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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62:136-148 (2007)
© 2007 The Gerontological Society of America

Impact of Aging on Rat Bone Marrow-Derived Stem Cell Chondrogenesis

Hongjun Zheng, James A. Martin, Yazan Duwayri, Gilbert Falcon and Joseph A. Buckwalter

1 University of Iowa Department of Orthopaedics and Rehabilitation, Iowa City.
2 Department of Surgery, Medical University of Ohio, Toledo.
3 North Dakota College of Medicine and Health Sciences, Grand Forks.

Address correspondence to Joseph A. Buckwalter, MD, 01008 Pappajohn Pavilion, Department of Orthopaedics, University of Iowa College of Medicine, Iowa City, IA 52242. E-mail: joseph-buckwalter{at}uiowa.edu


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Damaged articular cartilage rarely heals or regenerates in middle-aged and elderly adults, suggesting that the chondrogenic potential of mesenchymal stem cells declines with age. To test this hypothesis, we measured the responses of rat bone marrow-derived mesenchymal stem cells (BMSCs) to chondrogenic induction in vitro. BMSCs from immature rats (1 week old), young adult rats (12 weeks old), and old adult rats (1 year old) were analyzed for cartilage extracellular matrix (ECM) production. Histologic analysis showed strong cartilage ECM formation by BMSCs from 1-week-old rats, but not by BMSCs from 12-week-old or 1-year-old rats. Real-time polymerase chain reaction revealed age-related declines in messenger RNA encoding type II collagen, aggrecan, and link protein, three major cartilage ECM components. Microarray analysis indicated significant age-related differences in the expression of genes that influence cartilage ECM formation. These findings support the hypothesis that the chondrogenic potential of mesenchymal stem cells declines with age.

POSTTRAUMATIC osteoarthritis (PTOA) commonly develops rapidly after fractures that disrupt the articular surface (1–3). Despite aggressive surgical intervention, more than 25% of patients with fractures of the hip and knee joints develop PTOA, and the rate increases to 50% or more of patients with ankle fractures (4–7). Osteoarthritis with extensive cartilage degeneration and joint space narrowing is usually evident on radiologic examination within 2–5 years after fracture (6,7). Clinical experience and epidemiologic studies indicate that the incidence of PTOA after joint fracture is strongly dependent on patient age (8–10). Adults in their 20s and 30s are significantly less likely to develop PTOA in injured joints than are adults older than 50 years (8). The obvious clinical goal of improving recovery rates in older patients awaits a better understanding of the biology underlying this strong aging effect.

Recovery from serious articular injuries involves cartilage regeneration. Although cartilage itself shows a very limited capacity for self-repair, pluripotent bone marrow-derived mesenchymal stem cells (BMSCs) are capable of complete cartilage regeneration (11–14). A role for BMSCs in this process is supported by evidence for spontaneous cartilage and bone regeneration following microfracture, a surgical treatment designed to increase the access of MSCs to cartilage defects (15). Results from a number of animal models indicate that transplanted BMSCs improve the repair of damaged cartilage, bone, tendon, and ligament in vivo (11,16–22). Thus, given appropriate environmental conditions, BMSCs can differentiate into chondrocytes, osteoblasts, and tendon or ligament fibroblasts (23–25). In vitro studies show that inducers of chondrocyte differentiation include the bone morphogenic proteins-2 and -6 (BMP-2, BMP-6) (26–28), the transforming growth factors beta-1 and beta-3 (TGF-ß1, TGF-ß3) (23), and fibroblast growth factor-2 (FGF-2) (29). One or more of these factors may play a role in stimulating cartilage regeneration in vivo.

Pluripotent BMSCs can be isolated from other cell types present in bone marrow aspirates on the basis of their adhesion to plastic culture dishes (23). However, clonal analysis has demonstrated that "plastic-selected" BMSCs are a far from homogenous population. BMSCs vary with respect to morphology (30,31), growth characteristics (32), ability to differentiate (33,34), and expression of cell surface antigens (35). BMSC isolates contain large, slowly replicating cells and small, rapidly growing cells, which retain multipotential differentiation status longer than do larger cells (36). There is evidence to suggest that some BMSCs express telomerase and that telomerase expression enhances osteogenic and chondrogenic differentiation (37,38). However, the fraction of telomerase-expressing cells appears to be species-specific, and telomerase activity may be very rare in human BMSCs compared to mouse BMSCs (39). Cells with different growth and differentiation characteristics have been separated from bone marrow isolates on the basis of immunoreactivity for antigens such as Stro-1 and CD105 (35,40,41). A number of studies suggest that aging impairs various aspects of mesenchymal stem cell function, but the impact of these changes on osteochondral repair remains unclear (42–45). Interestingly, the ratio of small to large cells in BMSC populations declines as the cells approach replicative senescence in culture (36). However, whether senescent stem cells accumulate with age in vivo is uncertain. Stenderup and colleagues (32) found that proliferation rate and population doubling limits of human BMSCs were significantly higher in young individuals (18–29 years) than in old individuals (aged 68–81 years), but there were no age-related differences in adipogenic or osteogenic potential (32). Additional studies show an age-related decline in the proportion of BMSCs capable of in vitro osteogenesis (33,43,44). D'Ippolito and colleagues (44) reported that alkaline phosphatase-positive colonies declined from 66 per million BMSCs in young patients (13–36 years old) to 15 per million cells in older patients (41–70 years old). In contrast, a recent in vivo study demonstrated that autologous bone marrow-derived stem cells harvested when rabbits were 1 year old were no better at repairing a patellar tendon injury than were cells isolated when the rabbits were 3 years older (45). Thus, although many in vitro studies support the hypothesis that aging effects on stem cells impair their wound healing function, the evidence for this impairment is still largely circumstantial.

Little information is available concerning the molecular and cellular changes that occur in stem cell populations during aging. Furthermore, most aging studies have focused on osteogenesis or adipogenesis as an outcome measure, thus the specific effects of age on chondrogenic differentiation have not been elucidated. To determine if aging affects stem cell chondrogenesis, we investigated the effects of age on the in vitro chondrogenic differentiation of rat BMSCs. BMSCs harvested from skeletally immature animals (1 week old), young mature animals (12 weeks old), and old mature animals (1 year old) were compared to study the effects of maturation and adult aging on the expression of cartilage-specific genes and on hyaline cartilage matrix formation.


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Isolation and Culture of BMSCs
BMSCs were isolated from 1-week-old, 12-week-old, and 1-year-old male Sprague–Dawley rats. Femurs and tibias were removed, and soft tissues were detached aseptically. Metaphyses were resected from both ends, and the diaphyses were flushed with Hank's Balanced Salt Solution. A suspension of bone marrow cells was obtained by repeated aspiration of the cell preparation through a 20-gauge needle. The cell suspension was centrifuged at 1000 g for 5 minutes, resuspended in BMSC growth medium (Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 2% penicillin and streptomycin), aliquoted into T-225 tissue culture flasks, and cultured at 37°C in a 5% CO2 atmosphere. Three days later, nonadherent cells were washed out by replacing the medium. The remaining adherent BMSCs were fed every 2–3 days until they reached confluence (usually 7–10 days). BMSCs were passaged with 0.025% trypsin-EDTA for 5 minutes at 37°C and replated in 100-mm culture dishes at a density of 5 x 105 cells per dish (for RNA extraction) or 2.5 x 106 cells per dish (for in vitro cartilage formation).

Each experiment was performed with BMSCs pooled from 3–5 rats for each age group. Chondrogenesis experiments were repeated 3 times with different batches of BMSCs isolated at different times.

Chondrogenic Induction
Twenty-four hours after the first passage, the BMSC growth medium was replaced by chondrogenic medium (Dulbecco's modified Eagle medium, pyruvate [1 mM], ITS-Premix [transferrin at 6.25 µg/mL, selenous acid, linoleic acid at 5.35 µg/mL, insulin, bovine serum albumin at 1.25 µg/mL; Invitrogen, Carlsbad, CA], 100 nM dexamethasone, ascorbate 2-phosphate at 37.5 µg/mL, TGF-ß1 at 10 ng/mL, insulin like growth factor [IGF]-I at 100 ng/mL). Thereafter, the chondrogenic medium was changed every other day. RNA was isolated after 7 days. For in vitro cartilage formation experiments, the cells were cultured for 3 weeks. The cells aggregated spontaneously into blocks 3 or 4 days after the first feeding of chondrogenic medium.

Total RNA Extraction
Total RNA was isolated by extraction of cultures with Trizol Reagent (Invitrogen, Carlsbad, CA) followed by purification with an RNeasy kit (Qiagen, Chatsworth, CA). The concentration of RNA was measured with an ultraviolet spectrometer.

Reverse Transcription–Polymerase Chain Reaction
Reverse transcription–polymerase chain reaction (RT–PCR) was used to assess expression of mesenchymal marker genes in BMSCs. Complementary DNA (cDNA) was synthesized and amplified using a commercial kit according to the manufacturer's recommendations (Invitrogen). Reverse transcription was first performed during a 30-minute incubation at 55°C, followed by a 2-minute incubation at 94°C to inactivate the reverse transcriptase. PCR amplification was performed in a 50 µL volume with 35 cycles of 94°C for 15 seconds, 58°C for 30 seconds (60°C for GADPH), and 70°C for 30 seconds (extended to 5 minutes in the last cycle). RT–PCR products were subjected to electrophoresis on 2% agarose gels and visualized with ethidium bromide. Gene names, accession numbers, associated primer sequences, and expected PCR product lengths are given in Table 1.


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  		Table 1. Oligonucleotide Primers for RT-PCR Analysis of Mesenchymal Cell Markers Gene Names, Accession Numbers, Associated Primer Sequences, and Expected PCR Product Lengths Are Shown.

 
Real-Time PCR Analysis
Three total RNA samples for each age group were analyzed to measure aging effects on chondrocyte marker gene expression. cDNA was synthesized as described above for RT–PCR. Oligonucleotide primers were designed for amplification of messenger RNA (mRNA) encoding GAPDH, cartilage-specific matrix proteins (collagen type II, aggrecan, link protein), and the chondrogenesis-related transcription factor, Sox9 (Table 2). Real-time PCR amplification was performed on an Applied Biosystems International (ABI, Foster City, CA) PRISM 7700 Sequence Detection System. Expression levels were determined using the relative threshold cycle (CT) method as described by the manufacturer of the detection system. Expression levels are stated in terms of fold increase or decrease relative to BMSCs from 1-year-old rats. This was calculated for each gene by evaluating the expression 2–{Delta}{Delta}CT, where {Delta}{Delta}CT is the result of subtracting [CTgene – CTGAPDH](1-year-old) from [CTgene – CTGAPDH](1-week-old) or [CTgene – CTGAPDH](12-week-old). The error bars given represent the standard deviation of the {Delta}{Delta}CT values. One-way analysis of variance (ANOVA) and the Tukey test were used to determine the statistical significance of age-related differences in the expression of each gene.


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  		Table 2. Oligonucleotide Primers for Real-Time Polymerase Chain Reaction Analysis of Cartilage Matrix Gene Expression.

 
Microarrays
Total RNA from three different rat BMSC isolates was pooled for each age group and submitted to the DNA Core Facility at the University of Iowa for microarray analysis. Affymetrix Rat Expression Set 230 GeneChip (Santa Clara, CA) microarrays containing more than 28,000 rat genes were used for analysis of global gene expression. Microarray assays were performed using procedures adapted from the Affymetrix GeneChip Expression Analysis Technical Manual. GeneChip one-cycle target labeling and control reagents kit (Affymetrix), which contained all the required reagents, was used. Briefly, 5 µg of total RNA was used to synthesize double-stranded cDNA using the One-Cycle cDNA Synthesis Kit. The DNA was purified by the Sample Cleanup Module (Affymetrix, Santa Clara, CA). In vitro transcription was performed to produce biotin-labeled copy RNA (cRNA) by using the IVT Labeling Kit (Affymetrix). Biotinylated cRNA was purified with the Sample Cleanup Module, fragmented to 50–200 nucleotides at 95°C in the presence of high magnesium concentration, and hybridized to Affymetrix Rat Expression Set 230 GeneChip at 45°C overnight (Hybridization Control Kit). After being washed, the array was stained with streptavidin–phycoerythrin. The staining signal was amplified by biotinylated antistreptavidin antibody, and final staining with streptavidin–phycoerythrin was performed on GeneChip fluidics station 450. Scans were performed with an Affymetrix GeneChip scanner 3000, and the expression value for each gene was calculated using Affymetrix Microarray Analysis Suite software. The expression data were analyzed by using GeneSpring software (Silicon Genetics, Santa Clara, CA).

Safranin-O Histology and Immunofluorescence
Aggregates were cryoembedded, sectioned, and fixed in 2% paraformaldehyde for histochemical and immunofluorescence staining. Some sections were stained with safranin-O to detect sulfated glycosaminoglycans in cell aggregates. The percentage of the cells that were stained by safranin-O was determined by manually counting cells in microscope fields representing.024 mm2 areas (0.6 mm w x 0.4 mm h) of aggregate cross-sections (2–3 mm diameter). Cells were counted as positive if the hematoxylin-stained nucleus was within a cell diameter of safranin-O-stained matrix. No attempt was made to distinguish between different stain intensities. Each section was sampled by taking images at six locations beginning at the outer edge of a section and passing through its middle. The means and standard deviations for each of the age groups were calculated based on a total of three sections (18 microscope fields) from three different aggregates. The results were analyzed by one-way ANOVA on ranks and Dunn's test to determine the significance of differences among the age groups (p <.05). The expression of the cartilage matrix proteins was detected using monoclonal antibodies to type II collagen (II-II6B3), aggrecan (12/21/1-C-6), and link protein (9/30/8-A-4) (Developmental Studies Hybridoma Bank, Iowa City, IA). Reduction and alkylation were performed for antiaggrecan and antilink protein staining. Briefly, sections were reduced in dithiothreitol at 10 mg/mL in 0.1 M Tris ph 8.0 for 2 hours at room temperature, then washed twice with 0.1 M Tris. Sections were then incubated for 20 minutes in iodoacetic acid at 60 mg/mL (in 0.1 M Tris ph 8.0), followed by two washes with phosphate-buffered saline [PBS]). Sections were blocked for 1 hour with PBS containing 0.1% Tween-20 and 1% bovine serum albumin, then incubated for 2 hours in undiluted hybridoma supernatants. Negative controls were incubated in PBS block (no primary antibody). Sections were washed in PBS containing 0.1% Tween-20 (PBST) and blocked for 30 minutes in PBST containing 10% goat serum. A Cy-3–conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA) was diluted 1:250 in PBST + 10% goat serum and applied to the sections. After a 1-hour incubation, the sections were washed in PBST and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Stained sections were imaged on an Olympus BX60 epifluorescence microscope.


   RESULTS
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Mesenchymal Cell Marker Expression in Rat Bone Marrow Cells
Prior to chondrogenic induction, BMSCs from one isolate were subjected to RT–PCR analysis for genes associated with mesenchymal cells (c-Kit, CD105, BMPR1a, and BMPr1b) (Figure 1). There were no obvious age-related differences in expression levels for CD105, BMPR1a, or BMPR1b. However, the level of c-Kit, a protein tyrosine kinase receptor for stem cell factor (46) appeared to decrease with increasing age. The hematopoietic stem cell marker Sca-1 (47) was undetectable in all age groups. These data indicated that the bone marrow cells from all age groups shared a common mesenchymal origin.


Figure 01
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  		Figure 1. Expression of mesenchymal markers in rat bone marrow-derived mesenchymal stem cells (BMSCs). A, Ethidium bromide–stained agarose gel shows reverse transcription–polymerase chain reaction products representing bone morphogenic protein receptors a and b (BMPR1a, BMPR1b), CD105, Sca-1, c-Kit, and GAPDH. RNA samples were obtained from cultures of BMSCs from 1-week-old (1w), 12-week-old (12w), or 1-year-old (1y) rats. MW = 100 bp molecular weight ladders. B, Densitometric analysis of the gel in A. Intensities of the bands representing marker genes were normalized to GAPDH intensity (Relative Intensity)

 
Cell Morphology and Aggregate Formation
In serum-containing medium, BMSCs in first passaged monolayer culture displayed a fibroblast-like morphology, and there was no obvious age-related variation in cell shape or size (Figure 2A). When plated at low density in chondrogenic medium, BMSCs became more polygonal in shape after 2–3 days. Some cells formed cobblestone-like nodes reminiscent of those formed by primary chondrocytes (Figure 2B). When plated at high density in chondrogenic medium, BMSCs spontaneously formed large aggregates after 48–72 hours. Single aggregates were formed from entire monolayers, which rolled up from the edge of the culture dish and contracted to form irregularly shaped masses. After 10 days in chondrogenic medium, these initially loosely structured aggregates became firmer in texture and more spherical in shape (Figure 2C). This process occurred in high-density cultures of BMSCs regardless of the age of the rats from which they were isolated.


Figure 02
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  		Figure 2. Spontaneous aggregation of bone marrow-derived mesenchymal stem cells (BMSCs) in monolayer culture. A, Monolayer culture of BMSCs in serum-containing medium 2 days after the first passage of cells from primary culture. There were no apparent age-related differences in cell shape. Bar = 200 µm. B, Low-density monolayer culture after 3 days in serum-free chondrogenic medium. Micrograph shows the polygonal chondrocyte-like shape acquired by BMSCs exposed to chondrogenic medium. C, Low-magnification image of an aggregate formed by 1-week-old BMSCs after 10 days in chondrogenic medium. The original monolayer culture has contracted and consolidated into the single mass shown. This was the case for all aggregate cultures. The gross appearance of the aggregate is typical of aggregates formed by BMSCs from all ages. Bar = 2 mm

 
Cartilage Matrix Formation In Vitro
BMSC aggregates were cultured in chondrogenic medium for 2–3 weeks for histologic and biochemical evaluation of cartilage ECM formation (Figure 3). Safranin-O staining revealed extensive proteoglycan accumulation in aggregates formed by BMSCs from 1-week-old rats (Figure 3A). This accumulation was particularly evident at the center of the aggregates where the spaces between cells were heavily stained (Figure 3D). In contrast, sections from aggregates composed of BMSCs from 12-week-old or 1-year-old rats were largely safranin-O negative (Figure 3B, E, C, and F). The numbers of BMSCs that stained with safranin-O were counted in sections of aggregates (Figure 3G). These data indicated a significantly higher percentage of positive cells in aggregates from 1-week-old rat BMSCs than in aggregates from adult rat BMSCs (>12-week-old, >1-year-old rats; p >.05). The slight difference between BMSCs from 12-week-old and 1-year-old adult rats was not statistically significant.


Figure 03
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  		Figure 3. Effects of age on cartilage matrix formation in aggregate cultures. Low-magnification images show the overall distribution of safranin-O staining in sections of aggregate cultures after 2 weeks in chondrogenic medium. Bone marrow-derived mesenchymal stem cells (BMSCs) were obtained from 1-week-old (A), 12-week-old (B), and 1-year-old (C) rats. Bar in A = 500 µm. High-magnification images show cell morphology and pericellular safranin-O staining in BMSCs from 1-week-old (D), 12-week-old (E), and 1-year-old (F) rats. Bar in D = 50 µm. Histogram shows the results of image analysis to determine the percentage of safranin-O–positive cells in different BMSC cultures (G). Columns show means for each age based on analysis of at least three sections from three different aggregate cultures (n = 9). Error bars indicate standard deviations. Bars and asterisks show statistically significant differences in positive cell counts (p <.05)

 
Replicate frozen sections from aggregate cultures were stained for collagen type II, aggrecan, and cartilage link protein using immunofluorescence (Figure 4). All three of these proteins were easily detected by this method in aggregates formed by BMSCs from 1-week-old rats (Figure 4A, D, and G) but not in aggregates formed by BMSCs from 12-week-old rats (Figure 4B, E, and H) or by BMSCs from 1-year-old rats (Figure 4C, F, and I). These findings were consistent with the safranin-O stains, which showed that BMSCs from only 1-week-old rats were capable of building a cartilage-like ECM under these conditions.


Figure 04
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  		Figure 4. Immunofluorescence staining for cartilage matrix proteins in aggregate cultures. Results are shown for cartilage link protein (A–C), type II collagen (D–F), and aggrecan (G–I). First column: staining on a culture from 1-week-old rats (A,D, and G); second column: staining on a culture from 12-week-old rats; third column: staining on a culture from 1-year-old rats. All micrographs are the same magnification. Bar = 200 µm

 
Chondrocyte Marker Gene Expression
Real-time PCR revealed significant age-related differences in the expression of genes encoding the cartilage matrix protein type II collagen, aggrecan, and link protein, and the chondrogenic transcription factor, Sox9 (Figure 5). Expression levels for all three matrix proteins were lowest in BMSCs from 1-year-old rats. Type II collagen gene expression was similar in BMSCs from 1-week-old and 12-week-old rats, but mean expression levels in both these groups were more than twice that measured in BMSCs from 1-year-old rats. Aggrecan expression peaked in BMSCs from 1-week-old rats and declined significantly thereafter: Expression in BMSCs from 1-week-old animals was nearly 4-fold greater than in BMSCs from 12-week-old animals and 9-fold greater than in BMSCs from 1-year-old animals. In contrast, link protein expression peaked in the 12-week-old age group: Expression in these BMSCs was more than 7-fold greater than in BMSCs from 1-week-old rats, and more than 14-fold greater than in BMSCs from 1-year-old rats. Sox9 expression also varied in an age-related manner, but levels were lowest in BMSCs from 1-week-old rats.


Figure 05
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  		Figure 5. Effects of age on chondrocyte marker gene expression by rat bone marrow-derived mesenchymal stem cells (BMSCs). Real-time polymerase chain reaction analysis for expression of type II collagen (A), aggrecan (B), link protein (C), and Sox9 (D). Columns show mean {Delta}{Delta}Ct values, which indicate expression levels in BMSCs from 1-week-old and 12-week-old rats relative to BMSCs from 1-year-old rats. Error bars indicate standard deviations

 
Microarray Analysis
Total RNA from BMSCs cultured for 1 week in chondrogenic medium was used for microarray hybridization analysis. The results showed differences in the expression of a variety of genes that may have affected in vitro cartilage ECM formation (Table 3). Age-related variations in the pattern of cartilage ECM gene expression were apparent (Table 3). The highest hybridization signal intensities for aggrecan and type II collagen were associated with BMSCs from 1-week-old rats, and the highest signal intensities for link protein were in BMSCs from 12-week-old rats. These results indicated a rank ordering of age groups similar to that indicated by PCR analysis. However, microarray and PCR results differed in terms of the apparent magnitudes of age-related changes in type II collagen and aggrecan gene expression. Signal intensities for type II collagen were approximately 2-fold greater in BMSCs from 1-week-old rats than in either adult group of BMSCs, whereas the difference between 1 week and 12 weeks measured by PCR was minimal. The signal intensity for aggrecan in BMSCS from 1-week-old rats was 2.9-fold higher than in BMSCs from 12-weeks-old rats, and 7.8-fold greater than in 1-year-old rats (as opposed to ~ 4-fold and > 9-fold, respectively, as measured by PCR).


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  		Table 3. Microarray Analysis of Aging Effects on BMSC Gene Expression Results are Shown for BMSCs from 1 Week-Old (1W), 12-Week-Old (12W), or 1-Year-Old (1Y) Rats, and Are Reported in Terms of Signal Intensities After Gene Chip Normalization.

 
A number of other cartilage-related genes were included in the analysis. Expression of cartilage oligomeric matrix protein (Comp) was 1.5-fold higher in BMSCs from young, 1-week-old rats compared to 12-week-old rats and 2.8-fold higher than in BMSCs from 1-year-old rats. Collagen type IX peptide expression ({alpha}-1 and {alpha}-3) declined by 2-6-fold in BMSCs from 12-week-old and 1-year-old rats compared with BMSCs from 1-week-old rats. No aging effects were observed for type XI collagen expression, which was expressed at the same high level in all three age groups. Type X collagen expression was slightly higher (1.5-fold) in BMSCs from 1-week-old rats than in BMSCs from adult animals.

There was a striking age-related rise in the expression of decorin, which was 10-fold lower in BMSCs from 1-week-old rats than in BMSCs from 12-week-old rats or 1-year-old rats. Biglycan mRNA levels, in contrast, decreased slightly with maturation. BMSCs from all age groups expressed noncartilage-specific ECM proteins (collagen types I, III, V, VII, and XII and fibronectin) at high levels, indicating incomplete differentiation to the chondrocyte phenotype even in cultures of BMSCs from 1-week-old rats.

Analysis of growth factor genes showed several significant age-related differences in expression (Table 3B). Of the bone morphogenic proteins, BMP-2 and BMP-6 were the most highly expressed in BMSCs from all age groups. Whereas BMP-2 expression was similar among the different ages, there was a slight (< 2-fold) increase in signal intensity for BMP-6 in BMSCs from 1-week-old compared to BMSCs from 1-year-old rats or 12-week-old rats. Growth and differentiation factors 1 and 15 were expressed at higher levels in BMSCs from 1-week-old rats than in BMSCs from either adult age group, whereas stem cell growth factor was expressed at higher levels in BMSCs from 12-week-old and 1-year-old rats than in BMSCs from 1-week-old rats. mRNA encoding IGF-I and its receptor were slightly more abundant in BMSCs from 1-week-old rats than in BMSCs from adult rats. The IGF binding protein IGFBP-5 was markedly more abundant in BMSCs from 12-week-old and 1-year-old rats than in BMSCs from 1-week-old rats. No consistent age-related patterns of expression were observed for connective tissue growth-related peptide (CTGRP); epidermal growth factor (EGF); FGF-1 and -2; TGF-ß-1, -2, and -3; platelet-derived growth factor (PDGF-{alpha}); or vascular endothelial factor (VEGF). Receptors for most of these factors were also expressed at similar levels in all age groups with the exception of the FGF-2 receptor, which was expressed at a 5-fold greater level in BMSCs from 12-week-old rats than in BMSCs from 1-week-old rats. This FGF receptor was also expressed at relatively high levels in BMSCs from 1-year-old rats (3-fold > 1-week-old). There were no striking age-related differences in signal intensity for the major catabolic cytokines interleukin-1ß and tumor necrosis factor-{alpha}, which were expressed at low levels by all BMSCs. However, expression of the inducible nitric oxide synthase gene, a mediator of interleukin-1ß effects, was markedly higher in BMSCs from 12-week-old and 1-year-old rats than in BMSCs from 1-week-old rats.

Some age-related differences were observed in the expression of genes encoding ECM proteases that are known to affect proteoglycan and collagen integrity and stability (Table 3). mRNA levels for several secreted matrix metalloproteinases (MMP-2, MMP-3, MMP-13), and the tissue metalloproteinase, disintegrin, and cysteine-rich protease-I (tMDCI) were slightly higher in BMSCs from mature rats than in BMSCs from immature rats. Expression of the plasminogen activator and urokinase receptor Plaur, which controls extracellular serine protease activity, was slightly greater in BMSCs from 12-week-old rats than in BMSCs from 1-week-old rats, but was 4-fold greater in BMSCs from 1-year-old rats than in BMSCs from 1-week-old rats. These findings suggest that ECM accumulation in cultures of adult cells might be inhibited by the degradation of secreted proteins caused by excess collagenase and aggrecanase activities.


   DISCUSSION
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the present study we found strong evidence for aging effects on the in vitro chondrogenic potential of rat BMSCs. The ability of BMSCs to produce a hyaline cartilage-like ECM in high-density aggregate cultures was used as the primary measure of chondrogenic potential. ECM accumulation over 2 weeks in culture was assessed by histologic analysis with safranin-O, a stain for cartilage proteoglycans. Positive safranin-O staining signifies a complex and ordered process of ECM development because proteoglycan accumulation under these conditions depends on coordinate expression of link protein, collagens, hyaluronan, and a number of accessory proteins and processing enzymes.

Examination of safranin-O-stained sections from aggregate cultures revealed that chondrogenic culture conditions induced BMSCs from 1-week-old rats to produce a hyaline cartilage-like ECM, whereas the same conditions failed to induce ECM development by BMSCs from 12-week-old or 1-year-old rats. Image analysis to determine the percentage of safranin-O–stained cells showed that nearly 85% of BMSCs from 1-week-old rats were positive whereas less than 3% of BMSCs from 12-week-old or 1-year-old rats were positive. Although there were significantly higher numbers of positive cells in cultures from 1-week-old rats, there was a still a substantial proportion of unstained cells (~15%) on the outer edges of the aggregates. It has been suggested that this lack of staining is due to differences in oxygen tension or cell density at the periphery versus the interior of three-dimensional BMSC cultures (23,24).

Immunofluorescence staining confirmed abundant proteoglycan (aggrecan), link protein, and type II collagen in 1-week-old cultures, but these cartilage markers were barely detectable in 12-week-old and 1-year-old cultures. Similar results were obtained when the incubation in chondrogenic medium was extended from 2 weeks to 6 weeks, indicating that the failure of adult cells to establish a matrix was not attributable simply to a lower rate of matrix synthesis (data not shown).

Real-time PCR analysis showed that aggrecan and type II collagen were expressed at the highest levels in BMSCs from 1-week-old rats and declined significantly with maturation and aging. However, link protein and Sox9 were expressed at higher levels in BMSCs from 12-week-old rats than in BMSCs from either 1-week-old or 1-year-old rats. Taken together, these findings demonstrate that (at the RNA level) cartilage ECM protein expression changes with maturation and aging but is never entirely lost. This finding would seem to be at odds with the apparent absence of cartilage ECM proteins in aggregate cultures of BMSCs from 12-week-old and 1-year-old rats as determined by histologic analysis. This disparity might be explained by the fact that RNA was extracted from BMSCs in monolayer culture, whereas protein analysis was performed on aggregates. Gene expression in BMSCs is likely to be affected by changes in cell shape and other differences inherent in the two culture conditions. The results might also be explained by failures in any of the many posttranscriptional and posttranslational processing steps required for proper ECM assembly. Thus, despite evidence of appreciable aggrecan, link protein, and type II collagen expression at the RNA level, the assembly of these molecules into a stable ECM cannot be taken for granted. The failure to accumulate proteoglycans in culture could also be related to excessive extracellular protease activities. Such enzymes are secreted by chondrocytes and have been shown to play a role in normal cartilage ECM turnover. However, excessive extracellular proteinase activity, such as occurs in degenerative joint disease, causes rapid ECM degradation and depletion even with high rates of synthesis and secretion. This suggests excessive ECM proteolysis could also play a role in the failure of BMSCs from adult rats to form cartilage ECM.

RT–PCR was used to determine if the BMSC populations were similar with respect to expression of stem cell markers. These analyses showed minimal age-related differences in the expression of BMP receptors 1a and 1b, or CD105, genes associated with osteo- and chondro- progenitors in BMSC populations (40,41). However, levels of c-Kit, a protein tyrosine kinase receptor for stem cell factor (46), appeared to decrease with increasing age, suggesting that this pathway might be less active in cells from older animals. As expected, we failed in multiple attempts to detect expression of the hematopoietic stem cell marker Sca-1 (47). These data confirmed that the cell populations used in these experiments shared a common mesenchymal origin regardless of the age of the donor animal.

Microarray analysis of chondrocyte-specific gene expression generally confirmed real-time PCR results and identified a number of other age-related differences in the expression of cartilage-specific genes. Among the many intriguing findings were the age-related declines in expression of the alpha 1 and 3 chains of collagen type IX, a minor collagen that plays an important role in the assembly of type II collagen fibrils (48). This decline, together with low type II collagen synthesis, might have led to deficient collagen fiber formation, a mechanism that could explain how, despite appreciable expression of aggrecan and link protein mRNAs by adult BMSCs, the proteins failed to accumulate around chondrocytes in aggregate cultures. Another relevant finding from microarray analyses involves the TGF-ß-inducible early response genes (TIEG), a subfamily of Sp1-like transcription factors that regulate the expression of tissue-specific genes (49). Our analysis showed that TIEG expression in rat BMSCs decreased with maturation from 1 week to 12 weeks by more than 50% but did not decline further with aging to 1 year. This result suggested that TIEG-dependent TGF-ß responses limited chondrogenesis in adult cells, a deficiency that might be overcome with higher TGF doses. However, we found no increased chondrogenic activity with TGF-ß1 up to 20 ng/mL, indicating that the amount of growth factor in the medium was not limiting (data not shown).

Another chondrogenic TGF superfamily member, BMP-6 (27,28), was expressed at higher levels in young rat BMSCs than in mature rat BMSCs, suggesting that age-related differences in the expression of factors secreted by BMSCs themselves might account for some of the loss of chondrogenic activity. The recent finding that FGF-2 increased the in vitro chondrogenic potential of adult stem cells suggests the growth factor milieu needed for efficient chondrogenesis may be more complex than previously thought (29).

The microarray findings also showed that type I and type III collagen were expressed at high levels by BMSCs from rats of all ages. The expression of these noncartilage-specific collagens, together with high levels of fibronectin expression, indicates that rat BMSCs had not undergone full conversion to a chondrocyte phenotype after 1 week under chondrogenic conditions. In fact, the mixed expression of these noncartilage-specific and cartilage-specific matrix proteins suggests an intermediate phenotype typical of that found in fibrocartilaginous tissues. It is unknown at this point whether noncartilage-specific gene expression declines with longer times in aggregate culture.

We acknowledge that our aggregate culture system differs in some details from many previously published micromass or pellet culture systems for chondrogenic induction. However, despite different starting conditions, the aggregate and conventional micromass or pellet approaches generate high-density, three-dimensional cultures that maintain cells in a rounded shape. Moreover, the spontaneous aggregate system has some advantage over micromass or pellet cultures, which are typically established using cells collected by trypsinization immediately prior to plating. This stressful and potentially damaging step is avoided in the aggregate system, in which a three-dimensional culture is formed by the cells with no manipulation other than feeding in serum-free chondrogenic medium. Finally, we observe that monolayer-cultured BMSCs from each age group readily and consistently formed aggregates, and that the aggregates they formed had similar cell densities. We believe that this behavior indicated that there was no age-related deficit in the ability to form aggregates that could explain the deficits in subsequent matrix production.

Our in vitro findings demonstrate an age-related loss of chondrogenic potency of BMSCs. However, the impact of these aging effects on the repair of osteochondral injuries in vivo remains to be proven. The age-related decline in osteochondral repair could be explained by age-related changes in the microenvironment encountered by stem cells at wound sites. For example, Kume and colleagues (50) found that glycation end products, which accumulate with aging in cartilage and other tissues, induce stem cell apoptosis and have an inhibitory effect on chondrogenesis. Another obvious possibility is age-related changes in the factors secreted by damaged tissues that could affect stem cell recruitment, proliferation, or differentiation independently of direct aging effects on stem cells. Although there is abundant in vitro evidence that stem cells are exquisitely sensitive to such environmental clues (51), there is as yet no direct experimental evidence for aging effects on injury-induced cytokine or growth factor levels in vivo. Nevertheless, this hypothesis must be tested before any firm conclusions can be drawn regarding the stem cell–specific effects of aging on osteochondral repair.

The culture system we used involves nonphysiologic conditions and potential stresses to cells that they would not encounter in vivo. Thus, it is possible that our results reflect age-related differences in the responses of cells to stressful culture conditions rather than to preexisting differences in gene expression patterns. Moreover, although we used a standard protocol for BMSC isolation and worked with early passaged cells, it is difficult to tell for certain whether the cells were representative of BMSC populations in vivo. Finally, though our in vitro study provided evidence for maturation and aging effects on the chondrogenic potential of rat BMSCs, due to the complexity of conditions in fractured joints we cannot conclude at present that such effects significantly impair cartilage regeneration in vivo. Despite these caveats, we believe that our findings justify further studies in an in vivo model to directly test the role of stem cell aging on cartilage repair and on long-term recovery from cartilage injury.


   Acknowledgments
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by award P50 AR48939 from the National Institutes of Health Specialized Center on Research for OA (http://poppy.obrl.uiowa.edu/Specialized Center of Research/SCOR.htm).


   Footnotes
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Decision Editor: Huber R. Warner, PhD

Received January 20, 2006

Accepted August 4, 2006


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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