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. 2000;20:11–16.

Transforming Growth Factor Beta One (TGF-β1) Enhancement of the Chondrocytic Phenotype in Aged Perichondrial Cells

An In Vitro Study

Myung C Lee *,*, Randal S Goomer *,*, Kenji Takahashi *,*, Frederick L Harwood *,*, Michael Amiel *,*, David Amiel *,*,✉
PMCID: PMC1888754  PMID: 10934619

Abstract

Background

Perichondrium is recognized as a tissue with chondrogenic potential yielding cells which can be used for osteochondral repair. Factors which influence the proliferative ability and chondrocytic phenotype of such cells include age and presence of specific growth factors, i.e. TGF-β1. The present in vitro study assessed proliferation and markers of chondrocytic phenotype in cells extracted from the rib perichondrium of four- to five-year-old aged rabbits, and assessed the effects of exogenously added TGF-β1 on those cells.

Methods

Assays included 3H-thymidine incorporation (cell proliferation), 35S-sulfate incorporation (proteoglycan synthesis) and quantitative RT-PCR for determination of type II collagen gene expression.

Results

The results demonstrated that addition of TGF-β1 to the culture media stimulated thymidine incorporation and proteoglycan synthesis up to fourand five-fold, respectively, in aged perichondrium-derived cells. Moreover, the exogenous addition of TGF-β1 to the culture media resulted in an up-regulation of transcriptional expression of the type II collagen gene.

Conclusions

In summary, the present study has demonstrated that exogenously added TGF-β1 can stimulate proliferation and chondrocytic phenotype in aged perichondrium-derived cells in vitro.

INTRODUCTION

The repair of articular cartilage injuries remains a challenge, with several current therapeutic modalities based on the grafting of chondrocytic tissues1,27 or the implantation of cells capable of generating a cartilage-like matrix.5,33 One such tissue, perichondrium, is recognized as having chondrogenic potential14,19,28,32 and one from which cells with a chondrocytic phenotype can be isolated for implantation into osteochondral defects.7–9 Among the factors that may influence the success of repair models utilizing chondrocytic cell transplantation is the age of the donor tissues. Specifically, parameters important to chondrocytic phenotype that are influenced by age include rate of cell proliferation, rate of proteoglycan synthesis and type II collagen gene expression. 10,25,26 These parameters are also known to be affected by certain members of the growth factor family of regulatory proteins. In particular, TGF-β1 has been reported to influence the proliferation of a variety of cell types including articular chondrocytes,16,17,30 osteoblasts12 and periosteum-derived cells.11,18 TGF-β1 has also been shown to stimulate proteoglycan synthesis,23,24,30,31 induce type II collagen production in chondroblasts31 and periosteum-derived cells,3,4,21 and stabilize the chondrocytic phenotype in such cells.3

In a previous study, we demonstrated that markers of the chondrocytic phenotype, i.e. proteoglycan synthesis and type II collagen gene expression, were enhanced by the addition of TGF-β1 to explant cultures of mature perichondrium-derived cells.13 The primary goals of the present in vitro study were to assess proliferation, proteoglycan synthesis and type II collagen gene expression in cells extracted from aged rabbit perichondrium, and to determine if those parameters may be susceptible to modulation by the addition of TGF-β1.

MATERIALS AND METHODS

Cell Preparation and Culture System

Six aged rabbits four to five years old were sacrificed according to the animal subjects protocols at UCSD and used for preparation of six individual primary perichondrium cell cultures. Using sterile procedures the costal ribs were dissected, adhering tissue was removed and the perichondrium layer was isolated. The perichondrium was then washed three times in sterile buffered salt solution containing antibiotics and incubated overnight in 0.1% collagenase (CLS-2, Worthington Biochemical, Freehold, NJ) solution at 37° C under 5.0% CO2. The collagenase digest was passed through a sterile 0.45 micron filter and the remaining cells and tissue debris were further digested with 0.1 µg/ml hyaluronidase (Sigma) and trypsin (Irvine Scientific) for two hours. This digest was then passed through an 80 micron filter to isolate the cells. Primary cell cultures were established on 100 ml tissue culture plates by incubation in α-MEM (α-modified Earl's medium) containing 10% FBS and antibiotics at 37°C under 5% CO2. Culture media was changed every two to three days and the cultures were maintained until 100% confluency was achieved. The cells were then trypsinized and counted prior to passaging onto 6 - well (35 mm) plates at 0.5 x 105 cells per well.

3H-Thymidine Incorporation Assay for Cell Proliferation

Passaged cells were kept in α-MEM supplemented with 10% FBS and antibiotics for 24 hours. After this period the medium containing 10% FBS was removed and replaced with medium containing 0.5% FBS. After a 12 hour incubation, TGF-β1 was added to separate cultures at concentrations of 0.1, 0.5, 1.0, 5.0 and 10.0 ng/ml (n=6 for each TGF-β1 concentration) and incubation incubation was continued for 48 hours. Cell cultures containing only 0.5% FBS served as untreated controls. The cultures were then pulsed with 3H-thymidine (2 μCi/ml of media) and incubated for 12 hours. The medium was then removed and the plates were washed twice with cold sterile phosphate buffered saline (PBS) before the cells were released by digestion with trypsin-EDTA. After centrifugation, the cell pellets were washed twice more with cold PBS to remove any remaining unincorporated tritium. The cell pellets from individual wells were lysed by adding 1.1 ml of 1 N NaOH and incubating at 60°C for two hours. One hundred μl of cell lysates were then added to 10 ml of scintillation cocktail (Fisher) and neutralized by addition of 20μl of 6N HCl. Radioactivity was counted in a model 6800 liquid scintillation spectrometer (Beckman Instruments). The remaining one ml of cell lysate from each well was used for DNA measurement as described by Amiel et al.2 After trypsinization, the numbers of viable and dead cells were counted manually with a hemocytometer using trypan blue staining. There were no differences in cell viability among the different wells of different groups; therefore, thymidine incorporation was normalized to DNA content in each well. The relative cell proliferation (3H-thymidine incorporation) rates are thus expressed as cpm/μg of total cellular DNA.

35S-Sulfate Incorporation Assay for Proteoglycan Synthesis

Passaged cells (0.5 x 105 cells per well in 6-well plates) were kept in α-MEM supplemented with 10% FBS and antibiotics for 24 hours at 37°C and then treated with TGF-β1 at concentrations of 0.1, 0.5, 1.0, 5.0 ng/ml (n=6 for each TGF-β1 concentration). After four days the cells were made quiescent by replacing 10% FBS with 0.5% FBS and incubating for 24 hours. 35S-sulfate (20 μCi /ml of media) was then added to the cell cultures and incubation was continued for four hours. The medium was then removed and the plates were washed twice with cold sterile phosphate buffered saline (PBS) before the cells were released by digestion with trypsin-EDTA. After centrifugation the cell pellets were washed twice with cold PBS to remove unincorporated 35S-sulfate. The cell pellets from individual wells were lysed by adding 1.1 ml of 1 N NaOH and incubating at 60°C for two hours. One hundred μl of cell lysates were then taken for quantitation of radioactivity by liquid scintillation spectrometry. The remaining one ml of cell lysates from each well was used for DNA measurement. The relative rates of proteoglycan synthesis (35S-sulfate incorporation) are expressed as cpm per μg of total cellular DNA.

Collagen Gene Expression By Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Passaged cells were plated at 4 x 105 cells per 100 mm diameter tissue culture plate. After 24 hours of incubation at 37°C, the cells were treated with TGF-β1 at concentrations of 0.1, 0.5, 1.0, 5.0 ng/ml (n=6 for each TGF-β1 concentration). After four days of incubation, 10% FBS was removed and replaced with 0.5% FBS and the cells were allowed to incubate for another 24 hours. Total cellular RNA was then extracted using the acid guanidinium-thiocyanate-phenol-chloroform extraction procedure.6 To ensure that PCR amplification of contaminating genomic DNA would not affect the data, total extracted RNA was treated with RNase free DNase (RQ1, Promega, Madison WI). Five micrograms of total RNA were converted to cDNA using oligo(dT) and MMLV reverse transcriptase (Promega, Madison, WI). First strand cDNA for type II collagen was then amplified by polymerase chain reaction. In order to monitor the quality of the RNA preparation and to normalize the reverse transcription RT-PCR protocol, the glyceraldehyde-6-phosphate dehydrogenase (GAPDH) housekeeping gene was also subjected to RT-PCR in all extracts. Ten microliters of the reverse transcription solution was then taken for PCR in a total reaction volume of 50 μl: 5 μl of a 10x Mg-free PCR buffer, 2.5 mM MgCl2, 5 units taq DNA polymerase (Promega), and 20 picomoles of each primer. PCR primers specific to selected coding regions of GAPDH (5'-TCCATGCCATCACTGCCA-3' & 5'-CATACCAGGAAATGAGCT-3') and type II collagen (5'-GACCCCATGCAGTACATG-3' & 5'-GACGGTCTTGCCCCACTT-3') were constructed (Retrogen, San Diego CA) based on the published sequences for those genes (36). PCR was performed using a DNA thermal cycler (GeneAmp PCR System 2400, Perkin Elmer, Norwalk, CT). For GAPDH and type II collagen transcripts a cycle profile consisted of 25 sec at 94°C for denaturation, 30 seconds at 58°C for annealing and 30 seconds at 72°C for extension. Following electrophoresis on 1% agarose gels containing ethidium bromide for visualization, photo-documentation and the Image Tool Analysis Program (NIH image version 1.6, Natl Inst of Health USA, http://rsb.info.nih.gov/nih-image/) were used to quantitate PCR products. PCR was performed for 12-32 cycles at two-cycle intervals in order to determine the linear range of PCR amplification. The midpoint of linear amplification, 20 cycles, was calculated from a linear regression curve of log mean density vs. cycle number. This number of cycles was subsequently used for all PCR reactions. Integrated band density values for the type II collagen gene were then normalized to GAPDH. All treatment groups of passaged cells (n=6 for each concentration of TGF-β1 + 0.5% FBS control) were analyzed in triplicate for a total of 18 RT-PCRs for each group. Absolute numbers of type II collagen gene transcripts were determined as previously described15,29 from a standard PCR curve derived from a 20-cycle amplification of serially diluted solutions of a plasmid (pGEM T-vector) containing the type II collagen PCR insert. Results are expressed as number of type II collagen transcripts per microgram of total RNA.

Statistical Analysis

Statistical significance was determined by analysis of variance (ANOVA) and all data are presented as mean ± standard deviation.

RESULTS

Effect of TGF-β1 on Thymidine Incorporation

The results of experiments exploring the effect of TGF-β1 on 3H-thymidine incorporation by aged perichondrium cells are illustrated in Figure 1. Treatment of aged cells with TGF-β1 caused a significant (p < 0.05) increase in 3H-thymidine incorporation over the entire range of growth factor concentrations applied, i.e. 0.1 - 10.0 ng TGF-β1 per ml of tissue culture media. TGF-β1 stimulation reached a peak at a concentration of 0.5 ng/ml where the incorporation rate was approximately four times greater than in untreated cells; an apparent plateau occured between a concentration of 5.0 and 10.0 ng/ml.

Figure 1.

Figure 1

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The effect of TGF-β1 on 3H-thymidine incorporation in cultured aged perichondrium - derived cells. Each column represents the mean ± standard deviation of six cultures. Asterisk (*) refers to statistical significance of p < 0.05 relative to the 0.0 ng/ml TGF-β1 control.

Effect of TGF-β1 on Proteoglycan Synthesis

Figure 2 shows the relative rates of in vitro proteoglycan synthesis (35S-sulfate incorporation) in aged perichondrium cells treated with TGF-β1. This figure shows that, relative to untreated controls, 35S-sulfate incorporation into aged cells was stimulated by addition of TGF-β1 to the culture media. This effect was significant (p < 0.05) over the entire range of growth factor concentrations tested. The greatest 35S-sulfate incorporation was achieved with the addition of 5.0 ng/ml of TGF-β1

Figure 2.

Figure 2

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The effect of TGF-β1 on 35S-sulfate incorporation in cultured aged perichondrium - derived cells. Each column represents the mean ± standard deviation of six cultures. Asterisk (*) refers to statistical significance of p < 0.05 relative to the 0.0 ng/ml TGF-β1 control.

Effect of TGF-β1 on Type II Collagen Gene Expression

PCR products for GAPDH were uniformly expressed in all groups (Figure 3). A typical result for RT-PCR of type II collagen mRNA is shown in the top panel of Figure 3; this figure clearly shows that the type II collagen band intensity was increased in aged perichondrium cells after treatment with TGF-β1. Type II collagen-specific transcripts were quantitated as number of type II collagen transcripts per μg of total RNA, and the results are illustrated in Figure 4. Relative to untreated controls (0.5% FBS), the level of transcription of the type II collagen gene in aged perichondrium-derived cells was significantly (p < 0.01) up-regulated with the addition of TGF-β1 to the culture media at concentrations ranging from 0.5 to 5.0 ng/ml. The greatest effect of the growth factor was observed at a concentration of 5.0 ng/ml.

Figure 3.

Figure 3

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Representative result for RT-PCR of RNA extracted from TGF-B1 treated cultured aged perichondrium - derived cells. Top: Type II collagen; Bottom: GAPDH.

Figure 4.

Figure 4

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Results of quantitative image analysis following RT-PCR of type II collagen mRNA after extraction of total RNA from TGF-β1 treated cultured aged perichondrium - derived cells. Each column represents the mean ± standard deviation of six cultures. Asterisk (*) refers to a statistical significance of p < 0.05 relative to the 0.0 ng/ml TGF-β1 control.

DISCUSSION

The present in vitro study has demonstrated that addition of TGF-β1 to cultured aged perichondrium-derived cells can stimulate cell proliferation and enhance the chondrocytic phenotype, i.e. proteoglycan synthesis and type II collagen gene expression. To our knowledge this is the first study to report such effect of a specific growth factor, i.e. TGF-β1 on aged perichondrial cells. Several studies have reported on age-related changes in proliferative ability and phenotypic expression in various cell types derived from such tissues as bone marrow and periosteum. For instance, O'Driscoll26 reported an age-related decline in chondrocytic markers in periosteum-derived cultured cells and suggested that such decline might be caused by a reduction in the pool of chondrocyte precursor cells. In a similar study, Nakahara25 showed that that periosteal cells with osteochondrocytic potential could be liberated from human donors up to the age of 22 years, but that the population of such cells decreased thereafter. With regard to the osteochondrocytic potential of human bone marrow cells, it has also been suggested that there is a difference between young and aged human donors in terms of the ability of such cells to undergo terminal osteochondral differentiation29 and in an in vivo study, Cohen and Lacroix reported that the osteochondrocytic potential of autogenous periosteal grafts in rabbits varied considerably depending on age.10

In a previous in vitro study carried out in our laboratory13 we reported that exogenous addition of TGF-β1 to media containing 0.5% fetal bovine serum resulted in a marked increase of tritiated thymidine uptake by mature (eight to ten months old) perichondrium-derived cells with optimum proliferative effects at 0.1 ng/ml. The present study showed that thymidine incorporation in aged perichondrium-derived cells was similarly increased by TGF-β1 over a wide range of growth factor concentrations, i.e. 0.1 to 10.0 ng/ml of media. This study also showed that proteoglycan synthesis (35S-sulfate incorporation) in aged perichondrium-derived cells was stimulated by TGF-β1 over a similar concentration range, while phenotypic expression of the chondrocytic collagen phenotype, i.e. type II collagen, was dramatically increased in aged cells treated with TGF-β1. The exact mechanisms of such effects remain to be elucidated, but since growth factor activities are mediated through their respective receptors, it is likely that those receptors undergo age related changes in number and/or responsiveness to growth factors. Further studies to explore the relationships between age and growth factor effect and mediator responsiveness would be appropriate.

Balance between cell proliferation and chondrocytic potential is important with regard to utilizing TGF-β1 in clinically related studies such as bioengineered cartilage repair. In this regard, TGF-β1 has been demonstrated to exhibit duality in its effects on chondrogenicity. It has been reported, for instance, that TGF-β1 can stimulate synthesis of cartilage-specific type II collagen in rat mesenchymal cells,22,31 while, at relatively higher concentrations, this growth factor inhibited synthesis of type II collagen in vitro in ar ticular chondrocytes.20 In the present study, 35S-sulfate incorporation and type II collagen gene expression were increased over a concentration range of 0.1 to 5.0 ng/ml of TGF-β1. This concentration range was also effective for increasing cell proliferation rates.

In conclusion, the present study has demonstrated that exogenously added TGF-β1 had a strong morphogenic effect and stimulated the chondrocytic phenotype in aged perichondrium-derived cells in vitro. These results are encouraging with regard to enhancing the repair of osteochondral defects in the aged using autologous transplantation of aged perichondrium-derived cells.

ACKNOWLEDGEMENT

This research was supported by NIH grants AG07996, AR28467, and the Orthopaedic Research and Education Foundation (OREF) Bristol Myers Awards.

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