Regulation of articular chondrocyte aggrecan and collagen gene expression by multiple growth factor gene transfer

Gene transfer is a promising approach to the delivery of chondrotrophic growth factors to promote cartilage repair. It is unlikely that a single growth factor transgene will optimally regulate these cells. The aim of this study was to identify those growth factor transgene combinations that optimally regulate aggrecan, collagen type II and collagen type I gene expression by articular chondrocytes. We delivered combinations of the transgenes encoding fibroblast growth factor-2, insulin-like growth factor I, transforming growth factor beta1, bone morphogenetic protein-2, and/or bone morphogenetic protein-7 and assessed chondrocyte responses by measuring changes in the expression of aggrecan, type II collagen and type I collagen genes. These growth factor transgenes differentially regulated the magnitude and time course of all three-matrix protein genes. In concert, the transgenes regulated matrix gene expression in an interactive fashion that ranged from synergistic to inhibitory. Maximum stimulation of aggrecan (16-fold) and type II collagen (35-fold) expression was with the combination of IGF-I, BMP-2, and BMP-7 transgenes. The results indicate that the optimal choice of growth factor genes for cell-based cartilage repair cannot be predicted from observations of individual transgenes. Rather, such gene therapy will require an empirically based selection of growth factor gene combinations.     

In vitro stimulation of articular chondrocyte mRNA and extracellular matrix synthesis by hydrostatic pressure

This study tested the effects of hydrostatic pressure (10 MPa) on adult articular chondrocyte mRNA and extracellular matrix synthesis in vitro. High density primary cultures of bovine chondrocytes were exposed to hydrostatic pressure applied intermittently at 1 Hz or constantly for 4 hours in serum-free medium or in medium containing 1% fetal bovine serum, mRNAs for aggrecan, types I and II collagen, and β-actin were analyzed by Northern blots and quantified by slot blots. Proteoglycan synthesis was quantified by ³⁵SO₄ uptake into cetylpyridinium chloride-precipitable glycosaminoglycans, and cell-associated aggrecan and type-II collagen were detected by immunohistochemical techniques. In serum-free medium, intermittent pressure increased aggrecan mRNA signal by 14% and constant pressure decreased type-II collagen mRNA signal by 16% (p < 0.05). In the presence of 1% fetal bovine serum, intermittent pressure increased aggrecan and type-II collagen mRNA signals by 31% (p < 0.01) and 36% (p < 0.001), respectively, whereas constant pressure had no effect on either mRNA. Intermittent and constant pressure stimulated glycosaminoglycan synthesis 65% (p < 0.001) and 32% (p < 0.05), respectively. Immunohistochemical detection of cell-associated aggrecan and type-II collagen was increased in response to both intermittent and constant pressure. These data support the hypothesis that physiologic hydrostatic pressure directly influences the extracellular matrix metabolism of articular chondrocytes.     

Effects of intermittent hydrostatic pressure and BMP‐2 on osteoarthritic human chondrocyte metabolism in vitro

Purpose

This study examined effects of intermittent hydrostatic pressure (IHP) and a chondrogenic growth factor, bone morphogenetic protein‐2 (BMP‐2), on anabolic, catabolic, and other metabolic markers in human osteoarthritic (OA) chondrocytes in vitro.

Methods

Articular chondrocytes, isolated from femoral OA cartilage and maintained in high‐density monolayer culture, were examined for effects of BMP‐2 and IHP on gene expression of matrix‐associated proteins (aggrecan, type II collagen, and SOX9) and catabolic matrix metalloproteinases (MMP‐2 and MMP‐3) and culture medium levels of the metabolic markers MMP‐2, nitric oxide (NO), and glycosaminoglycan (GAG). The results were analyzed using a mixed linear regression model to investigate the effects of load and growth factor concentration.

Results

IHP and BMP‐2 modulated OA chondrocyte metabolism in accordance with growth factor concentration independently, without evidence of synergism or antagonism. Each type of stimulus acted independently on anabolic matrix gene expression. Type II collagen and SOX9 gene expression were stimulated by both IHP and BMP‐2 whereas aggrecan was increased only by BMP‐2. IHP exhibited a trend to decrease MMP‐2 gene expression as a catabolic marker whereas BMP‐2 did not. NO production was increased by addition of BMP‐2 and IHP exhibited a trend for increased levels. GAG production was increased by BMP‐2.

Conclusions

This study confirmed the hypothesis that human OA chondrocytes respond to a specific type of mechanical load, IHP, through enhanced articular cartilage macromolecule gene expression and that IHP, in combination with a chondrogenic growth factor BMP‐2, additively enhanced matrix gene expression without interactive effects. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:361–368, 2011     

Modulation of bovine articular chondrocyte gene expression in vitro by oxygen tension

OBJECTIVE: Adult articular cartilage is a physiologically hypoxic tissue with a proposed gradient of oxygen tension ranging from <10% oxygen at the cartilage surface to <1% in the deepest layers. This gradient may be disturbed during diseases of the joint, for example in rheumatoid arthritis when synovial fluid pO(2)falls. We investigated whether changes in oxygen tension modulate gene expression in articular chondrocytes. DESIGN: Bovine articular chondrocytes were cultured in alginate beads in medium maintained at <0.1, 5, 10 or 20% oxygen. A modified RNA arbitrarily primed polymerase chain reaction (RAP-PCR) technique was used to identify several genes whose mRNA abundance in articular chondrocytes was dependent upon oxygen tension. Northern hybridization slot blots were used to quantify changes in mRNA level relative to a housekeeping gene, beta-actin. RESULTS: Genes found by RAP-PCR to undergo up-regulation in hypoxia included TIMP-1 and integrin-linked kinase. Collagen V mRNA levels were down-regulated in hypoxic chondrocytes. This led us to examine mRNA levels for various cytokines, matrix structural molecules and beta1 integrin. Interleukin 1beta, transforming growth factor beta and connective tissue growth factor were all up-regulated by low oxygen tensions, as was beta1 integrin. Collagen II (COL2A1) was down-regulated by hypoxia but aggrecan mRNA levels remained unchanged. The mRNA levels for GAPDH, the archetypal hypoxia responsive gene, were not modulated in articular chondrocytes by changes in oxygen tension. CONCLUSIONS: Oxygen tension modulates the abundance of mRNAs encoding structural molecules, several cytokines, beta1 integrin and integrin-linked kinase in articular chondrocytes. This may be important during disease progression. Chondrocytes are unusual in their response to hypoxia, presumably because they exist physiologically in a low oxygen environment.     

The use of debrided human articular cartilage for autologous chondrocyte implantation: maintenance of chondrocyte differentiation and proliferation in type I collagen gels.

Autologous chondrocyte implantation (ACI) is the most promising surgical treatment for large full thickness knee joint articular cartilage (AC) defects where cells from healthy non-weight bearing area AC are multiplied in vitro and implanted into such defects. In the routine surgical procedure for symptomatic knee full thickness AC defects, damaged AC surrounding the edge and the base of such defects is usually debrided and discarded. The purpose of this study was to examine if chondrocytes from this 'debrided' AC can proliferate, synthesize a cartilage specific matrix and thus can be used for ACI. METHODS: Biopsies were retrieved from 12 patients (debrided articular cartilage: DAC, aged 35-61) and from two autopsies (normal articular cartilage: NAC, aged 21 and 25). Chondrocytes were isolated, seeded at low density in type I collagen gels and as monolayer cultures for 4 weeks without passage. RESULTS: After 4 weeks cultures in type I collagen gels, cell proliferation from DAC (18.34 +/- 1.95 fold) was similar to cells from NAC (11.24 +/- 1.02 fold). Syntheses of proteoglycan and collagen in DAC were also similar to NAC. Newly synthesized matrices in gel cultures consisted predominantly of type II collagen as shown by immuno-labelling and SDS-PAGE followed by fluorography. Chondrocytes from 'debrided human AC' cultured at low density in type I collagen gels may be used for the ACI procedure as they provide sufficient viable cell numbers for ACI and maintain their chondrocyte phenotype as they synthesize a cartilage-like matrix.     

Down-regulation of chondrocyte aggrecan and type-II collagen gene expression correlates with increases in static compression magnitude and duration.

The goal of this study was to examine the simultaneous effects of mechanical compression of chondrocytes on mRNA expression and macromolecular synthesis of aggrecan and type-II collagen. Bovine cartilage explants were exposed to different magnitudes and durations of applied mechanical compression, and levels of aggrecan and type-IIa collagen mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase were measured and quantified by Northern blot analysis. Synthesis of aggrecan and type-II collagen protein was measured by radiolabel incorporation of [35S]sulfate and [3H]proline into macromolecules. The results showed a dose-dependent decrease in mRNA levels for aggrecan and type-II collagen, with increasing compression relative to physiological cut thickness applied for 24 hours. Radiolabel incorporation into glycosaminoglycans and collagen also decreased with increasing compression in a dose-related manner similar to the changes seen in mRNA expression. The modulation of aggrecan and type-II collagen mRNA and protein synthesis were dependent on the duration of the compression. Aggrecan and type-II collagen mRNA expression increased during the initial 0.5 hours of static compression; however, 4-24 hours after compression was applied total mRNA levels had significantly decreased. The synthesis of aggrecan and collagen protein decreased more rapidly than did mRNA levels after the application of a step compression. Together, these results suggest that mechanical compression rapidly alters chondrocyte aggrecan and type-II collagen gene expression on application of load. However, our results indicate that the observed decreases in biosynthesis may not be related solely to changes in mRNA expression. The mechanisms by which mechanical forces affect different segments of the biosynthetic pathways remain to be determined.     

Protective effects of intermittent hydrostatic pressure on osteoarthritic chondrocytes activated by bacterial endotoxin in vitro.

The role of continuous passive motion (CPM) in the management of septic arthritis and inflammatory arthritis remains of interest. CPM produces cyclic variations in intraarticular pressure that facilitates transport of fluid, nutrients, and solutes within and/or across the joint and stimulates chondrocyte metabolism. However, the precise mechanisms mediating the responses of chondrocytes to joint motion remain unclear. This study tested the hypothesis that dynamic mechanical loading counteracts effects of bacterial lipopolysaccharide (LPS), an inflammatory mediator, on chondrocyte metabolism. Intermittent hydrostatic pressure (IHP) (10 MPa for 4 h) was applied to human chondrocytes pretreated with LPS (1 microg/ml for 18 h). LPS activation of chondrocytes decreased mRNA signal levels of type II collagen by 67% and aggrecan by 56% and increased nitric oxide by 3.1-fold, monocyte chemotactic protein-1 mRNA signal levels by 6.5-fold, and matrix metalloproteinase-2 mRNA signal levels by 1.3-fold. Application of IHP to LPS-activated chondrocytes decreased nitric oxide synthase mRNA signal levels and nitric oxide levels in the culture medium. Exposure of LPS-activated chondrocytes to IHP upregulated type II collagen and aggrecan mRNA signal levels by 1.7-fold, relative to chondrocytes activated by LPS and maintained without loading. In addition, application of IHP decreased the upregulation in signal levels of monocyte chemotactic factor-1 and matrix metalloproteinase-2 following LPS activation by 45% and 15%, respectively. These data show that mechanical loading counteract effects of inflammatory agents, such as bacterial LPS, and suggest that postinfection sequelae are influenced by the presence or absence of joint loading.     

循环静水压力对维持兔软骨细胞表型的影响

目的：本研究将兔软骨细胞放在静水压力负载装置中培养，并传代，施加不同频率和大小的静水压，观察其对兔软骨细胞表型的影响。方法：用酶消化法获取兔关节软骨细胞，分别在不同的压力和频率下作普通培养瓶贴壁的单层培养，并传代。40kPa循环静水加压为实验组，40kPa持续静水加压培养和常压培养作为对照。Ⅰ型胶原和Ⅱ型胶原免疫组化，苏木素套核染色，倒置显微镜观察摄像。以RT—PCR方法检测软骨细胞中蛋白聚糖、聚集蛋白聚糖、Ⅰ型胶原和Ⅱ型胶原mRNA的表达。结果：在循环静水压力下培养，软骨细胞有较高的细胞增殖率，培养至第7代未见明显表型改变，仍然表达Ⅱ型胶原、蛋白聚糖和聚集蛋白聚糖等。常压培养，软骨细胞传代至第5代以后，逐渐失去软骨细胞的特有表型。而持续静水压力培养的软骨细胞传代至第3代以后就开始逐渐失去软骨细胞的特有表型。结论：一定的循环静水压力能维持软骨细胞的合成分泌功能，有助于软骨细胞特有表型的稳定。     

Flow cytometric analysis of the human articular chondrocyte phenotype in vitro

OBJECTIVE: To develop flow cytometry for the study of human articular cartilage cell phenotype and to validate the method on chondrocytes cultured in different in-vitro systems. METHODS: Chondrocyte phenotype was modulated by culturing the cells under different in-vitro conditions: i.e. in monolayer and in suspension culture in gelled agarose. Monolayer cultured chondrocyte phenotype was assayed by immunohistochemical staining with monoclonal antibodies against chondrocyte-specific aggrecan, type II and I collagen. Flow cytometry was used to quantify the proportions of chondrocytes expressing these extracellular matrix molecules in both culture conditions. To exclude the effects of cell-harvesting methods on the presence of cell-bound ECM molecules, non-proteolytic isolation procedures were used to obtain the chondrocytes for flow cytometry. Subconfluent cells from monolayer cultures were detached with EDTA. Chondrocytes cultured in gelled agarose were obtained after the agarose was enzymatically digested with agarase. RESULTS: Immunohistochemical staining showed that monolayer-cultured chondrocytes, in the presence of serum, gradually lost the expression of chondrocyte-specific aggrecan and type II collagen, while type I collagen was increasingly expressed. Flow cytometry allowed monolayer cultured chondrocyte phenotype to be assessed reproducibly. Chondrocyte phenotype was characterized through the cell membrane-associated extracellular matrix antigens. EDTA, used to obtain single cells from monolayer cultures, did not affect the cell-associated matrix. Where the chondrocytes had been cultured in gelled agarose, flow cytometry allowed quantification of the percentages of chondrocytes maintaining or reexpressing their original phenotype. The agarase digestion procedure used to isolate the cells from the agarose gel did not affect the plasma membrane-associated extracellular matrix antigens. CONCLUSION: Flow cytometry allows quantification of cells expressing aggrecan, type II and I collagen in their cell-associated extracellular matrix. A continuously increasing number of specific monoclonal antibodies will broaden the range of applications offered by this method.     

Excessive degradation of type II collagen in articular cartilage in equine osteochondrosis.

Articular osteochondrosis (OCD) occurs in both man and animals. The etiology remains to be determined. Studies of OCD lesions in animals may provide clues as to its pathogenesis. The aim of our study was to determine whether there was evidence for increased degradation namely proteoglycan (PG) release and type II collagen cleavage in articular cartilage harvested from OCD lesions. We examined ex vivo explants at post-mortem from equine OCD lesions and macroscopically normal site and age matched cartilage. These were cultured over a 10 day period in serum-free medium. Type II collagen cleavage was measured in articular cartilage and media using an Elisa assay to detect the COL2-3/4C(short) epitope, which is generated on cleavage of the triple helix of type II collagen by collagenases. PG release was measured by a dye-binding assay. Cumulative release of PG and COL2-3/4C(short) and their contents in cartilage at the end of the culture period were determined. In OCD lesions there was a significant increase in type II collagen cleavage by collagenase but no evidence for increase of PG degradation. These findings point to a selective increase in type II collagen cleavage by collagenases, in OCD lesions of the kind observed in osteoarthritis. Further work is needed to determine whether changes represent primary or secondary events in the pathogenesis of OCD.     

A type XI collagen mutation leads to increased degradation of type II collagen in articular cartilage

OBJECTIVE: To determine in articular cartilage whether degraded type II collagen is more abundant in Col11a1 mutant cho/+ than in age-matched +/+ mice and whether collagen degradation occurs in a generalized or localized fashion. DESIGN: Knee joints from cho/+ and +/+ mice at 6, 9, 12 and 15 months of age were dissected, fixed, cryosectioned, and stained with antibody COL2-3/4m against denatured type II collagen using a FITC-conjugated secondary antibody. Sections were viewed and photographed under a fluorescence microscope and areas of staining were quantified. RESULTS: Before 12 months of age, little degraded collagen staining was detectable in +/+ or cho/+ mice. By 15 months, however, cho/+ mice showed significantly more degraded type II collagen than age-matched controls. Degraded collagen staining was localized at the articular surface, not distributed generally throughout the articular cartilage. CONCLUSIONS: The results suggest a model in which cumulative biomechanical stresses trigger increased collagen synthesis and degradation in both +/+ and cho/+ mice at around 12 months of age. Cho/+ mice, however, are less able to synthesize and assemble normal replacement collagen fibrils because of the Col11a1 mutation. Degradation is further activated, resulting in the accumulation of degraded type II collagen in the articular cartilage extracellular matrix. Similar mutations that do not overtly affect skeletal development may likewise predispose humans to increased collagen degradation and resultant osteoarthritis.     

Distribution of type VI collagen in chondrocyte microenvironment: study of chondrons isolated from human normal and degenerative articular cartilage and cultured chondrocytes

The chondron is the microanatomical unit composed of a chondrocyte and its pericellular microenvironment (PCME), including the pericellular matrix and capsule. In the present study, we extracted chondrons from human articular cartilages and investigated the relationship between the distribution of the matrix molecules, including type VI collagen, and the degeneration of articular cartilage. We also investigated the effects of interleukin-1 (IL-1) and transforming growth factor -1(TGF-₁) on the distribution of type VI collagen in cultured chondrocytes. Chondrons were extracted by low-speed homogenization from cartilage pieces obtained from forensic autopsies and from patients with knee osteoarthritis (OA) undergoing total knee arthroplasty. Cartilage sections were classified into three groups (normal, slight degeneration, and moderate degeneration) based on the degree of degeneration according to Mankins score. Extracted chondrons were immunostained, and the distribution of the matrix molecules, including type VI collagen, was investigated using a confocal laser scanning microscope (CLSM). The chondrocytes isolated by enzymic treatment were subjected to three-dimensional culture in agarose gel and then treated with IL-1 or TGF-₁. The distribution of newly synthesized type VI collagen in agarose gel was also investigated using the CLSM. Type VI collagen was localized specifically within the PCME of chondrons. The volume ratio of PCME to chondrocyte (P/C ratio) was significantly higher in the moderate degeneration group than in the other two groups. The accumulation of type VI collagen around a chondrocyte was obviously increased by the addition of TGF-₁. The P/C ratio significantly increased as the severity of the OA progressed, suggesting that type VI collagen distributed specifically in the PCME was playing a protective role for chondrocytes by maintaining the pericellular microenvironment in OA.     

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.     

Type II collagen synthesis in the articular cartilage of a rabbit model of osteoarthritis: expression of type II collagen C-propeptide and mRNA especially during early-stage osteoarthritis

Background The aim of this study was to observe time course changes in type II collagen synthesis in various regions of articular cartilage affected with osteoarthritis (OA) by examining the expression of type II collagen C-propeptide (pCOL II-C) and mRNA in a rabbit OA model. Methods Osteoarthritis was experimentally induced by partial lateral meniscectomy in the knees of Japanese white rabbits. The cartilage of the animals was then examined histologically over time. The degenerative area of articular cartilage was divided into three areas, according to the degree of degeneration. The ability to synthesize type II collagen was estimated by the immunohistological staining of pCOL II-C and the in situ hybridization of mRNA in type II collagen. Results The positive rate of pCOL II-C immunostaining in chondrocytes was highest in the central-degenerative region 1 week after surgery, and the highest rate in the para-degenerative region was observed 2 and 4 weeks after surgery. The percentage of pCOL II-C positive cells increased as the histological degeneration score increased to moderate degeneration and then decreased with further progression of the severity of cartilage degeneration. Examination by in situ hybridization revealed that the regions marked by strong pCOL II-C mRNA expression were similar to those indicated by the immunohistology results. Conclusions These results suggest that the type II collagen-synthesizing potential of chondrocytes is highest in moderately degenerated areas of OA articular cartilage. Cartilage repair continues to be seen even as OA advances, although the reaction varies depending on the stage of OA.