocytes cultured in a rotary bioreactor is comparable to the deposition achieved under static culture conditions. (2) To demonstrate that the biomechanical properties of human septal chondrocytes cultured in a bioreactor are enhanced with time and are analogous to beads cultured under static culture. Study Design Prospective, basic science. Setting Research laboratory. Methods Human septal chondrocytes from 9 donors were expanded in monolayer and seeded in alginate beads. The beads were cultured in a rotary bioreactor for 21 days in media supplemented with growth factors and human serum, using static culture as the control. Biochemical and biomechanical properties of the beads were measured. Results Glycosaminoglycan (GAG) accumulation significantly increased during 2 measured time intervals, 0 to 21 days and 10 to 21 days (P < .01). No significant difference was seen between the static and bioreactor conditions. Substantial type II collagen production was demonstrated in the beads terminated at day 21 of culture in both conditions. In addition, the biomechanical properties of the beads were significantly improved at 21 days in comparison to beads from day 0. Conclusion Human septal chondrocytes cultured in alginate beads exhibit significant matrix deposition and improved biomechanical properties after 21 days. Alginate bead diameter and stiffness positively correlated with GAG and type II collagen accretion. Matrix production in beads is supported by the use of a rotary bioreactor.     

Platelet-rich plasma stimulates porcine articular chondrocyte proliferation and matrix biosynthesis

OBJECTIVE: Platelet-rich plasma (PRP) is a fraction of plasma that contains high levels of multiple growth factors. The purpose of this study was to examine the effects of PRP on cell proliferation and matrix synthesis by porcine chondrocytes cultured in alginate beads, conditions that promote the retention of the chondrocytic phenotype, in order to determine the plausibility of using this plasma-derived material for engineering cartilage. DESIGN: PRP and platelet-poor plasma (PPP) were prepared from adult porcine blood. Adult porcine chondrocytes were cultured in the presence of 10% PRP, 10% PPP or 10% fetal bovine serum (FBS) for 3 days. Cell proliferation, proteoglycan (PG) and collagen synthesis were quantified, and the structure of newly synthesized PG and collagen was characterized. RESULTS: Treatment with 10% PRP resulted in a small but significant increase in DNA content (+11%, vs FBS; P<0.01; vs PPP; P<0.001). PG and collagen syntheses by the PRP-treated chondrocytes were markedly higher than those by chondrocytes treated by FBS or PPP (PG; PRP: +115% vs FBS; +151% vs PPP, both P<0.0001, collagen; PRP: +163% vs FBS; +163% vs PPP, both P<0.0001). Biochemical analyses revealed that treatment with PRP growth factors did not markedly affect the types of PGs and collagens produced by porcine chondrocytes, suggesting that the cells remained phenotypically stable in the presence of PRP. CONCLUSION: PRP isolated from autologous blood may be useful as a source of anabolic growth factors for stimulating chondrocytes to engineer cartilage tissue.     

Repair of porcine articular cartilage defect with autologous chondrocyte transplantation.

Articular cartilage is known to have poor healing capacity after injury. Autologous chondral grafting remains the mainstay to treat well-defined, full-thickness, symptomatic cartilage defects. We demonstrated the utilization of gelatin microbeads to deliver autologous chondrocytes for in vivo cartilage generation. Chondrocytes were harvested from the left forelimbs of 12 Lee-Sung pigs. The cells were expanded in monolayer culture and then seeded onto gelatin microbeads or left in monolayer. Shortly before implantation, the cell-laden beads were mixed with collagen type I gel, while the cells in monolayer culture were collected and re-suspended in culture medium. Full-thickness cartilage defects were surgically created in the weight-bearing surface of the femoral condyles of both knees, covered by periosteal patches taken from proximal tibia, and sealed with a porcine fibrin glue. In total, 48 condyles were equally allotted to experimental, control, and null groups that were filled beneath the patch with chondrocyte-laden beads in gel, chondrocytes in plain medium solution, or nothing, respectively. The repair was examined 6 months post-surgery on the basis of macroscopic appearance, histological scores based on the International Cartilage Repair Society Scale, and the proportion of characteristic chondrocytes. Tensile stress-relaxation behavior was determined from uniaxial indentation tests. The experimental group scored higher than the control group in the categories of matrix nature, cell distribution pattern, and absence of mineralization, with similar surface smoothness. Both the experimental and control groups were superior to the null group in the above-mentioned categories. Viable cell populations were equal in all groups, but the proportion of characteristic chondrocytes was highest in the experimental group. Matrix stiffness was ranked as null > native cartilage > control > experimental group. Transplanted autologous chondrocytes survive and could yield hyaline-like cartilage. The application of beads and gel for transplantation helped to retain the transferred cells in situ and maintain a better chondrocyte phenotype.     

A novel two-step method for the formation of tissue-engineered cartilage by mature bovine chondrocytes: the alginate-recovered-chondrocyte (ARC) method.

Most attempts to tissue-engineer cartilage have involved seeding of cultured cells into a biological or synthetic scaffold. We have developed a novel two-step culture approach that makes possible the in vitro formation of cartilaginous-like tissue by mature adult bovine chondrocytes without the aid of a synthetic matrix. The first step consists of culturing chondrocytes under conditions that maintain their rounded shape and their molecular phenotype as assessed by type II collagen and aggrecan production. This step was accomplished by culturing the isolated chondrocytes in alginate beads until the cells have reestablished a proteoglycan-rich cell-associated matrix (CM). The second step consists of culturing the cells with their CM, after recovery from the beads, on a tissue culture insert with a porous membrane. In this study, young adult bovine articular chondrocytes were cultured in alginate beads in the presence of 10% or 20% fetal bovine serum (FBS). After 7 days of culture, the alginate beads were dissolved by incubating the beads for 20 min in sodium citrate buffer, a calcium chelator. Following a brief centrifugation, the cells with their CM were recovered, resuspended in medium containing 10% or 20% FBS and seeded onto a tissue culture insert. After 1 week of culture on the insert, the individual cells with their CM progressively became incorporated into a mass of cartilaginous tissue. Culture with 20% FBS resulted in the best formation of tissues. These tissues, easily recovered from the insert, were then subjected to biochemical and histological analyses. The biochemical results showed that the chondrocytes remain phenotypically stable in the tissues. The de novo tissue has a relatively high ratio of PG/collagen. Histological examination of the tissue revealed it contained a cartilage-like matrix strongly stained with toluidine blue. This scaffold-free system appears ideal to study, in vitro, the development of transplantable cartilaginous tissue.