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.     

In vivo internalization of Staphylococcus aureus by embryonic chick osteoblasts

Staphlylococcus aureus is the primary pathogen associated with osteomyelitis, an acute and recurrent bone disease. Internalization of S. aureus by cultured embryonic chick calvarial osteoblasts has been observed. The purpose of this study was to demonstrate that internalization of bacteria by embryonic chick calvarial and tibial osteoblasts occurs in vivo. In initial experiments, 10(8) colony forming units (cfu) of S. aureus, strain UAMS-1 or Cowan 1, were injected subcutaneously under the scalp skin of 17 day chick embryos. After 45 min, calvariae were harvested and processed for transmission electron microscopy (TEM). In subsequent experiments, 10(9) cfu of UAMS-1 were injected into the allantoic sac of 17 day chick embryos via a small opening in the egg shell. After 48 h, calvariae and tibiae were harvested for TEM. S. aureus cells were found in approximately 14% of the calvarial osteoblasts after subcutaneous injection and in 11% of calvarial and tibial osteoblasts following intraallantoic injection. Endosomes were observed in some cells, but most bacteria internalized appeared to be free in the cytoplasm. Osteoblasts with as few as five bacteria had a greater loss of cytoplasmic integrity and a more heterochromatic nucleus than osteoblasts with fewer bacteria or than uninfected osteoblasts. S. aureus cells in calvariae and tibiae were also observed in the cytoplasm of approximately 4% of the osteocytes in mineralized bone matrix. Thus, internalization of S. aureus by osteoblasts in vivo augments the previous observation in vitro. This study has also shown that osteoblasts with few bacteria continue differentiating into osteocytes. Results of these experiments support the hypothesis that internalization of S. aureus by osteoblasts may play a role in the etiology of osteomyelitis.     

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.     

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 inmonolayer 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.     

Transient chondrogenic phase in the intramembranous pathway during normal skeletal development.

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments.     

Microcinematographic and autoradiographic kinetic studies of bone cell differentiation in vitro: matrix formation and mineralization

Matrix formation and mineralization have been reported in vitro with cells isolated from rat calvaria bones by collagenase digestion (Nefussi et al., 1985). In the current study, kinetics of bone nodule formation and osteoblastic cell differentiation were studied in this in vitro system using an improved microcinematographic device and flash and follow-up labeling autoradiographic techniques. Microcinematographic analysis showed the formation of bone nodules within 24 h. The initial event observed was the change in the top cells layer which became alkaline phosphatase positive. Matrix synthesis occurred a few hours after this. The autoradiographic results demonstrated the formation of an integrated system where osteoblasts and osteocytes were active and synthesized a collagen matrix and mineralized it in a similar time sequence than in vivo.     

Procollagenase associated with the noncalcified matrix of bone and its regulation by parathyroid hormone

Collagenolytic enzyme activity associated with the noncalcified pool of collagen was studied using calvarial matrices from which the periosteal cell envelope had been removed. Aminophenylmercuric acetate (APMA) stimulated degradation of about 5% of the noncalcified collagen in matrices prepared from freshly dissected bone. Significantly more activity was detected if intact calvaria were cultured 24 h before removal of the cells, in which case 20-30% of the noncalcified collagen was degraded following treatment with APMA. Trypsin elicited a similar response. The collagen being degraded was representative of the entire pool of noncalcified collagen and was not underhydroxylated. Treatment of intact calvaria with parathyroid hormone (PTH) before removal of the cells increased the level of both active collagenase and procollagenase activity associated with the matrix. Enhanced 3H release was noted for PTH treated intact bone in the prior 24 h. Inactivation of endogenous procollagenase by phenanthroline had no effect on the ability of isolated calvarial cells to resorb the bone upon treatment with resorptive agents. The data show that PTH-stimulated collagenolysis of noncalcified collagen involves increased deposition of procollagenase onto the noncalcified matrix in addition to activation of the enzyme.     

Recombinant osteogenic protein-1 upregulates extracellular matrix metabolism by rabbit annulus fibrosus and nucleus pulposus cells cultured in alginate beads.

This study was performed to determine if recombinant human osteogenic protein-1 (rhOP-1) is effective in promoting matrix synthesis and matrix formation by rabbit nucleus pulposus (NP) and annulus fibrosus (AF) cells cultured in alginate beads. The effects of culturing the cells in the presence of various concentrations of rhOP-1 were assessed by measuring changes in cell proliferation, proteoglycan (PG) and collagen synthesis and mRNA expression, and in the matrix contents of PG and collagen, as indicators of matrix accumulation. At high concentrations, rhOP-1 had a moderate mitogenic effect on both NP and AF cells. It also stimulated the synthesis of PG and collagen in a dose-dependent manner: this was associated with a corresponding increase in the expression of mRNA for aggrecan core protein and collagen type II. The stimulatory effect of rhOP-1 on PG synthesis was more pronounced than that on collagen synthesis. Continuous treatment with rhOP-1 led to an increase in the total DNA, PG and collagen contents in both NP and AF cultures. The results presented here provide evidence of the ability of rhOP-1 to stimulate the metabolism of both rabbit AF and NP cells cultured in alginate beads.     

Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development.

We examine clonal murine calvarial MC3T3-E1 cells to determine if they exhibit a developmental sequence similar to osteoblasts in bone tissue, namely, proliferation of undifferentiated osteoblast precursors followed by postmitotic expression of differentiated osteoblast phenotype. During the initial phase of developmental (days 1-9 of culture), MC3T3-E1 cells actively replicate, as evidenced by the high rates of DNA synthesis and progressive increase in cell number, but maintain a fusiform appearance, fail to express alkaline phosphatase, and do not accumulate mineralized extracellular collagenous matrix, consistent with immature osteoblasts. By day 9 the cultures display cuboidal morphology, attain confluence, and undergo growth arrest. Downregulation of replication is associated with expression of osteoblast functions, including production of alkaline phosphatase, processing of procollagens to collagens, and incremental deposition of a collagenous extracellular matrix. Mineralization of extracellular matrix, which begins approximately 16 days after culture, marks the final phase of osteoblast phenotypic development. Expression of alkaline phosphatase and mineralization is time but not density dependent. Type I collagen synthesis and collagen accumulation are uncoupled in the developing osteoblast. Although collagen synthesis and message expression peaks at day 3 in immature cells, extracellular matrix accumulation is minimal. Instead, matrix accumulates maximally after 7 days of culture as collagen biosynthesis is diminishing. Thus, extracellular matrix formation is a function of mature osteoblasts. Ascorbate and beta-glycerol phosphate are both essential for the expression of osteoblast phenotype as assessed by alkaline phosphatase and mineralization of extracellular matrix. Ascorbate does not stimulate type I collagen gene expression in MC3T3-E1 cells, but it is absolutely required for deposition of collagen in the extracellular matrix. Ascorbate also induces alkaline phosphatase activity in mature cells but not in immature cells. beta-glycerol phosphate displays synergistic actions with ascorbate to further stimulate collagen accumulation and alkaline phosphatase activity in postmitotic, differentiated osteoblast-like cells. Mineralization of mature cultures requires the presence of beta-glycerol phosphate. Thus, MC3T3-E1 cells display a time-dependent and sequential expression of osteoblast characteristics analogous to in vivo bone formation. The developmental sequence associated with MC3T3-E1 differentiation should provide a useful model to study the signals that mediate the switch between proliferation and differentiation in bone cells, as well as provide a renewable culture system to examine the molecular mechanism of osteoblast maturation and the formation of bone-like extracellular matrix.     

Characterization of mitogenic activities extracted from bovine bone matrix

The mitogenic activity in the unfractionated mixture of proteins released from adult bovine bone matrix during demineralization with ethylenediaminetetraacetate (EDTA) has been examined. Bovine bone extract (BBE) from 1 to 25 μg protein per ml stimulated proliferation of chick embryo calvaria bone cells, newborn mouse bone cells, and osteoblastlike cell lines MMB-1 and ROS 17/2.8. BBE also stimulated DNA synthesis in cells from chick embryo cartilage, skin and skeletal muscle tissues and fibroblastlike BALB/c 3T3 and NRK cells. BBE contained β transforming growth factor (TGF) activity (NRK cell colony formation in soft agar in the presence of epidermal growth factor EGF). The cell specificity results suggest that (1) BBE contains more than one growth factor, including a βTGF and a factor that is not specific for bone cells, and (2) all of the bone derived growth factor activities that have been described previously, including SGF, are apparently present in BBE.

Maximal stimulation of chick embryo calvarial cell DNA synthesis by BBE was equal to or exceeded maximal stimulation by nonosseous growth factors that have been reported to stimulate DNA synthesis in bone organ cultures (EGF, fibroblast growth factor, platelet-derived growth factor, insulinlike growth factor I, and multiplication stimulating activity). Combinations of BBE with maximally stimulatory concentrations of each growth factor stimulated DNA synthesis to a greater magnitude than did each growth factor alone. These results suggest that combinations of bone derived and systemic factors can coordinately stimulate bone cell proliferation.     

Three-dimensional cultures of normal human osteoblasts: proliferation and differentiation potential in vitro and upon ectopic implantation in nude mice

We report the establishment in vitro of three-dimensional (3D) cultures of human osteoblasts (hOB) derived from normal adults and supported uniquely by the extracellular matrix (ECM) they deposit. Osteoblasts were cultured in 3D cultures in vitro for up to 120 days. The 3D cultures, examined at 25, 31, and 48 days, expressed protein markers of osteoblastic cells, namely osteonectin, collagen type I, fibronectin, osteopontin, bone sialoprotein, biglycan, and decorin. Sequentially, alkaline phosphatase (AP) and then Ca incorporation, mineralization of matrix (monitored by histochemistry and transmission electron microscopy), and finally osteocalcin expression, were detected in the 3D cultures. Ultrastructurally, morphology progressed from early to mature osteoblast and to osteocyte-like. Cells were embedded in a matrix with organized collagen type I fibers containing, increasingly with time of culture, needle-shaped crystals, often associated with matrix vesicles, characteristic of those in bone. During the culture (up to 120 days) there was an outgrowth of proliferating osteogenic cells from the 3D structure. Subcutaneous implantation in nude mice for 20 days of osteoblasts cultured in 3D culture for different lengths of time in vitro, showed progression of mineralization from the inner region of the implant outward, with peripheral cells being embedded in nonmineralized, collagen-rich matrix. The 3D implants were invaded by vessels derived from the host.     

Characterization of endosteal osteoblastic cells isolated from mouse caudal vertebrae

We have developed a reliable procedure for isolating endosteal osteoblasts from mouse trabecular bone. Endosteal osteoblasts were obtained by migration and proliferation of the cells from the metaphyseal bone surface of caudal vertebrae onto nylon meshes. The isolated cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The cell population consisted of 95% alkaline-phosphatase-positive cells. The cell level of alkaline phosphatase was elevated (1.19 +/- 0.26 (SD) mumol PNP/mn/mg protein) and the enzyme activity was heat-inhibitable, indicating its skeletal origin. Light and electron microscopic observation revealed that cells have morphologic and ultrastructural appearance of typical osteoblasts with high protein synthesis activity. Osteoblasts grown in multilayers in the presence of 50 micrograms/ml ascorbic acid produced within 4 days an abundant fibrous intercellular collagenous matrix forming nodules in which osteocyte-like cells were embedded. Immunolabeling revealed synthesis of type I collagen but no detectable type III collagen. In presence of 7 mM beta-glycerophosphate the matrix became mineralized after 14-21 days of culture. Mineralization could not be induced by mouse skin fibroblasts cultured under similar conditions. The mineral deposits were closely associated with the collagen matrix, consisted of EDTA-removable, Von Kossa and alizarin red S stainable material and were composed of hydroxyapatite crystals identified by X-ray electron probe microanalysis. The isolated endosteal osteoblasts also displayed an intense (+457%) increase in intracellular cAMP production in response to human (1-34) PTH (2 x 10(-8) M) stimulation. The confluent cells responded to 20 nM 1,25(OH)2D3 by a significant 45% reduction in heat labile alkaline phosphatase activity. This procedure allowed us to isolate from trabecular bone a cell population that differentiates into osteoblasts in vitro, respond to calcitropic hormones and that retains its capacity to form a calcified bone tissue in culture. This method provided us a culture system for investigating the differentiation and metabolism of endosteal osteoblastic bone forming cells.     

Subchondral bone osteoblasts induce phenotypic changes in human osteoarthritic chondrocytes

OBJECTIVE: To determine the influence of osteoarthritic (OA) phenotype of subchondral osteoblasts on the phenotype of human chondrocytes. METHODS: Human chondrocytes were isolated from OA cartilage and cultured in alginate beads for 4 or 10 days in the absence or in the presence of osteoblasts in monolayer. The osteoblasts were either isolated from non-sclerotic (N) or sclerotic (SC) zones of human subchondral bone. Before co-culture, osteoblasts were incubated for 72 h with or without 1.7 ng/ml interleukin (IL)-1beta, 100 ng/ml IL-6 with its soluble receptor (50 ng/ml) or 10 ng/ml oncostatin M. SOX9, type I, II and X collagen (COL1, COL2, COL10), osteoblasts-stimulating factor (OSF)-1, bone alkaline phosphatase (ALP), parathyroid hormone related peptide (PTHrP) and its receptor (PTH-R) messenger RNA (mRNA) levels in chondrocytes were quantified by real-time polymerase chain reaction. RESULTS: In comparison with chondrocytes cultured alone in alginate beads, chondrocytes after 4 days in co-culture with N or SC osteoblasts expressed significantly less SOX9 and COL2 mRNA. The decrease of SOX9 and COL2 gene expression was significantly more pronounced in the presence of SC than in the presence of N osteoblasts (P<0.001). OSF-1 mRNA level in chondrocyte was increased by both N and SC osteoblasts, but to a larger extent by SC osteoblasts (P<0.001). PTHrP expression in chondrocytes was 21-fold increased by N osteoblasts but four-fold inhibited by SC osteoblasts. PTHrP secretion was also increased by N but reduced by SC osteoblasts. SC, but not N osteoblasts, induced a significant decrease of PTH-R gene expression in chondrocyte. In our experimental conditions, chondrocytes did not express COL1, COL10 or ALP, even after 10 days of co-culture with osteoblasts. CONCLUSIONS: In co-culture, SC subchondral osteoblasts decrease SOX9, COL2, PTHrP and PTH-R gene expression by chondrocytes but increase that of OSF-1. These findings suggest that SC osteoblasts could initiate chondrocyte phenotype shift towards hypertrophic differentiation and subsequently further matrix mineralization.     

Morphologic characterization of osteoblast-like cell cultures isolated from newborn rat calvaria

Summary Two methods for harvesting osteoblast-like cell populations from newborn (10 days) rat calvaria were compared. The first one consisted in culturing the periosteum-free bones and then trypsinizing the cells on the bone surface. The second one involved the migration of the osteoblasts on glass fragments before trypsinization. Since the plating efficiency, the proportion of alkaline phosphatase-positive cells, the population doubling time, and the calcium deposition were more adequate, the second method was used to further characterize the behavior of the cultures. During the first week of culture, the cells featured shapes similar to those observedin vivo on the surface of periosteum-free calvaria. They formed multilayers and, in the presence of ascorbic acid, synthetized an organic matrix containing exclusively type I collagen. Later, small amounts of type III collagen appeared. The cells were embedded in the matrix and progressively acquired the morphologic phenotype of osteocyte-like cells. The matrix mineralized in the presence of β-glycerophosphate. The technique of dropinoculation (high concentration of cells in a small volume of medium) promoted the multilayer formation and the achievement of large mineralized plates (about 1 cm²) in 3 weeks of culture.