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.     

Biomechanics of meniscus cells: regional variation and comparison to articular chondrocytes and ligament cells

Central to understanding mechanotransduction in the knee meniscus is the characterization of meniscus cell mechanics. In addition to biochemical and geometric differences, the inner and outer regions of the meniscus contain cells that are distinct in morphology and phenotype. This study investigated the regional variation in meniscus cell mechanics in comparison with articular chondrocytes and ligament cells. It was found that the meniscus contains two biomechanically distinct cell populations, with outer meniscus cells being stiffer (1.59 ± 0.19 kPa) than inner meniscus cells (1.07 ± 0.14 kPa). Additionally, it was found that both outer and inner meniscus cell stiffnesses were similar to ligament cells (1.32 ± 0.20 kPa), and articular chondrocytes showed the highest stiffness overall (2.51 ± 0.20 kPa). Comparison of compressibility characteristics of the cells showed similarities between articular chondrocytes and inner meniscus cells, as well as between outer meniscus cells and ligament cells. These results show that cellular biomechanics vary regionally in the knee meniscus and that meniscus cells are biomechanically similar to ligament cells. The mechanical properties of musculoskeletal cells determined in this study may be useful for the development of mathematical models or the design of experiments studying mechanotransduction in a variety of soft tissues.     

Characterization of a serum-free culture system comparing growth factor requirements of transformed and untransformed cells

We describe the first completely serum-free model culture system for comparing growth control in transformed and untransformed cells. Continuous maintenance of untransformed AKR-2B fibroblasts and chemically transformed AKR-MCA cells in the presence of serum-free medium containing epidermal growth factor (E), insulin (I), and transferrin (T) resulted in cell lines which proliferated with similar doubling times (14 h), comparable to parental lines maintained in 10% serum (16 h). The transformed MCA-SF cells and untransformed AKR-SF cells did not differ in their saturation densities in medium containing E + I + T. However, the monolayer proliferation of MCA-SF cells was significantly greater than that of the AKR-SF cells in the presence of E + T, I + T, or T alone. Both cell lines required T to proliferate in monolayer culture. [3H]-Thymidine incorporation experiments and autoradiographic analysis indicated that quiescent MCA-SF cells could reenter the cell cycle by addition of nutrients alone. The combination of E + I + T produced no additional stimulation of DNA synthesis. In contrast, individual polypeptide growth factors (E, I, IGF-I, PDGF, FGF a or b, or TGF-beta 1) were required to elicit a mitogenic response in the untransformed AKR-SF cells. Peak mitogenesis occurred from 18-20 h for all growth factors except TGF-beta 1 (32 h). Neither AKR-SF nor MCA-SF cells could grow with anchorage independence in serum-free medium, unless both TGF-beta 1 and FGF a or b were simultaneously present. The results indicate that this well-defined, serum-free model system can be utilized to detect growth factor-related alterations associated with the transformed state.     

Finite Element Modeling Predictions of Region-specific Cell-matrix Mechanics in the Meniscus

The knee meniscus exhibits significant spatial variations in biochemical composition and cell morphology that reflect distinct phenotypes of cells located in the radial inner and outer regions. Associated with these cell phenotypes is a spatially heterogeneous microstructure and mechanical environment with the innermost regions experiencing higher fluid pressures and lower tensile strains than the outer regions. It is presently unknown, however, how meniscus tissue mechanics correlate with the local micromechanical environment of cells. In this study, theoretical models were developed to study mechanics of inner and outer meniscus cells with varying geometries. The results for an applied biaxial strain predict significant regional differences in the cellular mechanical environment with evidence of tensile strains along the collagen fiber direction of ~0.07 for the rounded inner cells, as compared to levels of 0.02–0.04 for the elongated outer meniscus cells. The results demonstrate an important mechanical role of extracellular matrix anisotropy and cell morphology in regulating the region-specific micromechanics of meniscus cells, that may further play a role in modulating cellular responses to mechanical stimuli.     

Inhibition of normal rat kidney cell growth by transforming growth factor-beta is mediated by collagen

We have investigated the mechanism of inhibition of the serum-free monolayer growth of normal rat kidney (NRK) cells by transforming growth factor-beta (TGF-beta). NRK cells grown on fibronectin-coated dishes exhibited a biphasic response to TGF-beta. Monolayer growth was slightly stimulated by subpicomolar concentrations, while picomolar concentrations of TGF-beta inhibited NRK cell growth in the presence or absence of epidermal growth factor. NRK cells exhibited a similar biphasic growth response to exogenous type I collagen. TGF-beta induced a 3-5-fold increase in the deposition of type I collagen-like proteins into the extracellular matrix of NRK cells during serum-free growth. Type I collagen-like proteins were identified by their sensitivity to degradation by purified bacterial collagenase and by Western blot analysis. The TGF-beta dose-response curves for induction of extracellular matrix-localized collagen and inhibition of NRK cell growth were similar. Finally, the inclusion of a purified bacterial collagenase, which did not degrade TGF-beta or TGF-beta receptors, or alter control NRK growth, prevented exogenous collagen or TGF-beta from inhibiting the serum-free growth of NRK cells. Our results demonstrate that an increase in collagen secretion plays an important role in the inhibition of the growth of NRK cells by TGF-beta.     

Persistence of TGF-β1 induction of increased fibroblast contractility

Fibroblast contraction of collagen gels is regarded as a model of wound contraction. Transforming growth factor (TGF)-beta added to such gels can augment contraction consistent with its suggested role as a mediator of fibrotic repair. Since fibroblasts isolated from fibrotic tissues have been suggested to express a "fibrotic phenotype," we hypothesized that TGF-beta exposure may lead to a persistent increase in fibroblasts' contractility. To evaluate this question, confluent human fetal lung fibroblasts were treated with serum-free Dulbecco modified Eagle medium (DMEM), with or without 100 pM [corrected] TGF-beta1, TGF-beta2, or TGF-beta3 for 48 h. Fibroblasts were then trypsinized and cast into gels composed of native type I collagen isolated from rat tail tendons. After 20 min for gelation, the gels were released and maintained in serum-free DMEM. TGF-beta-pretreated fibroblasts caused significantly more rapid gel contraction (52.5+/-0.6, 50.9+/-0.2, and 50.3+/-0.5% by TGF-beta1, -beta2, and -beta3 pretreated fibroblasts, respectively) than control fibroblasts (74.0+/-0.3%, P < 0.01). This effect is concentration dependent (50-200 nM), and all three isoforms had equal activity. The effect of TGF-beta1, however, persisted for only a short period of time following the removal of TGF-beta, and was lost with sequential passage. These observations suggest that the persistent increase in collagen-gel contractility, mediated by fibroblasts from fibrotic tissues, would not appear to be solely due to previous exposure of these cells to TGF-beta.     

Combined effects of dynamic tissue shear deformation and insulin-like growth factor I on chondrocyte biosynthesis in cartilage explants

Biophysical forces and biochemical factors play crucial roles in the maintenance of the integrity of articular cartilage. In this study, we explored the effect of dynamic tissue shear deformation and insulin-like growth factor I (IGF-I) on matrix synthesis by chondrocytes within native cartilage explants. Dynamic tissue shear in the range of 0.5-6% strain amplitude at 0.1 Hz was applied to cartilage explants cultured in serum-free medium. Dynamic tissue shear above 1.5% strain amplitude significantly stimulated protein and proteoglycan synthesis, by maximum values of 35 and 25%, respectively, over statically held control specimens. In the absence of tissue shear, IGF-I augmented protein and proteoglycan synthesis up to twofold at IGF-I concentrations in the range of 100-300 ng/ml. When tissue shear and IGF-I stimuli were combined, matrix biosynthesis levels were significantly higher than the maximal effect caused by either stimulus alone. However, there was no significant interaction between tissue shear and IGF-I as determined by two-way ANOVA. We then quantified the effect of dynamic tissue shear on the transport of IGF-I into and within cartilage explants. [125I]IGF-I was added to the medium, and the levels of intratissue [125I]IGF-I were directly measured as a function of time over 48 h in the presence and absence of continuous dynamic shear strain. Dynamic shear did not alter the rate of uptake of [125I]IGF-I into the explants, suggesting that convective diffusion of [125I]IGF-I is negligible under the shear strain conditions used. This is in marked contrast to the enhancement of transport reported in response to uniaxial dynamic compression. Taken together, these data suggest that (1) the stimulatory effect of tissue shear is via mechanotransduction pathways and not by facilitated transport of biochemical factors and (2) chondrocytes may possess complementary signal transduction pathways for biophysical and biochemical factors leading to changes in metabolic activity.     

Angiotensin II-induced modulation of rat mesangial cell phenotype.

Inhibition of angiotensin II (AII) can ameliorate the severity of experimental radiation nephropathy. To determine the ability of AII to modulate mesangial cell phenotype, primary cultures of rat mesangial cells (passage number 6-11) were placed in serum-free medium 24 h prior to addition of AII (10(-9)-10(-5) M); control cells received serum-free medium alone. Cells were maintained in serum-free medium for a further 48 h. Addition of AII to quiescent mesangial cells resulted in significant (P < 0.05) time- and/or dose-dependent increases in Fn and Pail mRNA and/or immunoreactive protein. No significant change was observed in terms of Tgfb1 mRNA. A significant increase in total Tgfb1 protein (P < 0.01) secreted by AII-treated mesangial cells was noted; however, this increase was primarily in terms of latent TGF-beta; the relative proportion of active TGF-beta secreted decreased after AII incubation. AII had no effect on the activity of Mmp2 or Mmp9. However, AII-treated mesangial cells did show an increase in the amount of tissue inhibitor of metalloproteinase-2 (Timp2) immunoreactive protein secreted into the medium. The AII-mediated increase in Pail mRNA levels appeared due in part to activation of the AT1 receptor and was independent of TGF-beta; co-incubation with TGF-beta-neutralizing antibody failed to inhibit the AII-mediated increase in Pail mRNA. Thus mesangial cells treated with AII exhibit a pro-fibrosis phenotype.     

Enhanced boundary lubrication properties of engineered menisci by lubricin localization with insulin-like growth factor I treatment

In this study we analyzed the effects of IGF-I on the boundary lubricating ability of engineered meniscal tissue using a high density collagen gel seeded with meniscal fibrochondrocytes. Biochemical, histological, immunohistochemical, and tribological analyses were carried out to determine a construct's ability to functionally localize lubricin. Our study revealed that supplementation with IGF-I enhanced both the proliferation of cells within the construct as well as enhanced the anabolic activity of the seeded cells. Growth factor supplementation also facilitated the localization of ECM constituents (i.e. fibronectin and type II collagen) near the tissue surface that are important for the localization of lubricin, a boundary lubricant. Consequently, we found localized lubricin in the constructs supplemented with IGF-I. Tribologically, we demonstrated that lubricin serves as a boundary lubricant adsorbed to native meniscal surfaces. Lubricin removal from the native meniscus surface increased boundary friction coefficient by 40%. For the engineered constructs, the lubricin localization facilitated by growth factor supplementation also reduced friction coefficient by a similar margin, but similar results were not evident in control constructs. This study demonstrates that the use of growth factors in meniscal tissue engineering can enhance tribological properties by facilitating the localization of boundary lubricants at the surface of engineered tissue.     

Considerations on the use of ear chondrocytes as donor chondrocytes for cartilage tissue engineering

Articular cartilage is often used for research on cartilage tissue engineering. However, ear cartilage is easier to harvest, with less donor-site morbidity. The aim of this study was to evaluate whether adult human ear chondrocytes were capable of producing cartilage after expansion in monolayer culture. Cell yield per gram of cartilage was twice as high for ear than for articular cartilage. Moreover, ear chondrocytes proliferated faster. Cell proliferation could be further stimulated by the use of serum-free medium with Fibroblast Growth Factor 2 (FGF2) in stead of medium with 10% serum. To evaluate chondrogenic capacity, multiplied chondrocytes were suspended in alginate and implanted subcutaneously in athymic mice. After 8 weeks the constructs demonstrated a proteoglycan-rich matrix that contained collagen type II. Constructs of ear chondrocytes showed a faint staining for elastin. Quantitative RT-PCR revealed that expression of collagen type II was 2-fold upregulated whereas expression of collagen type I was 2-fold down regulated in ear chondrocytes expanded in serum-free medium with FGF2 compared to serum-containing medium. Expression of alkaline phosphatase and collagen type X were low indicating the absence of terminal differentiation. We conclude that ear chondrocytes can be used as donor chondrocytes for cartilage tissue engineering. Furthermore, it may proof to be a promising alternative cell source to engineer cartilage for articular repair.     

Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.     

Growth of H-35 rat hepatoma cells in unsupplemented serum-free media: Effect of transferrin,insulin and cell density

Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells. This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes of Health grants CA 24604-09 and CA 16463-14.     

Estimation of paracellular conductance of primary rat alveolar epithelial cell monolayers.

Freshly isolated rat type II pneumocytes, when grown on permeable tissue culture-treated polycarbonate filters, form confluent alveolar epithelial cell monolayers (RAECM). Cells in RAECM undergo transdifferentiation, exhibiting over time morphological and phenotypic characteristics of type I pneumocytes in vivo. We recently reported that transforming growth factor-beta(1) (TGF-beta(1)) decreases overall monolayer resistance (R(te)) and stimulates short-circuit current in a dose-dependent manner. In this study, we investigated the effects of TGF-beta(1) (50 pM) or 10% newborn bovine serum (NBS) on modulation of paracellular passive ion conductance and its contribution to total passive ion conductance across RAECM. On days 5-7 in culture, tight-junctional resistance (R(tj), kOmegacm(2)) of RAECM, cultured in minimally defined serum-free medium (MDSF) with or without TGF-beta(1) or NBS, was estimated from the relationship between observed transmonolayer voltage and resistance after addition of gramicidin D to apical potassium isethionate Ringer solution under open-circuit conditions. NaCl Ringer solution bathed the basolateral side throughout the experimental period. Results showed that transmonolayer conductance (1/R(te)) and tight-junctional conductance (1/R(tj)) are 0.59 and 0.14 mS/cm(2) for control monolayers in MDSF, 1.59 and 0.38 mS/cm(2) for monolayers exposed to TGF-beta(1), and 0.38 and 0.18 mS/cm(2) for monolayers grown in the presence of NBS. The contributions to total transepithelial ion conductance by the paracellular pathway are estimated to be 23, 23, and 47% for control, TGF-beta(1)-exposed, and newborn bovine serum (NBS)-treated RAECM, respectively.     

Enhancement of Metastatic and Invasive Capacity of Gastric Cancer Cells by Transforming Growth Factor-β1

Transforming growth factor-β (TGF-β),a multifunctional cytokine,exerts contradictory rolesin different kinds of cells.A number of studies have revealed its involvement in the progression of many typesof tumors.To investigate the effect of TGF-β on gastric carcinoma,SGC7901,BGC823 and MKN28 (aTGF-β-resistant cell line) adenocarcinoma clones were used.After pretreatment in serum-free medium withor without 10 ng/ml TGF-β1,their experimental metastatic potential,chemotaxis,and invasive and adhesiveability were measured.Furthermore,zymography for gelatinase was processed.Liver colonies were alsomeasured 4 weeks after inoculation of SGC7901,BGC823 and MKN28 in Balb/c nude mice,and an increasein the number of surface liver metastases was seen in SGC7901 (from 11.0±3.0 to 53.3±3.3) and BGC823(from 9.3±2.5 to 60.0±2.8) groups,whereas there was no difference between MKN28 groups (from 35.2±3.8 to 38.5±2.7).In vitro experiments showed that TGF-β1 increased the adhesion capacity of SGC7901and BGC823 cells to immobilized reconstituted basement membrane/fibronectin matrices and promoted theirpenetration through reconstituted basement membrane barriers.Zymography demonstrated that enhancedinvasive potential was partly due to the increased type Ⅳ collagenolytic (gelatinolytic) activity,but there wasno difference in type Ⅳ collagenolytic activity and other biological behaviors between MKN28 groups.Theseresults suggested that TGF-β1 might modulate the metastatic potential of gastric cancer cells by promotingtheir ability to break down and penetrate basement membrane barriers and their adhesive and motile activities.We speculated that TGF-β1 might act as a progression-enhancing factor in gastric cancer.Therefore blockageof TGF-β or TGF-β signaling might prevent gastric cancer cells from invading and metastasizing.     

Growth factor profile of irradiated human dermal fibroblasts using a serum-free method

Radiation therapy for cancer permanently damages tissue in the line of treatment. This study sought to establish a serum-free protocol to evaluate the growth of irradiated fibroblasts and to analyze the levels of basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) compared with normal fibroblasts. One irradiated cell line of human dermal fibroblasts was established from an intraoperative specimen obtained from a patient who had undergone radiation therapy for head and neck cancer. Irradiated and normal fibroblasts were then plated in UltraCULTURE (serum and growth factor free), modified Webber's medium (bFGF 50 ng/ml, insulin-like growth factor 100 ng/ml), and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum (serum with undefined basal growth factors). Irradiated cells were also seeded in UltraCULTURE with 50 and 100 ng/ml of bFGF. Cell counts were performed at 0, 1, 3, 5, and 7 days, and cell supernatants were assayed for bFGF and TGF-beta. Irradiated and normal fibroblasts exhibited stronger growth in modified Webber's medium than in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum. Growth of irradiated fibroblasts under bFGF modulation was similar to their growth in Webber's medium. Furthermore, irradiated fibroblasts remained viable in a serum-free and growth factor-free environment for at least 7 days; however, their growth and autocrine growth factor production was less than that of normal cells. This confirms the results of previous studies suggesting that cells from irradiated tissue undergo cellular changes. This study provides an effective model for the first-line evaluation of agents to improve wound healing, and it helps to establish standard levels of bFGF and TGF-beta production for irradiated fibroblasts.     

Transforming growth factor-beta and platelet-derived growth factor synergistically stimulate contraction by testicular peritubular cells in culture in serum-free medium

We report investigations on factors influencing contractility by testicular peritubular cells (PC) maintained in culture in a three-dimensional collagen gel system, and the behavior of PC in culture on a two-dimensional system. At low and moderate cell densities, PC embedded in collagen gels in serum-free Eagle's minimal essential medium (MEM) have a lesser degree of contractility than PC in culture in MEM containing calf serum. The contractility by PC, measured by determining changes in diameter of the collagen gel, was increased by addition of transforming growth factor-beta (TGF-beta) to serum-free MEM, and this was further enhanced by supplementing the medium with platelet-derived growth factor (PDGF). In the absence of TGF-beta, however, PDGF had no detectable effects on PC contractility. Other growth factors examined (epidermal growth factor, insulin, and fibroblast growth factor) did not influence the degree of contractility of PC in serum-free MEM in the presence or absence of TGF-beta. PC maintained in MEM supplemented with platelet-poor serum (PPS) have a lesser degree of contractility than their counterparts in MEM containing 2.5% calf serum. The addition of TGF-beta and PDGF to PPS-supplemented MEM restored contractility by PC to a level comparable to that observed by PC in MEM containing complete serum. The addition of nonpurified bovine serum albumin (BSA) to MEM greatly increased PC contractility. By contrast, highly purified BSA had no such effect, suggesting that one or more components adsorbed to the impure BSA was implicated. Polyclonal antibody against fibronectin did not influence the contractility of PC in collagen gels in the presence or absence of serum. Antiserum against TGF-beta partially blocked the enhancement of contractility of PC in MEM containing non-purified BSA. In PC plated on top of a collagen gel lattice, the attachment, spreading, and cell shape were greatly influenced by the presence of TGF-beta and PDGF, both singly and together. Data presented are interpreted to indicate that effects elicited by serum on the properties of PC in culture, and on the contractility of PC, can be attributed in part to the combined influences of TGF-beta and PDGF in serum.     

Meniscus maturation in the swine model: changes occurring along with anterior to posterior and medial to lateral aspect during growth

The meniscus plays important roles in knee function and mechanics and is characterized by a heterogeneous matrix composition. The changes in meniscus vascularization observed during growth suggest that the tissue‐specific composition may be the result of a maturation process. This study has the aim to characterize the structural and biochemical variations that occur in the swine meniscus with age. To this purpose, menisci were collected from young and adult pigs and divided into different zones. In study 1, both lateral and medial menisci were divided into the anterior horn, the body and the posterior horn for the evaluation of glycosaminoglycans (GAGs), collagen 1 and 2 content. In study 2, the menisci were sectioned into the inner, the intermediate and the outer zones to determine the variations in the cell phenotype along with the inner–outer direction, through gene expression analysis. According to the results, the swine meniscus is characterized by an increasing enrichment in the cartilaginous component with age, with an increasing deposition in the anterior horn (GAGs and collagen 2; P < 0.01 both); moreover, this cartilaginous matrix strongly increases in the inner avascular and intermediate zone, as a consequence of a specific differentiation of meniscal cells towards a cartilaginous phenotype (collagen 2, P < 0.01). The obtained data add new information on the changes that accompany meniscus maturation, suggesting a specific response of meniscal cells to the regional mechanical stimuli in the knee joint.     

Modulation of ion conductance and active transport by TGF-beta 1 in alveolar epithelial cell monolayers

Transforming growth factor-beta1 (TGF-beta 1) may be a critical mediator of lung injury and subsequent remodeling during recovery. We evaluated the effects of TGF-beta 1 on the permeability and active ion transport properties of alveolar epithelial cell monolayers. Rat alveolar type II cells plated on polycarbonate filters in defined serum-free medium form confluent monolayers and acquire the phenotypic characteristics of alveolar type I cells. Exposure to TGF-beta 1 (0.1-100 pM) from day 0 resulted in a concentration- and time-dependent decrease in transepithelial resistance (Rt) and increase in short-circuit current (Isc). Apical amiloride or basolateral ouabain on day 6 inhibited Isc by 80 and 100%, respectively. Concurrent increases in expression of Na+-K+-ATPase alpha 1- and beta 1-subunits were observed in TGF-beta 1-treated monolayers. No change in the alpha-subunit of the rat epithelial sodium channel (alpha-rENaC) was seen. Exposure of confluent monolayers to TGF-beta 1 from day 4 resulted in an initial decrease in Rt within 6 h, followed by an increase in Isc over 72-96 h. These results demonstrate that TGF-beta 1 modulates ion conductance and active transport characteristics of the alveolar epithelium, associated with increased Na+-K+-ATPase, but without a change in alpha-rENaC.     

TGF-beta enhances the production of hyaluronan in human lung but not in skin fibroblasts

Transforming growth factor-beta (TGF-beta) enhances the production of extracellular matrix components, such as type I and type III collagen, fibronectin, proteoglycans, in various cell types. The effect on hyaluronan synthesis in relation to proteoglycan synthesis has not been investigated. Human lung or skin fibroblast cultures were treated with TGF-beta in serum-free medium for various periods of time. 35SO4 or [3H]glucosamine was then added to the cultures in the absence of TGF-beta for up to 48 h. Hyaluronan and proteoglycans were isolated by ion-exchange chromatography and quantitated. TGF-beta induced a three- to fourfold increase in hyaluronan production by lung cells but had no effect on skin fibroblasts. In contrast, proteoglycan synthesis was enhanced in both cell types, although skin fibroblasts responded at lower concentrations of TGF-beta. Increased accumulation of hyaluronan was noted only in the cell medium, whereas proteoglycan accumulation was observed both in the medium and in the cell layer. The ED50 for TGF-beta on hyaluronan accumulation in lung cells was the same as that for proteoglycan accumulation, i.e., 40 pM. In skin fibroblasts the ED50 was considerably lower (4 pM). The induction time needed to attain full effect of TGF-beta was 6 h for both hyaluronan and proteoglycan synthesis. These results indicate that TGF-beta has tissue-specific effects on matrix production which may be of importance for control of cell proliferation in various disease states.