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Expression of Type X Collagen and Matrix Calcification in Three‐Dimensional Cultures of Immortalized Temperature‐Sensitive Chondrocytes Derived from Adult Human Articular Cartilage
Abstract
Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mutant of the simian virus 40 large tumor antigen and a linked geneticin (G418)-resistance marker. G418-resistant colonies were then isolated, ring-cloned, and expanded in serum-containing media. Several immortalized chondrocyte cell lines were established from the clones that survived, some of which have been maintained in continuous culture for over 2 years. Despite serial subcultures and maintenance as monolayers, these cells retain expression of markers specific for cells of the lineage, namely type II collagen and aggrecan, detected immunocytochemically. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet culture system, and in this report, we present evidence that a prototype of these lines (HAC-F cells) expresses markers normally associated with hypertrophic chondrocytes. When HAC-F cells were cultivated in centrifuge tubes, for periods of up to 63 days, at 39°C with mild and intermittent centrifugation they continued to express both lineage markers; total type II collagen/pellet remained stable, whereas there was a temporal decrease in cartilage-specific glycosaminoglycans content. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also begin to express a hypertrophic phenotype characterized by type X collagen synthesis and extensive mineralization of the extracellular matrix in late stage cultures. The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reaction as early as day 2, and anti-type X collagen immunoreactivity was subsequently localized in the matrix. The mineral was characterized by energy-dispersive X-ray microanalysis as containing calcium (Ca) and phosphorus (P) with a Ca:P peak height ratio close to that of mineralized bone tissue. The unexpected phenotype of this human chondrocyte cell line provides an interesting opportunity for studying chondrocyte maturation in vitro.
... In addition, these cell lines are known to be less sensitive than primary articular chondrocytes to inflammatory stimuli, 18 which is relevant when investigating OA pathology. Although a few chondrocyte cell lines were created from articular cartilage, they have associated drawbacks such as the requirement of unusual cell culture temperature for immortalization transgene expression, 19,20 lack of telomerase activity, [19][20][21] and/or low proliferation rates due to incomplete immortalization. 22,23 For these reasons, the aim of this study was the generation of two cell lines derived from articular chondrocytes from one patient with OA and one donor without OA. ...
... In addition, these cell lines are known to be less sensitive than primary articular chondrocytes to inflammatory stimuli, 18 which is relevant when investigating OA pathology. Although a few chondrocyte cell lines were created from articular cartilage, they have associated drawbacks such as the requirement of unusual cell culture temperature for immortalization transgene expression, 19,20 lack of telomerase activity, [19][20][21] and/or low proliferation rates due to incomplete immortalization. 22,23 For these reasons, the aim of this study was the generation of two cell lines derived from articular chondrocytes from one patient with OA and one donor without OA. ...
... 18 The few articular cartilagederived chondrocyte cell lines that have been generated also have associated drawbacks, some of which are likely due to incomplete immortalization with a single transgene. [19][20][21][22][23] Transduction with viral genes, such as SV40LT and human papillomavirus (HPV) E6/E7 genes, prevents cell growth arrest by interfering with p53 and Rb-mediated pathways, 34,35 but does not prevent replicative senescence driven by telomere shortening. On the contrary, transduction with hTERT prevents telomere shortening, 36,37 but cannot avoid stress-induced senescence. ...
Aims
After a few passages of in vitro culture, primary human articular chondrocytes undergo senescence and loss of their phenotype. Most of the available chondrocyte cell lines have been obtained from cartilage tissues different from diarthrodial joints, and their utility for osteoarthritis (OA) research is reduced. Thus, the goal of this research was the development of immortalized chondrocyte cell lines proceeded from the articular cartilage of patients with and without OA.
Methods
Using telomerase reverse transcriptase (hTERT) and SV40 large T antigen (SV40LT), we transduced primary OA articular chondrocytes. Proliferative capacity, degree of senescence, and chondrocyte surface antigen expression in transduced chondrocytes were evaluated. In addition, the capacity of transduced chondrocytes to synthesize a tissue similar to cartilage and to respond to interleukin (IL)-1β was assessed.
Results
Coexpression of both transgenes (SV40 and hTERT) were observed in the nuclei of transduced chondrocytes. Generated chondrocyte cell lines showed a high proliferation capacity and less than 2% of senescent cells. These cell lines were able to form 3D aggregates analogous to those generated by primary articular chondrocytes, but were unsuccessful in synthesizing cartilage-like tissue when seeded on type I collagen sponges. However, generated chondrocyte cell lines maintained the potential to respond to IL-1β stimulation.
Conclusion
Through SV40LT and hTERT transduction, we successfully immortalized chondrocytes. These immortalized chondrocytes were able to overcome senescence in vitro, but were incapable of synthesizing cartilage-like tissue under the experimental conditions. Nonetheless, these chondrocyte cell lines could be advantageous for OA investigation since, similarly to primary articular chondrocytes, they showed capacity to upregulate inflammatory mediators in response to the IL-1β cytokine. Cite this article: Bone Joint Res 2023;12(1):46–57.
... It has been reported that bovine chondrocytes grown in a monolayer culture show a low capacity of redifferentiation when transferred to pellets (Malda et al., 2003). However, some investigators have reported that human chondrocytes cultured in a monolayer redifferentiate when grown in pellet culture (Oyajobi et al., 1998). Such variation is likely due to species variability (Giannoni et al, 2005). ...
... Such variation is likely due to species variability (Giannoni et al, 2005). Furthermore, Oyajobi et al., (1998) reported that the redifferentiated chondrocytes express collagen type X, a gene typical of hypertrophy, but they have not shown any morphological evidence of the occurrence of hypertrophy in their system. ...
The book showed the existence of two different populations of hypertrophic chondrocytes in the growth cartilage of prenatal and postnatal horses, and they die by non-apoptotic modes of physiological death. An in vitro three-dimensional model for inducing hypertrophic chondrocytes to die by the same mechanisms seen in vivo has been established. The system produced in the current work has been used to study the gene expression differences between different populations of hypertrophic chondrocytes during their early and late stages.
... It has been reported that bovine chondrocytes grown in a monolayer culture show a low capacity of redifferentiation when transferred to pellets (Malda et al., 2003). However, some investigators have reported that human chondrocytes cultured in a monolayer redifferentiate when grown in pellet culture (Oyajobi et al., 1998). Such variation is likely due to species variability (Giannoni et al, 2005). ...
... Such variation is likely due to species variability (Giannoni et al, 2005). Furthermore, Oyajobi et al., (1998) reported that the redifferentiated chondrocytes express collagen type X, a gene typical of hypertrophy, but they have not shown any morphological evidence of the occurrence of hypertrophy in their system. ...
... Oyajobi et al (28) also found that a clonal cell line of tsTAg-immortalized human articular chondrocytes synthesized type II collagen and aggrecan in monolayer. ...
... The requirement of cell contact and extracellular matrix for expression of survival factors such as Bcl-2 (31-33) may explain why the pellet cultures of Oyajobi et al were able to survive at 37°C for at least 3 months (28). The poor survival of low-density alginate cultures of the tsT/ AC62 cells that were seeded and maintained at 39°C, compared with those allowed to form clusters for a few days at 32°C, may have been due partially to the lack of autocrine inhibitory signals for apoptosis produced in high-density chondrocyte cultures (34). ...
Objective
To develop a reproducible immortalized human chondrocyte culture model for studying the regulation of chondrocyte functions relevant to arthritic diseases in adult humans.Methods
Primary adult articular chondrocytes were immortalized with a retrovirus expressing a temperature-sensitive mutant of SV40–large T antigen (tsTAg). The established tsT/AC62 chondrocyte cell line was examined in monolayer and alginate culture systems. The levels of messenger RNA (mRNA) encoding cartilage matrix proteins and interleukin-1β (IL-1β)–inducible mRNA were analyzed by reverse transcriptase–polymerase chain reaction. Matrix protein synthesis was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis of 35S-sulfate–labeled proteoglycans and Western blotting of type II collagen and aggrecan. Type II collagen (COL2A1)–luciferase reporter gene expression was analyzed by transient transfection. Phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38 mitogen-activated protein kinase (p38 MAPK), and activating transcription factor 2 (ATF-2) were detected by Western blotting.ResultsThe tsT/AC62 cells expressed TAg at the permissive temperature (32°C), and the loss of TAg at 37°C and 39°C correlated with decreased cell proliferation. Cells in alginate culture deposited abundant alcian blue–stainable matrix and continued to proliferate at 32°C. Preferential retention of aggrecan was observed in the cell-associated matrix, while biglycan and decorin were secreted into the medium of monolayer and alginate cultures. The levels of COL2A1 and aggrecan mRNA were increased after transfer from monolayer to alginate culture at 32°C. Treatment with IL-1β decreased COL2A1 and aggrecan mRNA levels and increased the levels of matrix metalloproteinases 1, 3, and 13 mRNA, as well as those of cyclooxygenase 2, type I collagen, and secretory phospholipase A2 type IIA mRNA, but not those of inducible nitric oxide synthase mRNA. IL-1β also stimulated phosphorylation of p38 MAPK, SAPK/JNK, and ATF-2. The p38 MAPK–selective inhibitor, SB203580, partially reversed IL-1β–induced inhibition of COL2A1 mRNA levels and COL2A1–luciferase reporter gene expression.Conclusion
The tsT/AC62 cells provide a reproducible model that mimics the adult articular chondrocyte phenotype, particularly in alginate culture, and demonstrates characteristic responses to IL-1β. These studies also show, for the first time, that p38 MAPK is one of the signals required for IL-1β–induced inhibition of COL2A1 gene expression. Availability of this model will permit identification of signals that regulate cytokine responses, and will also provide rational strategies for targeting these pathways.
... Chondrocytes isolated from the deep zone of cartilage and grown on filter cultures express aggrecan core protein, type II, type I, and type X collagens, ALP, and osteopontin mRNA. This is similar to other mineralizing chondrocyte cultures (19,(28)(29)(30)(31)(32) and the deep zone of articular cartilage. (33,34) The cells did not express osteocalcin, which was expected since these cells are articular chondrocytes. ...
... drocyte cultures. (29) However, chondrocytes from other mineralizing tissues such as day 18 chick embryonic sternal chondrocytes, chick epiphyseal plate chondrocytes, or an immortalized human chondrocyte cell line show no change, (16,28,32) while embryonic chick vertebral chondrocytes, day 14 or 18 chick embryo sternal chondrocytes in the presence of ascorbic acid, (30,31,42) and the chondrogenic cell line ATDC5 (43) show an increase in type X collagen expression. The increase in osteopontin expression detected in mineralizing articular chondrocytes is in keeping with other studies using chondrocytes. ...
We have developed a method to form reconstituted mineralized articular cartilagenous tissue in vitro from isolated deep zone chondrocytes. The aim of this study was to characterize further these cultures prior to and during mineralization. Histologic examination of the cells up to 8 days in culture showed that the chondrocytes had formed cartilagenous tissue. Similar to the in vivo cartilage, the chondrocytes expressed aggrecan, types II, I, and X collagens, osteopontin, and alkaline phosphatase (ALP). No osteocalcin mRNA expression was detected in either the in vivo cartilage or in vitro-generated tissue. Addition of beta-glycerophosphate (beta-GP) to the medium on day 5 induced mineralization and changes in gene expression. Expression of type X collagen, type II collagen, aggrecan core protein, and ALP were inhibited significantly between 2 h and 24 h after the addition of beta-GP. At 72 h, expression of these genes were still significantly depressed. These changes correlated with a decrease in collagen and proteoglycan synthesis, and ALP activity. Osteopontin expression increased within 8 h but returned to constitutive levels by 72 h. No change in type I collagen expression was detected. The changes in gene expression were not due to a direct effect of beta-GP itself, because similar gene changes occurred in the presence of phosphoethanolamine, another agent which induces mineralization. No changes in gene expression were seen in nonmineralizing cultures. In summary, articular chondrocytes grown on filter culture show expression of similar genes to the chondrocytes in the deep zone of articular cartilage and that changes in expression of specific genes were observed during tissue mineralization, suggesting that it is a suitable model to use to study the mechanism(s) regulating the localized mineralization of articular cartilage.
... There are different methods to develop micromasses: They can be centrifuged in order to obtain a single pellet, the hanging drop culture method can be applied to form many small micromasses, continuous rotating culture flasks like spinner flasks may form micromasses and culture surfaces can be coated with non-adhesive substances like agarose or chitosan films. [5][6][7][8] Different cell types like osteoblasts, endothelial cells, or fibroblasts, but also ADSC were applied to form micromasses. [3,4,[9][10][11] The aim of the present study was to reveal the micromass-formation-potential of different hADSC types (subcutaneous, visceral and omental fat tissue derived cells) and to compare their histological attributes. Secondly we wanted to examine collagen membranes serving as scaffold for potential in-vivo application and related differences of subcutaneous, visceral and omental fat tissue derived micromasses in cultivation. ...
Background:
Adult stem cells appear to be a promising subject for tissue engineering, representing an individual material for regeneration of aged and damaged cells. Especially adipose derived stromal cells (ADSC), which are easily to achieve, allow an encouraging perspective due to their capability of differentiating into miscellaneous cell types. Here we describe the in vitro formation of human subcutaneous, visceral and omental ADSC micromasses and compare their histological attributes while being cultivated on collagen membranes.
Methods:
Subcutaneous, visceral and omental fat tissue derived cells were isolated and processed according to standard protocols. Positively stained cells for CD13, CD44 and CD90 were cultivated on agarose in order to study micromass formation using a special method of cell tracking. Stained paraffin-embedded micromasses were analysed morphologically before and after being plated on collagen membranes.
Results:
The micromass formation process was similar in all three tissue types. Subcutaneous fat tissue derived micromasses turned out to develop a more homogeneous and compact shape than visceral and omental tissue. Nevertheless all micromasses adhered to collagen membranes with visible spreading of cells. The immune histochemical (IHC) staining of subcutaneous, visceral and omental ADSC micromasses shows a constant expression of CD13 and a decrease of CD44 and CD 90 expression within 28 days. After that period, omental fat cells don't show any expression of CD44.
Conclusion:
In conclusion micromass formation and cultivation of all analysed fat tissues can be achieved, subcutaneous cells appearing to be the best material for regenerative concepts.
... In terms of using chondrocytes to form hypertrophic cartilage, previous research has shown that immortalised adult articular chondrocytes become hypertrophic and mineralised in pellet culture [25]. Also, immortalised chondrocytes derived human embryonic femurs were reported to produce osteoinductive signals in the culture medium [26]. There are, however, significant regulatory and safety concerns associated with the use of genetically modified cells for clinical applications. ...
The regeneration of large bone defects remains clinically challenging. The aim of our study was to use a rat model to use nasal chondrocytes to engineer a hypertrophic cartilage tissue which could be remodelled into bone in vivo by endochondral ossification.
... Previous studies have reported that centrifugation can stimulate proteoglycan synthesis as well as DNA content [Inoue et al. 1990; Maeda et al. 2001]. Others have used centrifugation every other day when studying different aspects of chondrocytes in pellet culture [Nakagawa et al. 1993; Oyajobi et al. 1998]. Whether the lack of detectable beneficial effect has to do with the absence of soluble factors described previously, shortages in the models themselves, or a combination of both, cannot be distinguished from the experimental data. ...
Tissue engineering is a multidisciplinary field that combines cells, biomaterial scaffolds and environmental factors to achieve functional tissue repair. This thesis focuses on the use of macroporous gelatin microcarriers as scaffolds in tissue engineering applications, with a special focus on cartilage and bone formation by human adult cells in vitro .
In our first study, human articular chondrocytes were seeded on macroporous gelatin microcarriers. The microcarriers were subsequently encapsulated in coagulated blood-derived biological glues and cultured under free-swelling conditions for up to 17 weeks. Even in the absence of recombinant chondrogenic growth factors, the chondrocytes remained viable and metabolically active for the duration of the culture period, as indicated by an increased amount of cell nuclei and extracellular matrix (ECM). The ECM showed several cartilage characteristics, but lacked the cartilage specific collagen type II. Furthermore, ECM formation was seen primarily in a capsule surrounding the tissue-engineered constructs, leading to the conclusion that the used in vitro models were unable to support true cartilage formation.
The capacity of human dermal fibroblasts to produce cartilage- and bone-like tissue in the previously mentioned model was also investigated. Under the influence of chondrogenic induction factors, including TGF-β1 and insulin, the fibroblasts produced cartilage specific molecules, as confirmed by indirect immunohistochemistry, however not collagen type II. Under osteogenic induction, by dexamethasone, ascorbate-2-phosphate and β–glycerophosphate, the fibroblasts formed a calcified matrix with bone specific markers, and an alkaline phosphatase assay corroborated a shift towards an osteoblast like phenotype. The osteogenic induction was enhanced by flow-induced shear stress in a spinner flask system.
In addition, four different types of gelatin microcarriers, differing by their internal pore diameter and their degree of gelatin cross-linking, were evaluated for their ability to support chondrocyte expansion. Chondrocyte densities on the microcarriers were monitored every other day over a twoweek period, and chondrocyte growth was analyzed by piecewise linear regression and analysis of variance (ANOVA). No differences were seen between the different microcarriers during the first week. However, during the second week of culture both microcarrier pore diameter and gelatin crosslinking had significant impacts on chondrocyte density.
Lastly, a dynamic centrifugation regime (f=12.5 mHz for 16 minutes every other day) was administered to chondrocyte-seeded microcarriers, with or without encapsulation in platelet rich plasma (PRP), to study the possible effect of dynamic stimuli on cartilage formation. Presence of PRP enhanced the structural stability of the tissue-engineered constructs, but we were not able to confirm any dose-response pattern between ECM formation and the applied forces. After 12 weeks, distinct gelatin degradation had occurred independent of both dynamic stimuli and presence of PRP.
In summary, this thesis supports a plausible use for gelatin microcarriers in tissue engineering of cartilage and bone. Microcarrier characteristics, specifically gelatin cross-linking and pore diameter, have been shown to affect chondrocyte expansion. In addition, the use of human dermal fibroblasts as an alternative cell source for cartilage and bone formation in vitro was addressed.
... Since chondrocyte pellet cultures are easy to reproduce, they are often used as a cartilage model for in vitro test systems. Chondrocyte pellets have been used to study the development of cartilaginous tissues ex vivo 41,42 , to evaluate matrix synthesis and turnover in the presence of growth factors and other pro-anabolic stimuli 1,41 and to investigate the response of chondrocytes to dynamic compression 16,35 . We used pellet cultures as the basis of an inflammatory co-culture system to study distinct aspects of destructive joint diseases like RA ex vivo. ...
The aim of this study was to evaluate the growth characteristics of freshly isolated porcine chondrocytes in high-density pellet cultures and to preliminary investigate their use in an interactive in vitro model with synovial fibroblast cell lines to study rheumatoid arthritis (RA).
1.8x10(6) chondrocytes/cm2 were seeded in 48-multiwell plates. Thickness, cell number and cell distribution in pellet cross sections were documented over a 22-day-long period. Alcian blue staining, type I and type II collagen staining, real-time reverse transcriptase polymerase polymerase chain reaction (RT-PCR) and high performance liquid chromatography (HPLC) were used to characterize cartilage extracellular matrix (ECM) formation, and cell proliferation was demonstrated by Ki67 staining. Furthermore, 2-week-old chondrocyte pellets were co-cultured for additional 2 weeks with two human synovial fibroblast cell lines derived from a normal donor (non-invasive cell line) and a RA patient (invasive-aggressive (IA) cell line), respectively.
Chondrocyte pellets from 11 individual preparations showed a significant increase in pellet thickness from 44+/-19 microm (day 3) to 282+/-19 microm (day 22). Calculation of chondrocyte distribution, cell number and pellet thickness indicated that pellet growth was due to ECM formation and not cell proliferation. This was also confirmed by low numbers of Ki67 positive chondrocytes and absence of cell clusters. HPLC, messenger RNA-analysis, histochemistry and antibody staining verified the expression of ECM components such as type II collagen, whereas type I collagen expression was very low. In contrast to the non-aggressive synovial fibroblast cell line, the IA synovial fibroblast cell line clearly showed cartilage invasion.
Pellet formation of freshly isolated chondrocytes followed a reproducible developmental kinetics and showed typical immature hyaline cartilage properties. Such uniform cartilage pellets are very useful as a substrate for interactive cell culture models that simulate diseases like RA.
... Articular chondrocytes are susceptible to de-differentiation, characterized by a fibroblastic morphology accompanied by loss of type II collagen and expression of type I collagen, when cultured in a monolayer [16][17][18]. Pellet culture systems have been widely used to reinitiate and maintain chondrogenic differentiation in monolayer expanded chondrocytes isolated from cartilage [48,49]. In the present study, although human articular chondrocyte pellets were larger in comparison to ATDC5 pellets at 21 days, histologically the pellets were similar. ...
Utilizing ATDC5 murine chondrogenic cells and human articular chondrocytes, this study sought to develop facile, reproducible three-dimensional models of cartilage generation with the application of tissue engineering strategies, involving biodegradable poly(glycolic acid) scaffolds and rotating wall bioreactors, and micromass pellet cultures. Chondrogenic differentiation, assessed by histology, immunohistochemistry, and gene expression analysis, in ATDC5 and articular chondrocyte pellets was evident by the presence of distinct chondrocytes, expressing Sox-9, aggrecan, and type II collagen, in lacunae embedded in a cartilaginous matrix of type II collagen and proteoglycans. Tissue engineered explants of ATDC5 cells were reminiscent of cartilaginous structures composed of numerous chondrocytes, staining for typical chondrocytic proteins, in lacunae embedded in a matrix of type II collagen and proteoglycans. In comparison, articular chondrocyte explants exhibited areas of Sox-9, aggrecan, and type II collagen-expressing cells growing on fleece, and discrete islands of chondrocytic cells embedded in a cartilaginous matrix.
... "Maturational arrest" of normal chondrocytes appears to be a dynamic state maintained by molecular constraints, whose failure results in their inappropriate maturation towards a hypertrophic phenotype (4). In a scale of propensity to hypertrophy, OA or aged chondrocytes are poles apart compared to normal chondrocytes, which however under particular "mineralizing" culture conditions can be conduced to progress towards hypertrophy and terminal differentiation (50). Conceivably IKKα activity could drive either OA or aged normal chondrocytes into a mature, "terminal" phenotype. ...
Osteoarthritic (OA) chondrocytes behave in an intrinsically deregulated manner, characterized by chronic loss of healthy cartilage and inappropriate differentiation to a hypertrophic-like state. IKKα and IKKβ are essential kinases that activate NF-κB transcription factors, which in turn regulate cell differentiation and inflammation. This study was undertaken to investigate the differential roles of each IKK in chondrocyte differentiation and hypertrophy.
Expression of IKKα or IKKβ was ablated in primary human chondrocytes by retro-transduction of specific short-hairpin RNAs. Micromass cultures designed to reproduce chondrogenesis with progression to the terminal hypertrophic stage were established, and anabolism and remodeling of the extracellular matrix (ECM) were investigated in the micromasses using biochemical, immunohistochemical, and ultrastructural techniques. Cellular parameters of hypertrophy (i.e., proliferation, viability, and size) were also analyzed.
The processes of ECM remodeling and mineralization, both characteristic of terminally differentiated hypertrophic cells, were defective following the loss of IKKα or IKKβ. Silencing of IKKβ markedly enhanced accumulation of glycosaminoglycan in conjunction with increased SOX9 expression. Ablation of IKKα dramatically enhanced type II collagen deposition independent of SOX9 protein levels but in association with suppressed levels of runt-related transcription factor 2. Moreover, IKKα-deficient cells retained the phenotype of cells in a pre–hypertrophic-like state, as evidenced by the smaller size and faster proliferation of these cells prior to micromass seeding, along with the enhanced viability of their differentiated micromasses.
IKKα and IKKβ exert differential roles in ECM remodeling and endochondral ossification, which are events characteristic of hypertrophic chondrocytes and also complicating factors often found in OA. Because the effects of IKKα were more profound and pleotrophic in nature, our observations suggest that exacerbated IKKα activity may be responsible, at least in part, for the characteristic abnormal phenotypes of OA chondrocytes.
Cartilage, at all sites of our body, has limited intrinsic capacity to repair. There are numerous methods to use when repairing cartilage, and the preferred method depends on the location of the cartilage defect, whether it is the nose, the ear, the airway, or a certain site in the joint. Different cell types with chondrogenic capacities are being used for cartilage regeneration. In this chapter we discuss the expansion in culture, the chondrogenic capacity, and the (clinical) application of articular chondrocytes, nasal chondrocytes, auricular chondrocytes, chondrocyte cell lines, mesenchymal stem cells, and pluripotent stem cells.
The regeneration of whole osteochondral constructs with a physiological structure has been a significant issue, both clinically and academically. In this study, we present a method using rabbit bone marrow stromal cells (BMSCs) cultured on a silk-RADA peptide scaffold in a specially designed two-chambered co-culture well for the generation of multilayered osteochondral constructs in vitro. This specially designed two-chambered well can simultaneously provide osteogenic and chondrogenic stimulation to cells located in different regions of the scaffold. We demonstrated that this co-culture approach could successfully provide specific chemical stimulation to BMSCs located on different layers within a single scaffold, resulting in the formation of multilayered osteochondral constructs containing cartilage-like and subchondral bone-like tissue, as well as the intermediate osteochondral interface. The cells in the intermediate region were found to be hypertrophic chondrocytes, embedded in a calcified extracellular matrix containing glycosaminoglycans and collagen types I, II and X. In conclusion, this study provides a single-step approach that highlights the feasibility of rabbit BMSCs as a single-cell source for multilayered osteochondral construct generation in vitro. Copyright © 2013 John Wiley & Sons, Ltd.
Introduction:
The zone of calcified cartilage (ZCC) anchors articular cartilage (AC) to subchondral bone through a layer of intermediate stiffness. The regulation and functional consequences of cartilage calcification may vary with depth from the articular surface. The hypothesis of this study was that the in vitro calcification of immature AC occurs selectively in the deep region and is associated with a local increase in stiffness.
Methods:
AC and growth plate cartilage (GPC) from calves were incubated in DMEM, 1% fetal bovine serum, 100 µg/mL ascorbate, and ±10 mM β-glycerophosphate (βGP) for up to 3 weeks. To assess the time course and effects of cell viability and βGP, full-depth strips of AC and GPC were analyzed by histology, indentation, and 45Ca++ uptake. To assess the effect of tissue zone, disks harvested from surface and deep zone AC and from reserve and hypertrophic zone of GPC were incubated independently and analyzed by compression and for 45Ca++ uptake and biochemical components.
Results:
The deep ~20% of immature AC calcified within 3 weeks, with calcification dependent on cell viability and βGP. Mineral was deposited continuously around cells in AC but only between cell columns in GPC. The deep zone of AC exhibited a compressive modulus of 0.53 MPa after βGP-induced calcification, ~4-fold stiffer than AC incubated without βGP.
Conclusions:
Cartilage explants exhibit inherent zone-specific calcification processes, resulting in an increase in stiffness associated with cartilage calcification. Such properties may be useful for engineering a biomimetic ZCC tissue to integrate cartilaginous tissue to bone, thereby forming a mechanically functional osteochondral unit.
A consistent observation in osteoporosis is bone volume reduction accompanied by increased marrow adipose tissue. No single
cause linking the two phenomena has yet been identified. In a human progenitor cell clone (hOP 7) derived from bone marrow,
however, we have demonstrated that rabbit serum can direct differentiation away from an osteoblast lineage to one of adipocytes.
We now report on whether human serum has a similar effect. Serum was collected from 10 pre- and 10 postmenopausal women and
from the 10 postmenopausal women before and following 6-week hormone replacement therapy (HRT). hOP 7 cells were cultured
with the various sera, and after 7-14 days adipocytogenesis was determined by oil red O staining and lipoprotein lipase (LPL)
and glycerol 3-phosphate dehydrogenase (G3PDH) expression. Incubation with 10% premenopausal serum led to labeling of 10.9%
of cells (P<0.05) with oil red O, whereas application of 10% postmenopausal serum led to a much larger effect, 43.5% labeling (P<0.001 with respect to premenopausal serum). Oil red O positivity was accompanied by loss of type I collagen expression
and increased LPL and G3PDH expression. HRT did not reverse the adipocytogenic effect of postmenopausal serum. In conclusion,
serum from postmenopausal women contains factors that steer hOP 7 bone progenitor cells toward an adipocytic phenotype, irrespective
of HRT. The study suggests a role for serum factors in the development of fatty marrow in postmenopausal osteoporosis.
To evaluate the effect of immortalized hypertrophic chondrocytes extracellular matrix (HCM) with or without the use of guided bone regeneration (GBR) on the healing of critical-size calvarial defects.
In 42 rats, 5 mm critical-size calvarial defects were surgically created. The animals were randomly allocated to six groups of seven rats each: Group A1: one defect was left untreated (control), while the contralateral defect was covered by a double non-resorbable membrane (GBR). Group B1: one defect was filled with calcium phosphate cement (CP), while the contralateral defect was treated with GBR and CP. Group C1: one defect was filled with a mixture of CP and HCM, while the contralateral defect was treated with GBR and CP+HCM. The healing period for all three groups was 30 days. The remaining three groups were treated in a similar manner but the healing period was 60 days. Five animals from each group were evaluated by maceration and two animals were analysed histologically.
At 30 days, all the control-treated defects did not present complete closure. When GBR was applied alone or combined with CP, 3/5 and 5/5 defects, respectively, presented complete closure. At 60 days, one defect from the control group presented complete closure. All the defects treated with GBR alone presented complete closure, whereas the combined use of GBR with CP or CP+HCM resulted in 4/5 and 3/5 defects with complete closure, respectively. The only treatment modality that did not present any specimen with defect closure at both 30 and 60 days was the combination of CP+HCM. The histological analysis indicated that when GBR was not used alone, the healing consisted of an amorphous acellular structure and loose granulation tissue, which, even though clinically resembled hard tissue, did not demonstrate the histological characteristics of bone.
The predictability of bone formation in critical-size defects depends mainly on the presence or absence of barrier membranes. The combined use of GBR with calcium phosphate alone or in combination with immortalized human HCM did not enhance the potential for osseous healing provided by the GBR procedure.
We have analyzed the distribution of type II collagen N- and C-propeptides in the cell layers and culture medium of bovine articular chondrocyte pellet cultures. Two splice variants of the type II collagen N-propeptide were detected by immunoblotting and immunoassay, using a new anti-peptide antibody, while the C-propeptide was detected using a monoclonal antibody. Type II collagen molecules containing the N-propeptide were detected weakly in cell layers, but not in tissue culture medium of chondrocyte pellet cultures, and both splice variants were observed. Free N-propeptide could not be detected in cell layers or medium. Type II procollagen molecules containing the C-propeptide were detected strongly in cell layers, but not in tissue culture medium, while the free C-propeptide was detected in both cell layers and medium. Since the N- and C-propeptides must be synthesized in a 1:1 molar ratio, we conclude that the N-propeptide is metabolized more quickly than the C-propeptide in this system. Our model can be used to study regulation of procollagen synthesis and propeptidase activity.
The mesoblastic clone, C1, behaves as a tripotential progenitor able to self-renew and to differentiate toward osteogenesis, chondrogenesis, or adipogenesis in response to specific inducers. In this study, expression and deposition by the C1 cells of essential components of the extracellular matrix, collagens type I, II, III, V, XI, VI, IX, and X were followed along the osteogenic and chondrogenic pathways, through biochemical, immunochemical, and electron microscopy analyses. Implementation of each program involves profiles of collagen synthesis and matrix assembly close to those documented in vivo. Depending on the applied inducers, cells adopt a defined identity and, controls acting at transcriptional and posttranslational levels adapt the set of deposited collagens to one particular cell fate. Osteogenic C1 cells selectively build a type I collagen matrix also containing type III, V, and XI collagens but selectively exclude type II collagen. Chondrogenic C1 cells first elaborate a type II collagen network and then acquire hypertrophic chondrocyte properties while assembling a type X collagen matrix as in the growth plate. This study provides an example of how a mesoblastic cell line can develop, in vitro, each of its genetic programs up to terminal differentiation. Intrinsic factors and time-dependent cell-matrix interactions might, as in vivo, underline the implementation of an entire differentiation program.
In vivo expression of the type III sodium-dependent phosphate transporter (NaPiT) Glvr-1 during endochondral ossification, suggests a functional role for inorganic phosphate (Pi) transport in cartilage calcification. For further analysis of this relationship, an in vitro model of endochondral ossification is required. In this context, we investigated the characteristics of Pi transport in the new chondrogenic cell line ATDC5 in relation to extracellular matrix (ECM) formation and mineralization. Pi uptake in ATDC-5 cells and in isolated matrix vesicles (MVs) is mediated by an Na-dependent Pi transporter with a pH dependency characteristic of a type III Pi carrier (lower activity at alkaline pH). Northern blot analysis indicated that ATDC-5 cells express Glvr-1 transcripts during the various stages of their maturation with a maximal level during the proliferating stage. In isolated MVs, Pi transport activity was maximal at day 21, concomitant with the beginning of type X collagen messenger RNA expression. These events preceded the initiation of matrix mineralization, which was apparent at day 25, and then gradually increased until day 47. This temporal relationship between maximal Pi transport activity in MVs and the expression of a marker of mineralizing chondrocytes is compatible with the possible involvement of Pi transport in the ECM calcification observed in ATDC-5 cell cultures. In conclusion, these observations suggest that ATCD-5 cells in culture represent a promising model for the analysis of a functional role of Pi transport in the initial events of endochondral ossification.
The inorganic component of bone and related hard tissues is generally described as sheets of uniform needle- and plate-like crystals. However, cryofixation has become the method of choice for ultrastructural studies of bone mineral when ladder-like arrangements of filaments contained within deformable microspheres about 1 microm in diameter are apparently the prime structural feature and are consistent with the optical image. The same methodology has now been applied to mature human dentine in caries-free juvenile and adult teeth. These were fixed, sliced, stained for mineral and examined optically or were snap frozen, fragmented under liquid nitrogen, freeze-substituted with methanol or acetone and embedded without thawing in Lowicryl K4M for electron microscopy. Others were processed by traditional transmission electron microscopy methods. To obtain maximum resolution, the electron micrographs were photographically printed as negatives and image-enhanced by digitisation using a Polaroid Sprint Scan 45 and laser printer. In both optical and cryopreparations of juvenile and adult dentine, mineral microspheres up to 1 microm in diameter, were present in the dentinal tubules and peritubular dentine. Within these objects, the mineral was primarily in the form of sinuous electron dense filaments, 5 nm thick, which had a characteristic periodicity. In these preparations needle-like and plate-like structures were rare. In contrast, after traditional transmission electron microscopy preparation although similar filamentous structures remained, the mineral more generally had the familiar form of needles measuring approximately 50 nm in the long axis. The cryopreserved calcified filaments were apparently particularly densely distributed in the intertubular dentine where their parallel ladder-like arrays often formed highly orientated struts and stays. It was concluded that early dentine mineral has the form of filamentous microspheres and as in bone (and other calcifying tissues and cells) has no specific association with collagen. It was also concluded that these structures compact and deform with maturity into a sub-structural framework which may relate to powerful biomechanical forces transmitted through the tissue. Needle- or plate-like mineral is probably rare in vivo in dentine, only becoming commonplace after extensive chemical processing.
Large and small proteoglycans are essential components of articular cartilage. How to induce chondrocytes to repair damaged cartilage with normal ratios of matrix components after their loss due to degenerative joint disease has been a major research focus. We have developed immortalized human chondrocyte cell lines for examining the regulation of cartilage-specific matrix gene expression. However, the decreased synthesis and deposition of cartilage matrix associated with a rapid rate of proliferation has presented difficulties for further examination at the protein level. In these studies, proteoglycan synthesis was characterized in two chondrocyte cell lines, T/C-28a2 and tsT/AC62, derived, respectively, from juvenile costal and adult articular cartilage, under culture conditions that either promoted or decreased cell proliferation. Analysis of proteo[36S]glycans by Sepharose CL-4B chromatography and SDS-PAGE showed that the large proteoglycan aggrecan and the small, leucine-rich proteoglycans, decorin and biglycan, were produced under every culture condition studied. In monolayer cultures, a high initial cell density and conditions that promoted proliferation (presence of serum for T/C-28a2 cells or permissive temperature for the temperature-sensitive tsT/AC62 cells) favored cell survival and ratios of proteoglycans expected for differentiated chondrocytes. However, the tsT/AC62 cells produced more proteoglycans at the nonpermissive temperature. Culture of cells suspended in alginate resulted in a significant decrease in proteoglycan production in all culture conditions. While the tsT/AC62 cells continued to produce a larger amount of aggrecan than small proteoglycans, the T/C-28a2 cells lost the ability to produce significant amounts of aggrecan in alginate culture. In addition, our data indicate that immortalized chondrocytes may alter their ability to retain pericellular matrix under changing culture conditions, although the production of the individual matrix components does not change. These findings provide critical information that will assist in the development of a reproducible chondrocyte culture model for the study of regulation of proteoglycan biosynthesis in cartilage.
Bone mineral morphology is altered by processing and this is rarely considered when preparing bone as a bioimplant material. To examine the degree of transformation, a commercial, coarsely particulate bone mineral biomaterial produced by prolonged deproteination, defatting, dehydration, and heating (donor material) was compared with similar particles of human bone (recipient material) prepared optimally by low-temperature milling. The two powders were freeze-substituted and embedded without thawing in Lowicryl K4M before sectioning for transmission electron microscopy (TEM) (other aliquots were processed by traditional TEM methods). To maximize resolution, electron micrographs were image-enhanced by digitization and printed as negatives using a Polaroid Sprint Scan 45. In addition to their morphology, the particles were examined for antigenicity (specific by reference to fluorescein isothiocyanate [FITC]-conjugated fibronectin, and nonspecific by reference to general FITC-conjugated immunoglobulins). Results showed that the optimally prepared human bone fragments stained discretely for fibronectin with negligible background autofluorescence. In contrast, the bioimplant fragments stained extensively with this and any other FITC-conjugated antibody and, unlike fresh bone, it also autofluoresced a uniform yellow. This difference was also expressed structurally and, although the bioimplant mineral consisted of rhomboidal plates up to 200 nm across and 10 nm thick, the optimally prepared bone mineral was composed of numerous clusters of 5-nm-wide sinuous calcified filaments of variable density and indeterminate length (which became straight needles 50 nm long and 5 nm thick following traditional chemical TEM fixation/staining). It was concluded that the inorganic phase of bone is both morphologically and immunologically transmutable and that, in biomaterials, the transformation is apparently so great that a broad indigenous antigenicity is unmasked, increasing the likelihood of resorption or rejection. This marked change may also provide preliminary insight into a more modest natural aging phenomenon with the localized lateral fusion of calcified filaments into less flexible, more immunologically reactive fenestrated plates.
To establish an immortalized normal human articular chondrocyte line which could be useful for a better understanding of cell molecular mechanisms relevant for the development of new therapeutic approaches in rheumatic diseases.
Chondrocytes from human adult articular healthy cartilage were transfected in primary culture with a plasmid containing two human papilloma virus type 16 (HPV-16) early function genes: E6 and E7, using the highly efficient cationic liposome-mediated (lipofection) procedure. The transfection was verified by reverse transcriptase-polymerase chain reaction analysis of E7 mRNA and by immunofluorence localization of the E7 protein in the cell cytoplasm. The established chondrocyte cell line was examined in monolayer and in two culture conditions that were described to re-induce differentiated characteristics: culturing in a serum-free defined medium supplemented with an insulin-containing serum substitute and seeding on a hyaluronan-based non-woven structured biomaterial. The expression of markers characteristic of cartilage was shown in the mRNA by reverse transcriptase-polymerase chain reaction. Immunohistological staining and Western blotting analysis were performed to evaluate type II collagen synthesis. Proteoglycans deposition was detected by Alcian Blue staining. A Field Emission In Lens Scanning Microscopy was used to look at the morphology of the immortalized cells at very high magnification.
Normal human articular chondrocytes were efficiently transfected leading to the establishment of an immortalized cell line as confirmed by HPV-16 E7 mRNA and protein detection. These cells were able to re-express type II collagen both at mRNA and protein levels under the two defined cultured conditions we used, still maintaining type I collagen expression. Collagen IX mRNA was present only in early primary culture while collagen type X and aggrecan transcripts were always detected. Alcian Blue staining showed a proteoglycan-rich matrix production. The ultrastructural analysis of the immortalized cells revealed that their morphology strictly resembled that of normal chondrocytes.
The cell line that we obtained may be a useful tool for increasing our knowledge of the genetic and biochemical events involved in the processes of cartilage growth and differentiation. Moreover, it appears to be a suitable model for pharmacological and toxicological studies related to rheumatic diseases relevant to humans.
Three clonal cell lines (MMR14, MMR17, and MMR32) were established from the costal cartilage derived from p53-/- mice. Expression profiles of cartilage-related molecules in MMR14 and MMR17 were compatible with those in cells of the hypertrophic zone. Prolonged in vitro culture induced the expression of calcification-related genes in both cell lines, but calcified nodules were observed only in MMR14. The expression profile of cartilage-related molecules in MMR32 was compatible with that of cells in the perichondrium, with high expression levels of decorin, bone morphogenetic protein-3, and parathyroid hormone-related peptide (PTHrP). When MMR14 was co-cultured with an equal amount of MMR32 without direct contact, the nodule formation was completely inhibited, whereas no such inhibition was observed when MMR14 was co-cultured with MMR17, indicating that soluble factors produced by MMR32 were responsible for the inhibition. Blocking the effects of PTHrP by either antagonizing peptide or neutralizing antibody against PTHrP failed to rescue the inhibitory effects of MMR32, and no increase of the cyclic adenosine monophosphate production in MMR14 was observed when co-cultured with MMR32, suggesting that soluble factors other than PTHrP produced by MMR32 were responsible for the inhibition of terminal differentiation of hypertrophic chondrocytes. This report is the first to show cell-to-cell interaction in the growth plate using cell lines, which will be useful material to investigate the regulatory mechanism of chondrocyte differentiation.
Maintenance of the articular surface depends on the function of articular chondrocytes (ACs) which produce matrix and are constrained from undergoing the maturation program seen in growth plate chondrocytes. Only during pathologic conditions, such as in osteoarthritis, are maturational constraints lost causing recapitulation of the process that occurs during endochondral ossification. With the aim of establishing a model to identify regulatory mechanisms that suppress AC hypertrophy, we examined the capability of 5-azacytidine (Aza) to have an impact on the maturational program of these cells. Primary ACs do not spontaneously express markers of maturation and are refractory to treatment by factors that normally regulate chondrocyte maturation. However, following exposure to Aza, ACs (i) were induced to express type X collagen (colX), Indian hedgehog, and alkaline phosphatase and (ii) showed altered colX and AP expression in response to bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), and parathyroid hormone-related protein (PTHrP). Since Aza unmasked responsiveness of ACs to BMP-2 and TGF-beta, we examined the effect of Aza treatment on signaling via these pathways by assessing the expression of the TGF-beta Smads (2 and 3), the BMP-2 Smads (1 and 5), and the Smad2 and 3-degrading ubiquitin E3 ligase Smurf2. Aza-treated ACs displayed less Smad2 and 3 and increased Smad1, 5, and Smurf2 protein and showed a loss of TGF-beta signaling on the P3TP-luciferase reporter. Suggesting that Aza-induction of Smurf2 may be responsible for the loss of Smad2 and 3 protein via this pathway, immunoprecipitation and metabolic labeling experiments confirmed that Aza accelerated the ubiquitination and degradation of these targets. Overall, Aza-treated ACs represent a novel model for the study of mechanisms that regulate maturational potential of articular cartilage, with the data suggesting that maturation of these cells may be due to up-regulation of Smad1 and 5 coupled with a Smurf2-dependent degradation of Smad2 and 3 and loss of TGF-beta signaling.
Immortalization of chondrocytes increases life span and proliferative capacity but does not necessarily stabilize the differentiated phenotype. Expansion of chondrocyte cell lines in continuous monolayer culture may result in the loss of phenotype, particularly if high cell density is not maintained. This chapter describes strategies for maintaining or restoring differentiated phenotype in established chondrocyte cell lines involving culture in serum-free defined culture medium, in suspension over agarose or polyHEMA, or within alginate or collagen scaffolds. Chondrocyte cell lines have been used successfully to develop reproducible models for studying the regulation of gene expression in experiments requiring large numbers of cells. Thus, approaches for studying transcriptional regulation by transfection of promoter-driven reporter genes and cotransfection of expression vectors for wild-type or mutant proteins are also described.
The role of the chondrocyte pericellular matrix (PCM) was examined in a three-dimensional chondrocyte culture system to determine whether retention of the native pericellular matrix could stimulate collagen and proteoglycan accumulation and also promote the formation of a mechanically functional hyaline-like neocartilage. Porcine chondrocytes and chondrons, consisting of the chondrocyte with its intact pericellular matrix, were maintained in pellet culture for up to 12 weeks. Sulfated glycosaminoclycans and type II collagen were measured biochemically. Immunocytochemistry was used to examine collagen localization as well as cell distribution within the pellets. In addition, the equilibrium compressive moduli of developing pellets were measured to determine whether matrix deposition contributed to the mechanical stiffness of the cartilage constructs. Pellets increased in size and weight over a 6-week period without apparent cell proliferation. Although chondrocytes quickly rebuilt a PCM rich in type VI collagen, chondron pellets accumulated significantly more proteoglycan and type II collagen than did chondrocyte pellets, indicating a greater positive effect of the native PCM. After 5 weeks in chondron pellets, matrix remodeling was evident by microscopy. Cells that had been uniformly distributed throughout the pellets began to cluster between large areas of interterritorial matrix rich in type II collagen. After 12 weeks, clusters were stacked in columns. A rapid increase in compressive strength was observed between 1 and 3 weeks in culture for both chondron and chondrocyte pellets and, by 6 weeks, both had achieved 25% of the equilibrium compressive stiffness of cartilage explants. Retention of the in vivo PCM during chondrocyte isolation promotes the formation of a mechanically functional neocartilage construct, suitable for modeling the responses of articular cartilage to chemical stimuli or mechanical compression.
Mesenchymal stem cells (MSCs) have a great therapeutic potential resulting from their ability to differentiate into multiple tissues when cultured under specific conditions. However, it has not been clearly demonstrated whether or not MSCs exhibit a multidifferentiation potential in three-dimensional collagen gel cultures. This study was conducted to explore the multidifferentiation potential of MSCs cultured in three-dimensional collagen gels. Human MSCs were cultured in 0.3% collagen gel for 20 days in chondrogenic differentiation medium (CDM), and for 14 days in osteogenic differentiation medium (ODM). Increases in GAG deposits, intensity of toluidine blue staining, and mRNA expressions of chondrogenic markers (type II collagen and type X collagen) were found in human MSCs cultured in the collagen gel maintained in CDM. Positive staining for alkaline phosphatase (ALP) activity and alizarin red, and increases in mRNA expressions of osteogenic markers (type I collagen, bone sialoprotein and ALP) were noted in the MSCs maintained in ODM. These findings emphasize that human MSCs have an ability to differentiate into both bone and cartilaginous tissues in three-dimensional collagen gel cultures, indicating potential clinical applications of MSC transplant therapy with collagen gel as a scaffold for bone or cartilage regeneration in complicated tissue defects.
Chondrocytes are useful as a cell culture system for studying arthritic degeneration in tissue engineered cartilage. However, primary chondrocytes have short in vitro lifespan and rapid shift of collagen phenotype. In this study, we used a high dosage of retroviral vector LXSN16E6E7 to transduce human primary chondrocytes and obtained an actively proliferating cell line, designated hPi, which expresses HPV-16 E6/E7 mRNA in early passages. Parental primary chondrocytes cease to grow after five passages, whereas hPi could be propagated beyond 100 passages without requiring additional cell elements in defined medium. After 48 passages, hPi can also give many profiles similar to those of parental primary chondrocyte, including type II collagen in mRNA and protein level, aggrecan in mRNA level, lacunae in type I collagen matrices, and morphology with GAG-specific Alcian blue staining. hPi has shown neoplastic transformation, as examined by NOD-SCID mice tumorigenicity assays for 3 months. Our results indicated that human primary chondrocytes could be immortalized by transduction with HPV-16 E6/E7, preserving stable cartilage-specific differentiation markers. The established chondrocyte cell line could provide a novel model to engineer cartilage in vitro and in vivo for cartilage repair research and clinical application.
Hypertrophic cartilage provides the morphological and biochemical template for orchestrating bone growth. To produce a bone-inductive material such as hypertrophic cartilage for clinical use, we have conditionally immortalized hypertrophic chondrocytic cells from human femur and expanded them in vitro through more than 145 divisions. The clonal cell lines generated by this process consistently express signals that induce both rat and human marrow cells to differentiate in vitro into osteoblastic cells. Further, implantation of the cell-free extracellular matrix from the immortalized chondrocytic cells causes vascularized bone to form in vivo in bony defects, but not in ectopic sites such as skeletal muscle. This study shows that molecular techniques can be used to generate bespoke human cell lines for bone tissue engineering. It also demonstrates that matrix material generated from human immortalized hypertrophic chondrocytic cells may provide an abundant, efficacious, and safer alternative to bone autograft--the currently preferred material for fracture repair.
We examined osteo-chondrogenic differentiation of a human chondrocytic cell line (USAC) by rhBMP-2 in vivo and in vitro. USAC was established from a transplanted tumor to athymic mouse derived from an osteogenic sarcoma of the mandible. USAC usually shows chondrocytic phenotypes in vivo and in vitro. rhBMP-2 up-regulated not only the mRNA expression of types II and X collagen, but also the mRNA expression of osteocalcin and Cbfa1 in USAC cells in vitro. In vivo experimental cartilaginous tissue formation was prominent in the chamber with rhBMP-2 when compared with the chamber without rhBMP-2. USAC cells implanted with rhBMP-2 often formed osteoid-like tissues surrounded by osteoblastic cells positive for type I collagen. rhBMP up-regulated Ihh, and the expression of Ihh was well correlated with osteo-chondrogenic cell differentiation. These results suggest that rhBMP-2 promotes chondrogenesis and also induces osteogenic differentiation of USAC cells in vivo and in vitro through up-regulation of Ihh.
A serum-free primary culture system for chicken growth plate chondrocytes has been developed which consistently undergoes mineral deposition. Upon attainment of confluency, the chondrocytes develop locally into multilayer cellular nodules leading to matrix calcification. Mineralization first occurs in matrix vesicles (MV) that are abundant in the extraterritorial matrix between the hypertrophic cells. Studies with ⁴⁵Ca reveal that significant accumulation of Ca²⁺ occurs as early as day 12, continuing progressively throughout the culture period. By day 24, the nodules become densely calcified. Fourier transform infrared spectroscopy reveals the mineral to be similar to apatite, with features essentially identical to those of mineral formed by MV in vitro. The presence of ascorbate is critical to the culture system; in its absence, calcification is rarely observed. Ascorbate stimulates MV formation and synthesis of cellular protein, alkaline phosphatase, and especially types II and X collagens. In addition, there is strong evidence that the types II and X collagens are associated with MV. 1) Electron microscopy reveals MV embedded in a type II collagenous network; 2) Western blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of MV using monospecific antibodies to types X and II collagen indicate that both collagens are present in specific MV fractions; 3) sucrose gradient purification of MV does not remove associated collagens; 4) graded salt extraction selectively releases type II collagen from MV; and 5) incubation of radiolabeled types II and X collagens with MV leads to their cosedimentation upon subsequent centrifugation. Taken together, the data suggest that coordinated synthesis of the collagens, alkaline phosphatase, MV formation, and Ca²⁺ accumulation by the cultures combine to induce mineral deposition in the multilayer nodules.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
In bone forming cartilage in vivo, cells undergo terminal differentiation, whereas most of the cells in normal articular cartilage do not. Chondrocyte hypertrophy can be induced also in vitro by diffusible signals. We have identified growth factors or hormones acting individually on 17-d chick embryo sternal chondrocytes cultured in agarose gels under strictly serum-free conditions. Insulin-like growth factor I or insulin triggered the first steps of chondrocyte maturation, i.e., cell proliferation and increased matrix deposition while the chondrocytic phenotype was maintained. However, cells did not progress to the hypertrophic stage. Proliferation and stimulated collagen production was preceded by a lag period, indicating that synthesis of other components was required before cells became responsive to insulin-like growth factor I or insulin. Very small amounts of FBS exerted effects similar to those of insulin-like growth factor I or insulin. However, FBS could act directly and elicited hypertrophy when constituting greater than 1% of the culture media. Basic FGF has been claimed to be the most potent chondrocyte mitogen, but had negligible effects under serum-free conditions. The same is true for PDGF, a major serum-mitogen. Under the direction of thyroxine, cells did not proliferate but became typical hypertrophic chondrocytes, extensively synthesizing collagen X and alkaline phosphatase.
The type X collagen is a short chain collagen associated with calcific cartilage and/or the expression of the hypertrophic chondrocyte phenotype. In articular cartilage, type X collagen is restricted to the basal zone of calcified cartilage adjacent to the subchondral bone. However, during pathological change such as in osteoarthritis, the synthesis of type X collagen becomes more widespread but never extends to the articular surface. Using immunocytochemistry and fluorography of newly synthesised collagens, we report that surface articular chondrocytes (which occupy the uppermost 10-15% of the tissue depth) from normal human cartilage initiate de novo synthesis of both type X collagen and alkaline phosphatase when maintained in suspension culture.
To investigate the appearance of hypertrophic chondrocytes in osteoarthritic (OA) cartilage, using type X collagen as a specific marker.
The biosynthesis of type X collagen was examined by metabolic labeling of freshly isolated articular chondrocytes with 3H-proline, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the synthesized collagens. Extracellular deposition of types X and II collagen was analyzed immunohistochemically.
Immunostaining revealed an irregular distribution of type X collagen, which was localized around chondrocyte clusters in fibrillated OA cartilage, but was absent from the noncalcified region of normal articular cartilage. Freshly isolated OA chondrocytes synthesized predominantly type X collagen, while control chondrocytes synthesized mostly type II collagen.
Our findings indicate focal premature chondrocyte differentiation to hypertrophic cells in OA cartilage.
In bone forming cartilage in vivo, cells undergo terminal differentiation, whereas most of the cells in normal articular cartilage do not. Chondrocyte hypertrophy can be induced also in vitro by diffusible signals. We have identified growth factors or hormones acting individually on 17-d chick embryo sternal chondrocytes cultured in agarose gels under strictly serum-free conditions. Insulin-like growth factor I or insulin triggered the first steps of chondrocyte maturation, i.e., cell proliferation and increased matrix deposition while the chondrocytic phenotype was maintained. However, cells did not progress to the hypertrophic stage. Proliferation and stimulated collagen production was preceded by a lag period, indicating that synthesis of other components was required before cells became responsive to insulin-like growth factor I or insulin. Very small amounts of FBS exerted effects similar to those of insulin-like growth factor I or insulin. However, FBS could act directly and elicited hypertrophy when constituting greater than 1% of the culture media. Basic FGF has been claimed to be the most potent chondrocyte mitogen, but had negligible effects under serum-free conditions. The same is true for PDGF, a major serum-mitogen. Under the direction of thyroxine, cells did not proliferate but became typical hypertrophic chondrocytes, extensively synthesizing collagen X and alkaline phosphatase.
Conditions were defined for promoting cell growth, hypertrophy, and extracellular matrix mineralization of a culture system derived from embryonic chick vertebral chondrocytes. Ascorbic acid supplementation by itself led to the hypertrophic phenotype as assessed by respective 10- and 15-fold increases in alkaline phosphatase enzyme activity and type X synthesis. Maximal extracellular matrix mineralization was obtained, however, when cultures were grown in a nutrient-enriched medium supplemented with both ascorbic acid and 20 mM beta-glycerophosphate. Temporal studies over a 3-wk period showed a 3-4-fold increase in DNA accompanied by a nearly constant DNA to protein ratio. In this period, total collagen increased from 3 to 20% of the cell layer protein; total calcium and phosphorus contents increased 15-20-fold. Proteoglycan synthesis was maximal until day 12 but thereafter showed a fourfold decrease. In contrast, total collagen synthesis showed a greater than 10-fold increase until day 18, a result suggesting that collagen synthesis was replacing proteoglycan synthesis during cellular hypertrophy. Separate analysis of individual collagen types demonstrated a low level of type I collagen synthesis throughout the 21-d time course. Collagen types II and X synthesis increased during the first 2 wk of culture; thereafter, collagen type II synthesis decreased while collagen type X synthesis continued to rise. Type IX synthesis remained at undetectable levels throughout the time course. The levels of collagen types I, II, IX, and X mRNA and the large proteoglycan core protein mRNA paralleled their levels of synthesis, data indicating pretranslational control of synthesis. Ultrastructural examination revealed cellular and extracellular morphology similar to that for a developing hypertrophic phenotype in vivo. Chondrocytes in lacunae were surrounded by a well-formed extracellular matrix of randomly distributed collagen type II fibrils (approximately 20-nm diam) and extensive proteoglycan. Numerous vesicular structures could be detected. Cultures mineralized reproducibly and crystals were located in extracellular matrices, principally associated with collagen fibrils. There was no clear evidence of mineral association with extracellular vesicles. The mineral was composed of calcium and phosphorus on electron probe microanalysis and was identified as a very poorly crystalline hydroxyapatite on electron diffraction. In summary, these data suggest that this culture system consists of chondrocytes which undergo differentiation in vitro as assessed by their elevated levels of alkaline phosphatase and type X collagen and their ultrastructural appearance.(ABSTRACT TRUNCATED AT 400 WORDS)
The effects of basic fibroblast growth factor (bFGF) on terminal differentiation of chondrocytes and cartilage-matrix calcification were investigated. Rabbit growth-plate chondrocytes maintained as a pelleted mass in a centrifuge tube produced an abundant proteoglycan matrix during the matrix-maturation stage, yielding a cartilage-like tissue. Thereafter, they terminally differentiated to hypertrophic chondrocytes which produced high levels of alkaline phosphatase. These cells induced extensive calcification of the matrix in the absence of additional phosphate (Kato, Y., Iwamoto, M., Koike, T., Suzuki, F., and Takano, Y. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9552-9556). Addition of bFGF to the chondrocyte cultures abolished the increases in alkaline phosphatase activity, 45Ca deposition, and the calcium content. These effects were dose-dependent, reversible, and observed in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. The inhibitory effects could be observed only when chondrocytes were exposed to bFGF in a transition period between the matrix-maturation and hypertrophic stages. As chondrocytes differentiated to hypertrophic cells, bFGF became less effective in inhibiting the expression of the mineralization-related phenotypes. The present study also shows that although the rate of [35S]sulfate incorporation into large, chondroitin sulfate proteoglycan in the cell-matrix fraction is very high during the matrix-maturation stage, it abruptly decreases by 90% after terminal differentiation. Furthermore, the terminal differentiation-associated decrease in proteoglycan synthesis was delayed by bFGF. These results provide evidence that bFGF inhibits terminal differentiation of chondrocytes and calcification.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
The thermolabile large T antigen, encoded by the simian virus 40 early-region mutant tsA58, was used to establish clonal cell
lines derived from rat embryo fibroblasts. These cell lines grew continuously at the permissive temperature but upon shift-up
to the nonpermissive temperature showed rapidly arrested growth. The growth arrest occurred in either the G1 or G2 phase of
the cell cycle. After growth arrest, the cells remained metabolically active as assayed by general protein synthesis and the
ability to exclude trypan blue. The inability of these cell lines to divide at the nonpermissive temperature was not readily
complemented by the exogenous introduction of other nuclear oncogenes. This finding suggests that either these genes establish
cells via different pathways or that immortalization by one oncogene results in a finely balanced cellular state which cannot
be adequately complemented by another establishment gene.
A serum-free primary culture system for chicken growth plate chondrocytes has been developed which consistently undergoes mineral deposition. Upon attainment of confluency, the chondrocytes develop locally into multilayer cellular nodules leading to matrix calcification. Mineralization first occurs in matrix vesicles (MV) that are abundant in the extraterritorial matrix between the hypertrophic cells. Studies with 45Ca reveal that significant accumulation of Ca2+ occurs as early as day 12, continuing progressively throughout the culture period. By day 24, the nodules become densely calcified. Fourier transform infrared spectroscopy reveals the mineral to be similar to apatite, with features essentially identical to those of mineral formed by MV in vitro. The presence of ascorbate is critical to the culture system; in its absence, calcification is rarely observed. Ascorbate stimulates MV formation and synthesis of cellular protein, alkaline phosphatase, and especially types II and X collagens. In addition, there is strong evidence that the types II and X collagens are associated with MV. 1) Electron microscopy reveals MV embedded in a type II collagenous network; 2) Western blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of MV using monospecific antibodies to types X and II collagen indicate that both collagens are present in specific MV fractions; 3) sucrose gradient purification of MV does not remove associated collagens; 4) graded salt extraction selectively releases type II collagen from MV; and 5) incubation of radiolabeled types II and X collagens with MV leads to their cosedimentation upon subsequent centrifugation. Taken together, the data suggest that coordinated synthesis of the collagens, alkaline phosphatase, MV formation, and Ca2+ accumulation by the cultures combine to induce mineral deposition in the multilayer nodules.
A clonal cell line with cartilage phenotypes and tumorigenicity during more than 3 years in culture was established from a human chondrosarcoma. In sparse cultures, the clonal line, named HCS-2/8, consisted of slightly elongated polygonal cells, which proliferated with a doubling time of 3.5 days. The cells became polygonal to spherical as they became confluent. After reaching confluence, the cells continued to proliferate slowly and formed nodules, which showed metachromasia when stained with toluidine blue. The nodules were three-dimensional in structure; cells were multilayered in the surface regions, overlying a thick layer of extracellular matrix, which showed metachromasia. Electron microscopically, the cells resembling chondrocytes in vivo were surrounded by an extracellular matrix consisting of thin collagen-like fibrils with numerous fine granules, presumably of proteoglycans. The cells actively synthesized proteoglycans as determined by [35S]sulfate incorporation. The hydrodynamic size of major proteoglycan monomers synthesized by the cells was that of so-called cartilage-specific proteoglycans, as determined by glycerol gradient centrifugation. Immunostaining identified type II collagen but not type I collagen. Fluorography and immunoblotting of collagens separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also demonstrated the synthesis of type II collagen but not type I collagen. Inoculation of HCS-2/8 cells into athymic mice resulted in the formation of chondrosarcomas that resembled the original tumor. Because of having these characters, HCS-2/8 cells should be useful not only in studies on the differentiated phenotypes of human chondrocytes but also in basic studies on the diagnosis, treatment, and etiology of human chondrosarcomas.
During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic chondrocytes, which then deposit a mineralized matrix to form calcified cartilage. Chondrocyte hypertrophy and matrix mineralization are associated with expression of type X collagen and the induction of high levels of the bone/liver/kidney isozyme of alkaline phosphatase. To determine what role vitamin C plays in these processes, chondrocytes derived from the cephalic portion of 14-day chick embryo sternae were grown in the absence or presence of exogenous ascorbic acid. Control untreated cells displayed low levels of type X collagen and alkaline phosphatase activity throughout the culture period. However, cells grown in the presence of ascorbic acid produced increasing levels of alkaline phosphatase activity and type X collagen mRNA and protein. Both alkaline phosphatase activity and type X collagen mRNA levels began to increase within 24 h of ascorbate treatment; by 9 days, the levels of both alkaline phosphatase activity and type X collagen mRNA were 15-20-fold higher than in non-ascorbate-treated cells. Ascorbate treatment also increased calcium deposition in the cell layer and decreased the levels of types II and IX collagen mRNAs; these effects lagged significantly behind the elevation of alkaline phosphatase and type X collagen. Addition of beta-glycerophosphate to the medium increased calcium deposition in the presence of ascorbate but had no effect on levels of collagen mRNAs or alkaline phosphatase. The results suggest that vitamin C may play an important role in endochondral bone formation by modulating gene expression in hypertrophic chondrocytes.
Activity of the bone/liver/kidney isozyme of alkaline phosphatase (AP) is known to be critical for mineralization in developing bone, although its role is unclear. The work now reported explores changes in the activity of this Zn2+-containing enzyme that occur during Ca2+ accumulation by matrix vesicles (MV). A marked loss (up to 65-70%) in AP activity was found to accompany Ca2+ accumulation by MV. These two events were highly correlated, both temporally and quantitatively. Investigation into possible causes revealed that the decline in AP activity during Ca2+ uptake was not due to action of proteases but rather resulted from interaction with the developing mineral phase, loss of metal ions (Zn2+ and Mg2+) from the active site of the enzyme, and concomitant irreversible denaturation of the enzyme. Protease inhibitors did not protect AP from loss of activity during mineralization; in contrast, protease treatments, which progressively destroyed the ability of MV to accumulate Ca2+ actually reduced loss of AP activity. These findings clearly demonstrate that AP is present at the site of MV mineralization and that its catalytic activity is profoundly reduced by the mineralization process.
Rabbit chondrocyte cultures on plastic dishes are capable of depositing a cartilaginous matrix, although the matrix does not calcify unless high levels of phosphate are added to the medium. In the present study, we cultivated a pelleted mass of rabbit growth-plate chondrocytes in the presence of Eagle's minimum essential medium supplemented with 10% fetal bovine serum and 50 micrograms of ascorbic acid per ml in a plastic centrifuge tube. These cells proliferated for several generations and then reorganized into a cartilage-like tissue that calcified without additional phosphate. The deposition of minerals was observed only after synthesis of a short-chain collagen and alkaline phosphatase. Serum factors were required for the increases in alkaline phosphatase and calcium contents. 5-Bromo-2'-deoxyuridine abolished the increases in uronic acid, alkaline phosphatase, and calcium contents. Transforming growth factor beta, at very low concentrations, suppressed the expression of the mineralization-related phenotype by chondrocytes. These results suggest that cartilage-matrix calcification can be controlled by growth factor(s) and that chondrocytes induce the mineralization of extracellular matrix when terminal differentiation is permitted in the absence of an artificial substrate.
A new immunoassay was developed to detect denaturation of type II collagen in osteoarthritis (OA). A peptide, alpha 1 (II)-CB11B, located in the CB11 peptide of type II collagen, was synthesized and used to produce a monoclonal antibody (COL2-3/4m) of the IgG1 (kappa) isotype. This reacts with a defined epitope in denatured but not native type II collagen and the alpha 3 chain of type XI collagen. The latter is present in very small amounts (about 1% wt/wt) in cartilage relative to the alpha 1 (II) chain. By using an enzyme-linked immunosorbent assay, type II collagen denaturation and total type II collagen content were determined. The epitope recognized by the antibody was resistant to cleavage by alpha-chymotrypsin and proteinase K which were used to extract alpha 1 (II)-CB11B from the denatured (alpha-chymotrypsin soluble) and residual native (proteinase K soluble) collagen alpha-chains, respectively, present in human femoral articular cartilage. Type II collagen content was significantly reduced from a mean (range) of 14% (9.2-20.8%) of wet weight in 8 normal cartilages to 10.3% (7.4-15.0%) in 16 OA cartilages. This decrease, which may result in part from an increased hydration, was accompanied by an increase in the percent denaturation of type II collagen in OA to 6.0% of total type II collagen compared with 1.1% in normal tissue. The percent denaturation was ordinarily greater in the more superficial zone than in the deep zone of OA cartilage.
Normal human adult articular chondrocytes were used to determine how the chondrocyte phenotype is modulated by culture conditions following long-term culture. We report here for the first time that human articular chondrocytes have a lifespan in the range of 34-37 population doublings. While chondrocytes cultured as monolayers displayed a fibroblastoid morphology and grew faster, those cultured as suspensions over agarose adopted a round morphology and formed clusters of cells reminiscent of chondrocyte differentiation in intact cartilage, with little or no DNA synthesis. These morphologies were independent of the age of the culture. Despite, these morphological differences, however, chondrocytes expressed markers at mRNA and protein levels characteristic of cartilage: namely, types II and IX collagens and the large aggregating proteoglycans, aggrecan, versican and link protein, but not syndecan, under both culture conditions. However, they also expressed type I collagen alpha 1(I) and alpha 2(I) chains. It has been suggested that expression of collagen alpha 1(I) by chondrocytes cultured as monolayers is a marker of the loss of the chondrocyte phenotype. However, we show here, using reverse transcriptase/polymerase chain reaction, that normal fresh intact human articular cartilage expresses collagen alpha 1(I). The data show that following long-term culture human articular chondrocytes retain their differentiated characteristics and that cell shape does not correlate with the expression of the chondrocyte phenotype. It is proposed that loss of the chondrocyte phenotype is marked by the loss of one or more cartilage-specific molecules rather than by the appearance of non-cartilage-specific molecules.
Immortalized human chondrocytes were established by transfection of primary cultures of juvenile costal chondrocytes with vectors encoding simian virus 40 large T antigen and selection in suspension culture over agarose. Stable cell lines were generated that exhibited chondrocyte morphology, continuous proliferative capacity (> 80 passages) in monolayer culture in serum-containing medium, and expression of mRNAs encoding chondrocyte-specific collagens II, IX, and XI and proteoglycans in an insulin-containing serum substitute. They did not express type X collagen or versican mRNA. These cells synthesized and secreted extracellular matrix molecules that were reactive with monoclonal antibodies against type II collagen, large proteoglycan (PG-H, aggrecan), and chondroitin-4- and chondroitin-6-sulfate. Interleukin-1 beta (IL-1 beta) decreased the levels of type II collagen mRNA and increased the levels of mRNAs for collagenase, stromelysin, and immediate early genes (egr-1, c-fos, c-jun, and jun-B). These cell lines also expressed reporter gene constructs containing regulatory sequences (-577/+3,428 bp) of the type II collagen gene (COL2A1) in transient transfection experiments, and IL-1 beta suppressed this expression by 50-80%. These results show that immortalized human chondrocytes displaying cartilage-specific modulation by IL-1 beta can be used as a model for studying normal and pathological repair mechanisms.
The thermolabile large T antigen, encoded by the simian virus 40 early-region mutant tsA58, was used to establish clonal cell lines derived from rat embryo fibroblasts. These cell lines grew continuously at the permissive temperature but upon shift-up to the nonpermissive temperature showed rapidly arrested growth. The growth arrest occurred in either the G1 or G2 phase of the cell cycle. After growth arrest, the cells remained metabolically active as assayed by general protein synthesis and the ability to exclude trypan blue. The inability of these cell lines to divide at the nonpermissive temperature was not readily complemented by the exogenous introduction of other nuclear oncogenes. This finding suggests that either these genes establish cells via different pathways or that immortalization by one oncogene results in a finely balanced cellular state which cannot be adequately complemented by another establishment gene.
Understanding the relationship between the structures and functions of calcified tissues is essential for an understanding of their pathology. This book emphasises aspects of calcification which are important to humans and presents aspects of current research, notably attempts to relate biological function to the structures and interactions, many chapters stress the importance of the extracellular matrix, and provide links with volumes 5 and 13 in the series Connective Tissue Matrix and Connective Tissue Matrix Part II respectively. Material selected for inclusion in this volume intentionally favour the more physical and physico-chemical aspects of contemporary research.
This chapter has two purposes. One is to provide background information on the minerals deposited in calcified tissues. The other is to summarise the techniques used to characterise them. An understanding of the range and scope of these techniques is essential for a critical evaluation of the material described in subsequent chapters. Furthermore, because of the complexity of many of the minerals, it is impossible to understand how their structures are defined without some appreciation of the techniques involved. Several techniques which are important subjects of current research are described in more detail in chapters devoted to them.
This is the 12th edition of the textbook of rheumatology first published in 1940. Like its predecessors, it presents voluminous and authoritative material on all facets of rheumatic diseases, from paleopathology to molecular biology to clinical features and treatment. The 143 contributors represent many of the top academic rheumatology divisions and related fields in the United States and abroad.It is impractical to read tomes like this from cover to cover. Instead, I tried to put the textbook through its paces by reading the sections that were pertinent to my clinical practice over a four-week period. Thus, while I did not see every condition described in the book, I found that I covered a surprising amount of it, either directly in my own patients, or during teaching sessions with fellows, house staff, and students, and in discussions with colleagues. My bias is that a subspecialty textbook should have two purposes:
A major goal of cell biologists is to have an in vitro cell model that simulates in vivo conditions. This cell model should be able to propagate in culture, express specialized tissue functions, and allow fundamental biological problems to be answered by a simple manipulation of the culture conditions. Unfortunately, normal differentiated cells generally do not proliferate in culture and often cease to express their specialized functions. As a result, studies have often been carried out on aberrant tumor cell lines that are capable of growth in culture and yet retain the ability to express the tissue-specific functions. However, because of their malignant state and in the absence of normal controls, conclusions from tumor cell studies may not be relevant to normal gene regulation. In the past decade, various attempts have been made to immortalize cells from normal tissues. Cell lines retaining differentiated functions have been established by transformation with chemical carcinogens (1), oncogenes (2, 3),...
Inorganic pyrophosphate (PPi) may be involved in the regulation of mineralization. The cell surface enzyme, ecto-NTP pyrophosphatase, could be a major source of extracellular PPi in bone, and agents that influence its activity in osteoblasts may modulate bone mineralization. We studied the effects of serum on the ecto-NTP pyrophosphatase activity of cultured human osteoblast-like cells. Enzyme activity was lowered when the concentration of fetal calf serum (FCS) was reduced from 10 to 2.5% (vol/vol) for 48 h, and a further decrease in activity was observed after 96 h. Relative to enzyme activity in cells cultured in serum-free medium for 96 h, adult human platelet-poor plasma (HPPP; 2.5-10% vol/vol) induced a small increase, similar concentrations of adult human serum (HS) induced much larger increases, and charcoal-depleted FCS was ineffective. In an attempt to identify the factor(s) present in serum that influence ecto-NTP pyrophosphatase activity, we examined transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF). PDGFs AA, AB, and BB (0.1-10 ng/ml) were ineffective, but both TGF-beta 1 and TGF-beta 2 increased enzyme activity. The increase was dose dependent between 0.001 and 10 ng/ml, was enhanced in the presence of 2% vol/vol FCS, and was not potentiated by PDGF or by 1,25-(OH)2D3. Furthermore, the increase was independent of cell density and was blocked by inhibitors of protein and RNA synthesis. Ecto-NTP pyrophosphatase of subject-matched human dermal fibroblasts was unaffected by TGF-beta (10 ng/ml), suggesting that modulation of activity by the growth factor may be tissue specific. Alkaline phosphatase (ALP) probably serves to hydrolyze extracellular PPi in bone. In contrast to effects on NTP pyrophosphatase activity is osteoblast-like cells, TGF-beta 1 and TGF-beta 2 (0.001-10 ng/ml) decreased ALP activity dose dependently after 72 h. By inducing opposing changes in ecto-NTP pyrophosphatase and ALP activities, TGF-beta may increase extracellular PPi concentrations in osseous tissues and consequently modulate bone mineral properties in vivo.
The optimal conditions for obtaining a calcified cartilage matrix approximating that which exists in situ were established in a differentiating chick limb bud mesenchymal cell culture system. Using cells from stage 21–24 embryos in a micro-mass culture, at an optimal density of 0.5 million cells/20 μl spot, the deposition of small crystals of hydroxyapatite on a collagenous matrix and matrix vesicles was detected by day 21 using X-ray diffraction, FT-IR microscopy, and electron microscopy. Optimal media, containing 1.1 mM Ca, 4 mM P, 25 μg/ml vitamin C, 0.3 mg/ml glutamine, no Hepes buffer, and 10% fetal bovine serum, produced matrix resembling the calcifying cartilage matrix of fetal chick long bones. Interestingly, higher concentrations of fetal bovine serum had an inhibitory effect on calcification. The cartilage phenotype was confirmed based on the cellular expression of cartilage collagen and proteoglycan mRNAs, the presence of type II and type X collagen, and cartilage type proteoglycan at the light microscopic level, and the presence of chondrocytes and matrix vesicles at the EM level. The system is proposed as a model for evaluating the events in cell mediated cartilage calcification.
Type II collagen and aggrecan are major components of the extracellular matrix of articular cartilage. Their biosynthesis and catabolism are regulated by chondrocytes. They may be used as markers of chondrocyte phenotype for cells cultured in vitro. Type II collagen gene expression was detected by amplification of type II collagen-specific sequences, using cDNA produced by reverse transcription of mRNA extracted from freshly isolated and cultured human articular chondrocytes by the polymerase chain reaction (PCR). The synthesis of gene product was confirmed by immunohistochemical localization of type II collagen in cartilage sections and in cultured chondrocytes. Aggrecan core protein was also immunolocalized in cartilage sections and in chondrocytes in culture. Expression of type II collagen or aggrecan was not detected immunohistochemically in skin or bone. These results demonstrate that human articular chondrocytes can be characterized in culture, by the combined application of PCR and immunohistochemistry. Interleukin-1β (IL-1β) may play an important role in the destruction of cartilage matrix in arthritis, whereas transforming growth factor-β (TGFβ) may have an opposing effect and their combined actions may modulate chondrocyte phenotype. The effect of rhIL-1β and rhTGβ on the production of type II collagen by chondrocytes in culture was investigated. It was shown that TGFβ enhanced the production of type II collagen, localized immunocytochemically, in cultured chondrocytes. IL-1β inhibited expression of mRNA for type II collagen. The implications of this study, in terms of a better understanding of degenerative cartilage disease, are discussed.
Summary Chondrocyte, matrix vesicle, and membrane fractions, as well as interstitial fluid samples from the proliferating and hypertrophic zones of chicken epiphyseal cartilage were analyzed for electrolyte content. Intracellular Ca levels were 1.4–2.1 mM, over 90% of which was nondiffusible. Isolated hypertrophic chondrocytes had higher intracellular Na and lower K than proliferating cells. Matrix vesicles contained 25 to 50 times higher concentrations of Ca than the adjacent cells. Vesicles from the zone of hypertrophy contained twice as much Ca as did those from the proliferating area. Ca/P1 molar ratios of matrix vesicles were much higher than those of cells or of later mineral deposits. These findings indicate that Ca is concentrated in matrix vesicles during formation, but acuumulation of Ca and P1 must continue in the matrix. X-ray diffraction of freeze-dried vesicle and membrane fractions failed to detect crystalline apatite, suggesting that crystals seen in electron micrographs of matrix vesicles may be artifacts. Interstitial fluid expressed from epiphyseal cartilage was higher in K, Pi, Mg and nucleotides, and lower in Na and Cl, than blood plasma. Fluid from the hypertrophic zone was higher in K and nucleotides, but not Pi or Mg, than that from the proliferating layer. These data suggest that selective leakage or extrusion of these constituents, which are normally intracellular, must occur, especially in the hypertrophic zone. More of the Ca and Mg, and less of the Pi, was protein-bound in cartilage fluid than in blood plasma. There was more binding of the divalent cations in fluid from proliferating than from hypertrophic cartilage. The presence of greater amounts of ultrafilterable peptides in fluid from hypertrophic than from proliferating cartilage or blood plasma, suggests that proteolytic activity may release bound divalent cations during mineralization.
We have developed a method to isolate RNA in high yield from adult articular cartilage. Homogenization of the articular cartilage with a freezer mill, extraction with 4 M guanidinium isothiocyanate/acid-phenol, and ultracentrifugation in cesium trifluoroacetate was found to be an effective and practical method for isolating a high yield of intact RNA from adult canine articular cartilage. The total RNA was suitable for Northern blot analysis. The mRNA that could then be isolated by oligo-dT affinity chromatography was found to be a suitable substrate for in vitro translation, for making a cDNA library, and for PCR amplification.
Articular cartilage is a permanent tissue whose cells do not normally take part in the endochondral ossification process. To determine whether articular chondrocytes possess the potential to express traits associated with this process such as cell hypertrophy and type X collagen, chondrocytes were isolated from adult chicken tibial articular cartilage and maintained in long-term suspension cultures. As a positive control in these experiments, we used parallel cultures of chondrocytes from the caudal portion of chick embryo sternum. Both articular and sternal chondrocytes readily proliferated and progressively increased in size with time in culture. Many had undergone hypertrophy by 4-5 weeks. Analysis of medium-released collagenous proteins revealed that both articular and sternal chondrocytes initiated type X collagen synthesis between 3 and 4 weeks of culture; synthesis of this macromolecule increased with further growth. Immunofluorescence analysis of 5-week-old cultures showed that about 15% of articular chondrocytes and 30% of sternal chondrocytes produced type X collagen; strikingly, there appeared to be no obvious relationship between type X collagen production and cell size. The results of this study show that articular chondrocytes from adult chicken tibia possess the ability to express traits associated with endochondral ossification when exposed to a permissive environment. They suggest also that the process of cell hypertrophy and initiation of type X collagen synthesis are independently regulated both in articular and sternal chondrocytes.
Several methods have been described for investigating chondrocyte metabolism in vitro. In this study, in-situ hybridization (ISH) using an oligonucleotide probe (i.e. a poly-d(T) probe) to detect total messenger RNA (mRNA) in cartilage explants has been compared with radiosulphate and radioleucine uptake studies in an attempt to assess the value of ISH in investigating chondrocyte metabolism. The relative results of the three parameters indicate qualitative similarities in cells in the intermediate, deep and calcified zones but differences in the superficial zone. The relative levels of mRNA and leucine and sulphate uptake in the midzone areas could be construed as indicating that the bulk of cellular activity was directed towards the synthesis of proteoglycans. A similar relation between the three parameters, but at a lower level, was seen in chondrocytes in the calcified zone demonstrating that these cells are viable and biosynthetic. Both quantitative and qualitative differences between the three methods were observed in the superficial chondrocytes regarding the amount of mRNA compared to sulphate and leucine uptake. The results suggest that ISH can detect differences in the amount of mRNA present in chondrocytes in differing zones of cartilage and, like the radioleucine and radiosulphate studies, particularly emphasizes their functional heterogeneity.
Communication among individual cell types that populate connective tissues such as cartilage or bone is of critical importance in determining the phenotypic properties of these tissues under both physiologic and pathologic conditions. Cytokines, which may be defined as soluble products released from one cell that can modulate the activity of other cells, play a critical role in this process of cell communication. The introduction of molecular biologic techniques has permitted identification of specific cytokines previously characterized on the basis of biologic activities. Cloning and sequencing of these products have provided formal evidence for their existence and allowed identification of the full spectrum of their biologic activities. These results have established that individual cytokines may have multiple biologic activities and that multiple cytokines share common functional properties. Based on these results, the term "cytokine" has been used more generally to include products originally described as growth or differentiation factors, e.g., interleukins, monokines, or lymphokines. Cytokines have an important role in the initiation and control of skeletal tissue growth and development and in regulating bone remodeling in the adult organism. As in other connective tissues, these effects are mediated via paracrine, autocrine, and endocrine mechanisms. In skeletal tissues, cytokines may modulate the activity of resident cells by an additional mechanism. Factors produced locally within bone or arriving via the circulation are incorporated into the mineralized bone matrix, and their release during skeletal remodeling could provide the basis for coupling the activity of bone resorbing and forming cells. The principal cytokines that have been shown to affect skeletal tissues include factors previously described as monokines or lymphokines such as interleukin-1 (IL-1), tumor necrosis factors (TNF-alpha and TNF-beta), and interferon-gamma (IFN-gamma); the colony-stimulating factors; and the so-called growth and differentiation factors including transforming growth factors-alpha and -beta (TGF-alpha and TGF-beta), insulinlike growth factor-I (IGF-I), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF). Although the effects of the individual cytokines are diverse, it is possible to classify individual factors based on their effects on specific aspects of bone formation or resorption. Significant progress has been made recently toward elucidating the mechanisms of action of the cytokines. Binding studies using radiolabeled ligands have characterized the specific cell surface receptors and defined their distribution and properties among skeletal tissue cells. Various so-called signal transduction pathways have been implicated in mediating these effects...
Chondrocytes express a well-characterized set of marker proteins making these cells useful for studies on differentiation and regulation of gene expression. Because of the inherent instability of primary rat chondrocytes in culture, and because several rat chondrocyte genes have been cloned and characterized (including the collagen II promoter and enhancer), a rat chondrocyte cell line would be especially useful. To obtain this line we infected primary fetal rat costal chondrocytes with a recombinant retrovirus (NIH/J-2) carrying the myc and raf oncogenes, which have been shown to have an "immortalizing" function. Following infection, a rapidly proliferating clonal line was isolated that maintained a stable phenotype through 45 passages (11/2 year in culture). This line, termed IRC, grows in suspension culture as multicellular aggregates and in monolayer culture as polygonal cells which accumulate an alcian blue-stainable matrix. IRC cells synthesize high levels of cartilage proteoglycan core protein, and link protein, but show reduced collagen II expression. In addition, the cells express virally derived myc mRNA and protein, but do not express v-raf. Retinoic acid, which is a known modulator of chondrocyte phenotype, down-regulates expression of chondrocyte marker proteins, while stimulating v-myc expression by IRC cells. These data suggest that v-myc expression by chondrocytes results in rapid cell division and maintenance of many aspects of the differentiated phenotype. These "immortalized" cells, however, remain responsive to agents such as retinoic acid which modulate cell phenotype. The potential exists for development of chondrocyte cell lines from diseased cartilage, as well as from human cartilage.
The dimethylmethylene blue assay for sulphated glycosaminoglycans has found wide acceptance as a quick and simple method of measuring the sulphated glycosaminoglycan content of tissues and fluids. The available assay methods have lacked specificity for sulphated glycosaminoglycans in the presence of other polyanions, however, and have not discriminated between the different sulphated glycosaminoglycans. We now describe a modified form of the dimethylmethylene blue assay that has improved specificity for sulphated glycosaminoglycans, and we show that in conjunction with specific polysaccharidases, the dimethylmethylene blue assay can be used to quantitate individual sulphated glycosaminoglycans.
Sub-populations of bovine articular chondrocytes derived from different depths of the cartilage showed differences in accumulation of proteoglycan-rich extracellular matrix in culture. To extend these morphological studies, the synthesis and catabolism of 35S-labeled proteoglycans have been examined in similar cultures. Chondrocytes from deep zones synthesized significantly more proteoglycans than cells from the superficial zone. While all populations of chondrocytes synthesized predominantly aggregating proteoglycans, a higher proportion of isotope was present in non-aggregating proteoglycans in cultures of superficial chondrocytes, by comparison with those of deep cells. Proteoglycans were degraded more rapidly by superficial cells than by chondrocytes from deeper layers. These results correlate both with previous histochemical studies of similar cultures, and with known depth-related variations in biochemical composition of intact articular cartilage.
Bovine articular chondrocytes cultured in agarose gel comprise a heterogeneous population when judged by morphological and histochemical criteria. The purpose of the present experiments was to compare, under the same conditions of culture, sub-populations of chondrocytes derived from different depths of articular cartilage. Sub-populations of chondrocytes were cultured separately following their isolation from slices of articular cartilage cut from successive depths of the tissue. Chondrocytes derived from superficial and deep zones differed significantly in morphology, rate of proliferation, and activity in secreting a proteoglycan-rich extracellular matrix. The differences are sufficient to account for the heterogeneity observed in cultures of the entire cell population, and the correlate well with known variations with depth in morphology and histochemistry of intact articular cartilage. These results demonstrate that articular chondrocytes continue in culture to express metabolic differences which reflect their original anatomical location; such differences may have important functional significance.
Electron microscopy, electron diffraction, and high-spatial-resolution electron probe X-ray microanalysis have been used to characterize certain ultrastructural components observed in undecalcified, unfixed, and unstained thin sections of epiphyseal growth plate cartilage from tibiae of normal 4-week-old rats. The tissue was prepared anhydrously by dry ultracryomicrotomy. Electron microscopy revealed electron-dense mitochondrial granules (500–1000 Å in diameter) within chondrocytes of proliferative and hypertrophic cartilage zones. The mitochondrial granules in frozen thin sections generated no distinct reflections on electron diffraction. X-ray spectra from individual granules showed both calcium and phosphorus with a molar Ca/P ∼0.8–1.1. There was no apparent decrease in the number of mitochondrial granules as extracellular mineralization increased toward distal portions of the growth plate. Electron microscopy of the extracellular regions of proliferative and hypertrophic cartilage zones showed matrix vesicles (1000–2500 Å in diameter) and small, electron-dense particles (200–800 Å in diameter). No mineral particles within or associated with the membrane-bound matrix vesicles could be observed by electron microscopy in any region of the growth plate examined, and only phosphorus could be detected in individual vesicles on X-ray microanalysis. The small, electron-dense particles were not membrane limited; they were distributed in a heterodispersed fashion and found in the same cartilage regions in which matrix vesicles were present, as well as in regions proximal to those in which matrix vesicles occurred. These small, heterodispersed structures were not apparent in sections of rat cartilage fixed and stained by aqueous methods. The electron-dense, heterodispersed particles in frozen sections produced no coherent pattern on electron diffraction and generated calcium and phosphorus with a molar Ca/P ∼0.9–1.0 following X-ray microanalysis. The electron optical and analytical data are consistent with the conclusion that the small heterodispersed particles represent the first solid phase of calcium phosphate deposited in the extracellular matrix of rat epiphyseal growth plate cartilage. When the same tissue was examined after preparation by conventional aqueous techniques, matrix vesicles were observed containing distinct and relatively large crystals characterized by weak calcium and phosphorus on electron probe microanalysis. The comparative results obtained from completely unfixed tissue and from tissue exposed to aqueous solvents raise serious doubts as to whether matrix vesicles contain a solid phase of calcium phosphate in vivo and consequently question the proposed role of the calcification of matrix vesicles in tissue mineralization.
For several decades simian virus 40 (SV40) early region genes have been used as a means of generating immortalized human cell lines; however, the molecular mechanisms of this process have begun to be understood only recently. SV40-induced immortalization proceeds via two phases. In the first phase ("lifespan extension"), cells continue proliferating for a limited number of population doublings beyond the point at which normal cells undergo senescence. This is mainly due to the ability of SV40 large T antigen (LTAg) to bind to the protein products of the p53 and retinoblastoma (Rb) genes. The second phase ("immortalization") occurs in only a small minority of cells, and cell hybridization analyses indicate that this is a gene inactivation event. The gene or genes involved are currently unknown, but chromosomal localization data are accumulating which should make their cloning and characterization possible in the near future.
Data obtained while investigating growth plate chondrocyte differentiation during endochondral bone formation both in vivo and in vitro indicate that initial chondrogenesis depends on positional signaling mediated by selected homeobox-containing genes and soluble mediators. Continuation of the process strongly relies on interactions of the differentiating cells with the microenvironment, that is, other cells and extracellular matrix. Production of and response to different hormones and growth factors are observed at all times and autocrine and paracrine cell stimulations are key elements of the process. Particularly relevant is the role of the TGF-beta superfamily, and more specifically of the BMP subfamily. Other factors include retinoids, FGFs, GH, and IGFs, and perhaps transferrin. The influence of local microenvironment might also offer an acceptable settlement to the debate about whether hypertrophic chondrocytes convert to bone cells and live, or remain chondrocytes and die. We suggest that the ultimate fate of hypertrophic chondrocytes may be different at different microanatomical sites.
To understand the growth, maturation, and regulation of growth plate chondrocytes, it is necessary to isolate the different chondrocytes into distinct subpopulations of maturational development. Five subpopulations (A-E) of bovine fetal growth plate chondrocytes were separated by discontinuous gradient centrifugation. Four subpopulations (B, C, D, and E, from low to high density) with good viability were cultured at high density in microwells for up to 30 days. They all established an extensive extracellular matrix composed of proteoglycan and collagen. The largest and last dense cells in subpopulation B were the first to synthesize (at days 5-6) type X collagen and to calcify this matrix. Matrix calcification (formation of hydroxyapatite in the presence of sodium beta-glycerophosphate) always followed the initiation of type X synthesis. All the other subpopulations synthesized type X collagen and calcified their extracellular matrix. Although these events occurred in the same order, they were delayed according to the order of increasing cell size. These observations indicate that these subpopulations represent different stages in cellular maturation that lead to expression of the hypertrophic phenotype. Once mineral formation was well established, there was an increase in the matrix content of the C-propeptide of type II collagen (which is known to bind to hydroxyapatite and accumulate in calcifying extracellular matrix). This was accompanied by a reduction in the total collagen content, which accompanied an abrupt reduction in type X collagen synthesis, whereas type II collagen synthesis was largely maintained. These reductions in collagen content and type II collagen synthesis were not observed in the absence of calcification (beta-glycerophosphate omitted from culture). This new culture system recreates many of the sequential cellular and extracellular changes exhibited in situ during the development of the physis and provides new information about cellular and extracellular matrix changes that occur before and at the time of calcification.
We have used the three-dimensional culture system in alginate beads to redifferentiate human articular chondrocytes which were first expanded on a plastic support. After 15 days in alginate beads, electron microscopy showed that cells had synthesized an extracellular matrix containing collagen fibrils. Electrophoretic analysis of proline-labeled cells demonstrated that redifferentiated chondrocytes synthesized mainly type II collagen and its precursors (pro alpha 1II, pc alpha 1II, and pn alpha 1II). After pepsin digestion a small amount of collagen type XI was also detected. These results were confirmed by Northern blot analysis of total RNAs. Hybridization with collagen cDNA probes coding for the alpha 1(II) and alpha 1(I) chains of collagen types II and I showed that chondrocytes cultured in alginate expressed mainly alpha 1(II) mRNA, whereas alpha 1(I) mRNA transcripts were almost undetectable. Such a result was observed even after several passages on plastic flasks, suggesting that dedifferentiated cells were able to revert to a chondrocytic phenotype in this three-dimensional system. However, SV40-transformed chondrocytes were not able to redifferentiate in alginate as no alpha 1(II) mRNAs were detected. Total RNA was converted into cDNA by reverse transcription and amplified by polymerase chain reaction. This technique was employed to amplify mRNAs specific for collagen type II and type X and the large aggregating proteoglycan aggrecan. Two transcripts resulting from an alternative splicing of the complement regulatory protein (CRP)-like domain of aggrecan were originally identified in chondrocytes in monolayers. Like intact cartilage, chondrocytes in alginate expressed only the larger transcript with the CRP domain, whereas the two transcripts were equally expressed in SV40-transformed chondrocytes. Thus, the alginate system appears to represent a relevant model for the redifferentiation of human chondrocytes, especially when only a small cartilage biopsy is available, and could prove useful for pulse-chase studies of patients with skeletal chondrodysplasias. However it was unable to restore the chondrocytic phenotype in virally transformed cells.
A panel of eight conditionally immortal lines derived by infection of human breast epithelial cells with an amphotropic retrovirus transducing a ts mutant of SV40 large T-antigen was analyzed with respect to individual retroviral integration patterns. Each line contained multiple integration sites which were clonal and stable over extended passage. Similar integration patterns were observed between individual lines arising separately from the same stock of pre-immortal cells, suggesting a common progenitor. Retroviral integration analysis of pre-immortal cells at different stages of pre-crisis growth showed changes indicative of a progressive transition from polyclonality to clonality as the cells approached crisis. Each of the immortal lines contained a sub-set of the integration sites of their pre-immortal progenitors, with individual combinations and copy numbers of sites. Since all the cell lines appeared to originate from single foci in separate flasks, it is likely that each set arose from a common clone of pre-immortal cells as the result of separate genetic events. There was no evidence from this analysis to suggest that specific integration sites played any part either in the selection of pre-crisis clones or in the subsequent establishment of immortal lines.
Chondrocyte cultures grown in centrifuge tubes with intermittent centrifugation differentiate into hypertrophic chondrocytes and form calcification. We examined chondrocytes cultured in this system electron microscopically. Rat growth-plate chondrocytes were seeded in a plastic centrifuge tube and cultured in the presence of Eagle's minimum essential medium supplemented with 10% fetal bovine serum and 50 micrograms of ascorbic acid per ml. Specimens were examined by using electron microscopy and selected-area electron-diffraction techniques. In the early stage of culture, a few chondrocytes were scattered and extracellular matrices were not observed. In the middle stage of the cultures, the chondrocytes resembled proliferative cells. Matrix vesicles appeared to be budding from the cell surfaces of chondrocytes and were observed sparsely in the extracellular matrices, which were well formed around the chondrocytes. Matrix vesicles increased substantially during the following cultures. In the mature stage of the cultures, crystal formation related to matrix vesicles was observed. In the 33-day cultures, several masses of calcified matrix were formed and it was confirmed to be apatite by selected-area electron diffraction analysis. The chondrocytes appeared hypertrophic during this same stage. The 56-day culture was similar to the 33-day culture. It was concluded that this culture system provides an extracellular-matrix mineralization which is produced by chondrocytes per se.
Chondrocytes are specialised cells which produce and maintain the extracellular matrix of cartilage, a tissue that is resilient and pliant. In vivo, it has to withstand very high compressive loads, and that is explicable in terms of the physico-chemical properties of cartilage-specific macromolecules and with the movement of water and ions within the matrix. The functions of the cartilage-specific collagens, aggrecan (a hydrophilic proteoglycan) and hyaluronan are discussed within this context. The structures of cartilage collagens and proteoglycans and their genes are known and a number of informative mutations have been identified. In particular, collagen fibrillogenesis is a complex process which can be altered by mutations whose effects fit what is known about collagen molecular structural functions. In other instances, mutations have indicated new functions for particular molecular domains. As cartilage provides the template for the developing skeleton, mutations in genes for cartilage-specific proteins often produce developmental abnormalities. The search for mutations amongst such genes in heritable disorders is being actively pursued by many groups, although mutation and phenotype are not always well correlated, probably because of compensatory mechanisms. The special nature of the chondrocyte is stressed in connection with its cell involvement in osteoarthritis, the most widespread disease of diarthrodial joints.
Many osteoblastic cell lines are currently in use, but these have limitations either in terms of their relevance to adult human biology and disease or in terms of their suitability for biochemical and molecular analyses. Consequently, we undertook the development of conditionally transformed adult human osteoblastic cell lines. Osteoblasts were obtained from a normal explant cancellous bone chip culture. These cells were infected with adenovirus-ori-SV40 tsA 209, which encodes a temperature-sensitive large T-antigen mutant. Cells immortalized with this virus express a transformed phenotype at the permissive temperature of 34 degrees C but revert to a normal phenotype at the nonpermissive temperature of 40 degrees C. Using this approach, we have isolated several cell clones and describe the characterization of one that was designated HOB-02-C1. Immunocytochemistry revealed that > 95% of the cells express the large T-antigen at both temperatures. These cells exponentially proliferate at 34 degrees C with a doubling time of approximately 2 days but irreversibly stop dividing at 40 degrees C. However, cell volume increases > 2-fold when the cells are maintained for 6 days at the higher temperature. This clone expresses alpha 1 type (I) procollagen mRNA and secretes type I procollagen C-peptide at both temperatures, although the levels were slightly elevated at 40 degrees C. The cell line expresses alkaline phosphatase activity at 34 degrees C, and the basal level of this enzyme increases 2- to 6-fold at 40 degrees C. Alkaline phosphatase activity is induced 4- to 8-fold by 1 alpha,25-dihydroxyvitamin D3 (vitamin D3) at both temperatures, but transforming growth factor-beta 1 (TGF-beta 1) suppresses enzyme expression > 90% at 40 degrees C. Vitamin D3 also induces a 10-fold increase in osteocalcin secretion when the clone is maintained at 34 degrees C, and this induction is enhanced > 8-fold at 40 degrees C. Parathyroid hormone and forskolin stimulate a 4- to 6-fold increase in the production of intracellular cyclic AMP (cAMP) by the cells at 34 degrees C, and this stimulation is enhanced 2- to 4-fold at 40 degrees C. In contrast, prostaglandin E2 stimulates a 7- to 8-fold increase in cAMP only when the cells are maintained at 34 degrees C. This cell line secretes TGF-beta 1 and interleukin-6 (IL-6) at 34 degrees C, but only the basal secretion of IL-6 increases 70% at 40 degrees C. Finally, alizarin red-S histochemical staining demonstrates that these cells produce mineralized nodules at both temperatures. In summary, the results of this study indicate that the HOB-02-C1 cells have a mature osteoblastic phenotype. Consequently, this new cell line and others obtained in a similar fashion should be valuable in vitro tools for cellular, biochemical, and molecular studies of adult human osteoblast biology.