• View in gallery

    Mean ± SD number of cells after MSC monolayer expansion for 6 days in cultures supplemented with 0 ng of FGF-2/mL (control treatment) or 100 ng of FGF-2/mL. Values did not differ significantly (P < 0.05).

  • View in gallery

    Mean ± SD DNA content in MSC pellets at 1, 7, and 14 days after initiation of monolayer expansion in medium supplemented with 0 ng of FGF-2/mL (control treatment [white bars]), 100 ng of FGF-2/mL (horizontal-striped bars), 10−7M dexamethasone (vertical-striped bars), or 100 ng of FGF-2/mL and 10−7M dexamethasone (black bars). *Within a treatment, value differs significantly (P < 0.05) from the value for day 1. †Within a day, value differs significantly (P < 0.05) from the value for the control treatment.

  • View in gallery

    Mean ± SD GAG content (A) and GAG content standardized on the basis of the total pellet DNA content (B) in MSC pellets at 1, 7, and 14 days after initiation of monolayer expansion with the control treatment, FGF-2, dexamethasone, or FGF-2–dexamethasone. *Standardized GAG content on days 7 and 14 differ significantly (P < 0.05) from the day 1 values (panel B); however, there was no significant difference among treatments.

    See Figure 2 for remainder of key for panel A.

  • View in gallery

    Mean ± SD collagen type II protein content (A) and collagen type II protein content standardized on the basis of the total pellet DNA content (B) in MSC pellets at 14 days after initiation of monolayer expansion with 0 ng of FGF-2/mL (control treatment), 100 ng of FGF-2/mL (FGF-2), 10−7M dexamethasone (DEX), or 100 ng of FGF-2/mL and 10−7M dexamethasone (FGF-2 + DEX). Notice that the scale on the y-axis differs between portions of the figure. *Value differs significantly (P < 0.05) from the value for the control treatment.

  • View in gallery

    Mean ± SD ALP activity (A) and ALP activity standardized on the basis of the total pellet DNA content (B) in MSC pellets at 14 days after initiation of monolayer expansion with the control treatment, FGF-2, dexamethasone, or FGF-2–dexamethasone. Notice that the scale on the y-axis differs between portions of the figure. See Figure 4 for remainder of key.

  • View in gallery

    Mean ± SD fold-difference in aggrecan mRNA (A), collagen type II mRNA (B), and ALP mRNA (C) in MSC pellets at 14 days after initiation of monolayer expansion with FGF-2, dexamethasone, or FGF-2–dexamethasone, compared with results for the control treatment. Results represent the fold-difference in mRNA after contents were standardized on the basis of mRNA expression of elongation factor-1α and logarithmically transformed. Notice that the scale on the y-axis differs among portions of the figure. See Figure 4 for remainder of key.

  • View in gallery

    Photomicrographs of sections of MSC pellets obtained on day 14 after initiation of monolayer expansion for MSC cells cultured in a control treatment (A), FGF-2 (B), dexamethasone (C), or FGF-2–dexamethasone (D). Toluidine blue stain; bar = 0.5 mm.

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Effect of dexamethasone supplementation on chondrogenesis of equine mesenchymal stem cells

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  • 1 Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.
  • | 2 Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.
  • | 3 Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.
  • | 4 Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

Abstract

Objective—To determine whether expansion of equine mesenchymal stem cells (MSCs) by use of fibroblast growth factor-2 (FGF-2) prior to supplementation with dexamethasone during the chondrogenic pellet culture phase would increase chondrocytic matrix markers without stimulating a hypertrophic chondrocytic phenotype.

Sample Population—MSCs obtained from 5 young horses.

Procedures—First-passage equine monolayer MSCs were supplemented with medium containing FGF-2 (0 or 100 ng/mL). Confluent MSCs were transferred to pellet cultures and maintained in chondrogenic medium containing 0 or 10−7M dexamethasone. Pellets were collected after 1, 7, and 14 days and analyzed for collagen type II protein content; total glycosaminoglycan content; total DNA content; alkaline phosphatase (ALP) activity; and mRNA of aggrecan, collagen type II, ALP, and elongation factor-1α.

Results—Treatment with FGF-2, dexamethasone, or both increased pellet collagen type II content, total glycosaminoglycan content, and mRNA expression of aggrecan. The DNA content of the MSC control pellets decreased over time. Treatment with FGF-2, dexamethasone, or both prevented the loss in pellet DNA content over time. Pellet ALP activity and mRNA were increased in MSCs treated with dexamethasone and FGF-2–dexamethasone. After pellet protein data were standardized on the basis of DNA content, only ALP activity of MSCs treated with FGF-2–dexamethasone remained significantly increased.

Conclusions and Clinical Relevance—Dexamethasone and FGF-2 enhanced chondrogenic differentiation of MSCs, primarily through an increase in MSC numbers. Treatment with dexamethasone stimulated ALP activity and ALP mRNA, consistent with the progression of cartilage toward bone. This may be important for MSC-based repair of articular cartilage.

Abstract

Objective—To determine whether expansion of equine mesenchymal stem cells (MSCs) by use of fibroblast growth factor-2 (FGF-2) prior to supplementation with dexamethasone during the chondrogenic pellet culture phase would increase chondrocytic matrix markers without stimulating a hypertrophic chondrocytic phenotype.

Sample Population—MSCs obtained from 5 young horses.

Procedures—First-passage equine monolayer MSCs were supplemented with medium containing FGF-2 (0 or 100 ng/mL). Confluent MSCs were transferred to pellet cultures and maintained in chondrogenic medium containing 0 or 10−7M dexamethasone. Pellets were collected after 1, 7, and 14 days and analyzed for collagen type II protein content; total glycosaminoglycan content; total DNA content; alkaline phosphatase (ALP) activity; and mRNA of aggrecan, collagen type II, ALP, and elongation factor-1α.

Results—Treatment with FGF-2, dexamethasone, or both increased pellet collagen type II content, total glycosaminoglycan content, and mRNA expression of aggrecan. The DNA content of the MSC control pellets decreased over time. Treatment with FGF-2, dexamethasone, or both prevented the loss in pellet DNA content over time. Pellet ALP activity and mRNA were increased in MSCs treated with dexamethasone and FGF-2–dexamethasone. After pellet protein data were standardized on the basis of DNA content, only ALP activity of MSCs treated with FGF-2–dexamethasone remained significantly increased.

Conclusions and Clinical Relevance—Dexamethasone and FGF-2 enhanced chondrogenic differentiation of MSCs, primarily through an increase in MSC numbers. Treatment with dexamethasone stimulated ALP activity and ALP mRNA, consistent with the progression of cartilage toward bone. This may be important for MSC-based repair of articular cartilage.

Arthritis is a major cause of reduced athletic performance, disability, and reduced quality of life in horses, dogs, and humans.1–6 The high incidence of osteoarthritis is attributable in part to the minimal intrinsic healing capacity of injured articular cartilage.7 Mesenchymal stem cells are pluripotential cells capable of differentiating into multiple types of connective tissue cells.8 Mesenchymal stem cells have been recognized as an attractive resource for the repair of musculoskeletal tissues, including articular cartilage.9–14 Postnatally derived MSCs are an autologous source of cells that can be harvested by aspiration without damage to an articular cartilage donor site.9,10,12,14 After isolation, MSCs can be expanded in culture with minimal loss of differentiation potential.15 Finally, when placed back into a patient, these cells typically suppress the host's immune response.16

Investigators have evaluated BM-MSCs for their chondrogenic potential in vitro.17–24 Despite the optimistic support for applications of stem cells in articular cartilage repair, no clear clinical benefit has been detected when BM-MSCs have been used for repair of cartilage defects in osteoarthritic knees of humans or femoropatellar joints of horses.10,14 Some of the aforementioned in vitro studies19,24 have indicated that BMMSCs stimulated to undergo chondrogenesis in pellet cultures can also develop a phenotype consistent with the hypertrophic lineage. Hypertrophic chondrocytes contribute to bone formation through endochondral ossification, which is in contrast to chondrocyte populations resident in permanent tissues such as articular cartilage. Clearly, more rigorous phenotypic control of MSC chondrogenic differentiation is warranted to optimize the use of MSCs for applications in joint resurfacing.9,10,12,14

Tissue engineering strategies that use MSCs for repair of articular cartilage attempt to recapitulate embryonic cartilage formation in a local, reparative context.11,25 From this perspective, the goal is to develop a system in which BM-MSCs develop into phenotypically stable hyaline cartilage without the endochondral progression to bone that is typical in the bulk of embryologically synthesized cartilage. In vitro MSC chondrogenesis is driven by culturing MSCs in high-density, 3-dimensional culture systems with TGF-β or a related bone morphogenetic protein.18–20,22,24 However, the specific stimuli that reliably confer a permanent chondrocytic phenotype in differentiating MSCs, as opposed to cartilage destined to be converted to bone, has yet to be determined.

Treatment of BM-MSCs with dexamethasone can stimulate cartilage and bone formation.8,19,24–29 However, this effect of dexamethasone varies considerably among species.26–29 For example, dexamethasone induces osteogenic differentiation of human BM-MSCs26,28; however, bone morphogenic protein-2, and not dexamethasone, is required for osteogenic differentiation of murine MSCs.26 Human BM-MSCs stimulated toward osteogenic cell lines actually required dexamethasone treatment to produce ALP, a hallmark for distinguishing osteogenic cells in culture.27,28 In another study,19 human BM-MSCs stimulated along chondrogenic cell lines and concurrently treated with TGF-β1 and dexamethasone, but not dexamethasone alone, had a significant increase in chondrogenesis and markers of bone formation.

The study reported here was performed to determine the effect of dexamethasone treatment on chondrogenesis of equine MSCs. Specifically, the study was designed to assess whether dexamethasone treatment during the 3-dimensional phase of chondrogenic differentiation increases expression of chondrogenic markers and biosynthesis of cartilaginous matrix components, compared with results for MSCs treated with TGF-β1 alone. In addition, phenotypic analyses were conducted to determine whether dexamethasone-treated equine BM-MSCs express an endochondral chondrocytic phenotype or markers consistent with a permanent or articular cartilage type. These issues were addressed in BM-MSC populations expanded in conventional serum-supplemented medium and in medium concurrently supplemented with 100 ng of FGF-2/mL, as determined on the basis of another study30 conducted by our laboratory group. Fibroblast growth factor-2 has been used in many MSC culture systems to stimulate proliferation31–36 and maintain self-renewal capacities of MSCs.32,33,36 In 2 studies,30,31 it was determined that FGF-2 administration during MSC monolayer expansion supports consequent chondrogenesis. Our hypothesis for the study reported here was that dexamethasone supplementation for differentiating MSCs during the 3-dimensional phase of culture would increase chondrogenesis but would lead to expression of an endochondral chondrocytic phenotype. In addition, we hypothesized that treatment with FGF-2 to induce expansion of equine BM-MSC monolayers followed by dexamethasone treatment of pellet cultures would also increase chondrogenesis without the expression of the endochondral phenotype.

Materials and Methods

Sample population—Bone marrow aspirates were obtained aseptically from the tuber coxae of 5 young (< 2 years of age) healthy horses. Bone marrow aspirates were obtained from horses donated to the University of Illinois or with consent of the owners before their horses were euthanatized. All bone marrow aspirates were obtained in accordance with guidelines reviewed and approved by the Institutional Animal Care and Use Committee of the University of Illinois.

Collection and preparation of samples—All horses were sedated with xylazine hydrochloride (0.5 to 1.0 mg/kg, IV). Horses were subsequently anesthetized by administration of ketamine hydrochloride (2.2 mg/kg, IV) and diazepam (0.1 mg/kg, IV), which were injected via a catheter placed in the left jugular vein. Anesthesia was maintained by IV administration of guaifenesin (5% solution/L) combined with 1,000 mg of ketamine and 1,000 mg of xylazine. The area over the right tuber coxa was clipped and aseptically prepared, and a bone marrow biopsy needlea was used to aspirate bone marrow into 30-mL syringes containing 1,000 units of heparin diluted to a volume of 5 mL by the addition of PBS solution. All bone marrow harvests were completed in < 30 minutes.

Thirty milliliters of bone marrow aspirate was diluted with 15 mL of PBS solution and centrifuged at 300 × g for 15 minutes. Supernatant was removed, the pellet was resuspended in PBS solution, and centrifugation was repeated. Pelleted cells were resuspended in 12 mL of low-glucose Dulbecco modified Eagle mediumb supplemented with 10% fetal bovine serum, 300 μg of L-glutamine/mL, 100 U of sodium penicillin/mL, 100 μg of streptomycin sulfate/mL, and 1 mmol sodium pyruvate/mL. Resuspended cells were placed in a 25-cm2 flask. Culture flasks were left undisturbed at 37°C in a 5% carbon dioxide atmosphere with 90% humidity for 5 days to allow cells to attach to the culture dishes.23,37,38 After the initial 5 days, medium was replaced every 2 to 3 days with 12 mL of fresh medium for 2 to 3 weeks until cells were near confluency.

Cell expansion—Nearly confluent monolayers of MSCs were treated with trypsin, resuspended, and plated at 18,000 cells/cm2 (450,000 cells/culture flask). These first-passage cells were supplemented with medium containing no FGF-2 (control culture) or 100 ng of FGF-2c/mL. Medium was changed every 2 to 3 days, and the appropriate FGF-2 supplementation was maintained. After expansion with FGF-2 for 6 days, confluent monolayers were treated with trypsin, counted, and transferred to pellet cultures containing 200,000 cells/pellet. Cell counts were performed as described elsewhere,23 and cell viability was determined by use of trypan blue dye exclusion.39,d

Cell differentiation—The MSCs were resuspended (400,000 cells/mL of basal chondrogenic medium). Basal chondrogenic medium consisted of high-glucose Dulbecco modified Eagle medium containing 5 ng of TGF-β1e/mL, 37.5 μg of ascorbic acid/mL, 1% insulin-transferrin-selenous acid supplement, 300 μg of L-glutamine/mL, 100 U of sodium penicillin/mL, and 100 μg of streptomycin sulfate/mL. Basal chondrogenic medium was supplemented with 0 or 10−7M dexamethasone.f Thus, there were 4 treatment groups (control treatment, FGF-2, dexamethasone, and FGF-2–dexamethasone). Suspended cells (200,000 cells in 0.5 mL of basal chondrogenic medium supplemented with 0 or 10−7M dexamethasone) were placed in a conical polypropylene microcentrifuge tubeg and centrifuged at 500 × g for 5 minutes to form a pellet. Pellet cultures were supplemented with chondrogenic medium containing 0 or 10−7M dexamethasone every 2 to 3 days. Each bone marrow aspirate yielded 68 pellets.

Twenty pellets were used for each treatment group. Three pellets were collected on each of days 1, 7, and 14 after initiation of monolayer expansion and stored at −80°C until used for measuring total pellet GAG and DNA contents. Six pellets were collected on day 14 and stored at −80°C until used for measuring collagen type II content and ALP activity. Three pellets were snap-frozen in liquid nitrogen and used for subsequent RNA analysis. Two pellets were placed in 4% paraformaldehyde and processed for histologic examination.

Total pellet DNA content—Total DNA content of each pellet was determined by fluorometric measurement of an incorporated dye,h as described elsewhere.40 All samples were assayed in duplicate, and a mean value was calculated to provide a single data point. Optical density values were converted to total DNA per pellet by reference to a standard curve generated by use of serial dilutions of calf thymus DNA.

Total pellet GAG content—Three pellets were digested in 0.1% papaini at 65°C for 16 hours.19,41 Dimethylmethylene blue binding assays were performed to measure sulfated GAG concentrations, which were compared against a standard curve generated by use of chondroitin sulfate.19 All samples were assayed in duplicate, and a mean value was calculated.

Total pellet collagen type II content—Collagen type II protein content in each pellet was determined by use of a commercially available ELISAj; the ELISA was used in accordance with the manufacturer's protocol. Three pellets from each treatment group were lyophilized and digested for collagen type II protein quantification.30 All samples were compared against a standard curve generated by use of a collagen type II standard. A mean value was calculated for each set of duplicates to provide 1 data point/aspirate.

Total pellet ALP activity—Activity of ALP was determined by use of a calorimetric kinetic assay with the substrate p-nitrophenyl phosphate from a commercially available kitk; the kit was used in accordance with the manufacturer's protocol. Three MSC pellets were homogenized in 300 μL of 0.2% Triton X-100 for sample preparation. All samples were assayed in duplicate and verified against a kit standard. A mean value was calculated for each set of duplicates to provide 1 data point/aspirate.

Pellet mRNA expression—Total RNA was extracted from 3 MSC pellets from each treatment group by use of a commercially available kit.l The mRNA in each sample was converted to cDNA by use of a commercially available reverse transcription kitm and oligo(dT) primers. The cDNA generated from the reverse transcription reactions was then amplified by a real-time PCR assay by use of Taq DNA polymerasen and gene-specific primers. Real-time quantitative PCR analysis was performed in duplicate for collagen II, aggrecan, and ALP; values for each were standardized on the basis of mRNA expression of elongation factor-1α.42,43 A PCR detection systemo was used to perform the assay.

Histologic examination—Pellets were kept in 4% paraformaldehyde for 24 hours. Then, the pellets were transferred to PBS solution and stored at 4°C. Pellets were dehydrated in alcohol, embedded in paraffin, sectioned, and stained with toluidine blue and H&E.

Statistical analysis—Mean values for monolayer-expanded cell numbers, pellet GAG content, pellet collagen type II content, pellet DNA content, and pellet ALP activity were calculated for each aspirate of all horses in the study. Aggrecan mRNA content, collagen type II mRNA content, and ALP mRNA content were standardized on the basis of expression of elongation factor-1α and logarithmically transformed prior to analysis. The effects of FGF-2, dexamethasone, or FGF-2–dexamethasone on these variables were evaluated by use of a 2-way ANOVA with aspirate as a covariate to control for the effects within and between horses. A 2-way ANOVA for repeated measures was used for analysis of pellet GAG and DNA contents on days 1, 7, and 14. Pairwise multiple comparisons by use of the Holm-Sidak method were used to evaluate differences in means to detect significant effects. Statistical analyses were performed by use of a commercial statistical program.p Values of P ≤ 0.05 were considered significant.

Results

Cell counts after monolayer expansion—Considerable variation was detected in cell numbers among aspirates and among horses. However, there was no overall significant (P = 0.844) effect on cell numbers with FGF-2 expansion of monolayer MSCs (Figure 1). Power for the performed t test was extremely low (0.20).

Figure 1—
Figure 1—

Mean ± SD number of cells after MSC monolayer expansion for 6 days in cultures supplemented with 0 ng of FGF-2/mL (control treatment) or 100 ng of FGF-2/mL. Values did not differ significantly (P < 0.05).

Citation: American Journal of Veterinary Research 69, 8; 10.2460/ajvr.69.8.1013

Total pellet DNA content—Mean ± SD total pellet DNA content did not differ significantly among treatment groups on day 1 (control treatment, 1.58 ± 0.46 μg; FGF-2, 1.51 ± 0.36 μg; dexamethasone, 1.41 ± 0.42 μg; and FGF-2–dexamethasone, 1.47 ± 0.35 μg; Figure 2). The power for the test performed to evaluate the interactions among treatments over days was 1.00. The control treatment in basal chondrogenic medium had a significant (P < 0.001) 1.9- to 2.0-fold decrease in pellet DNA content on days 7 (0.85 ± 0.57 μg) and 14 (0.81 ± 0.62 μg). Supplementation with FGF-2, dexamethasone, or FGF-2–dexamethasone ameliorated this decrease in DNA content. On day 7, pellet DNA content of the FGF-2–dexamethasone treatment (1.48 ± 0.67 μg) was significantly (P = 0.002) increased (1.7-fold increase), compared with the DNA content for the control treatment. On day 14, pellet DNA content of dexamethasone (1.47 ± 0.75 μg) and FGF-2–dexamethasone (1.67 ± 0.89 μg) treatments were significantly (P < 0.001) increased (1.8- to 2.1-fold increase), compared with the DNA content for the control treatment.

Figure 2—
Figure 2—

Mean ± SD DNA content in MSC pellets at 1, 7, and 14 days after initiation of monolayer expansion in medium supplemented with 0 ng of FGF-2/mL (control treatment [white bars]), 100 ng of FGF-2/mL (horizontal-striped bars), 10−7M dexamethasone (vertical-striped bars), or 100 ng of FGF-2/mL and 10−7M dexamethasone (black bars). *Within a treatment, value differs significantly (P < 0.05) from the value for day 1. †Within a day, value differs significantly (P < 0.05) from the value for the control treatment.

Citation: American Journal of Veterinary Research 69, 8; 10.2460/ajvr.69.8.1013

Total pellet GAG content—Mean ± SD total pellet GAG content did not differ among treatment groups on day 1 (control treatment, 2.84 ± 0.91 μg; FGF-2, 2.74 ± 0.53 μg; dexamethasone, 2.12 ± 0.98 μg; and FGF-2–dexamethasone, 2.36 ± 1.19 μg; Figure 3). Power for the test performed to evaluate the interactions among treatments over days was 0.97. The pellet GAG content in the control treatment remained unchanged over time, whereas treatment with FGF-2, dexamethasone, and FGF-2–dexamethasone caused a significant (P = 0.023) increase (1.4- to 1.7-fold increase) in pellet GAG content on days 7 (FGF-2, 3.94 ± 1.90 μg; dexamethasone, 3.42 ± 2.33 μg; and FGF-2–dexamethasone, 3.72 ± 1.86 μg) and 14 (FGF-2, 3.80 ± 2.06 μg; dexamethasone, 3.50 ± 2.39 μg; and FGF-2–dexamethasone, 3.71 ± 1.94 μg). On day 7, mean pellet GAG content of FGF-2 (3.94 ± 1.90 μg), dexamethasone (3.42 ± 2.33 μg), and FGF-2–dexamethasone (3.72 ± 1.86 μg) treatments were significantly (P = 0.009) increased (1.6- to 1.9-fold increase), compared with mean GAG content of the control treatment (2.12 ± 1.52 μg). On day 14, the pattern was similar to that on day 7, but only the FGF-2 treatment (3.80 ± 2.06 μg) resulted in a significant (P = 0.008) increase (1.5-fold increase) in pellet GAG content, compared with mean GAG content of the control treatment (2.49 ± 2.24 μg).

Figure 3—
Figure 3—

Mean ± SD GAG content (A) and GAG content standardized on the basis of the total pellet DNA content (B) in MSC pellets at 1, 7, and 14 days after initiation of monolayer expansion with the control treatment, FGF-2, dexamethasone, or FGF-2–dexamethasone. *Standardized GAG content on days 7 and 14 differ significantly (P < 0.05) from the day 1 values (panel B); however, there was no significant difference among treatments.

See Figure 2 for remainder of key for panel A.

Citation: American Journal of Veterinary Research 69, 8; 10.2460/ajvr.69.8.1013

When the total pellet GAG content was adjusted on the basis of DNA content of the pellet, there were no significant (P = 0.075) differences among treatment groups on the same day. However, there was a significant (P = 0.002) increase in adjusted pellet GAG content on days 7 (2.92 ± 1.84 μg) and 14 (2.82 ± 1.99 μg), compared with the adjusted pellet GAG content on day 1 (1.77 ± 0.91 μg; Figure 3).

Total pellet collagen II content—On day 14, mean ± SD total pellet collagen II protein content was significantly (P = 0.004) increased (2.2- to 2.5-fold increase) for treatments FGF-2 (0.031 ± 0.025 μg), dexamethasone (0.028 ± 0.024 μg), and FGF-2– dexamethasone (0.030 ± 0.026 μg), compared with content for the control treatment (0.013 ± 0.018 μg; Figure 4). Similar to the results for total pellet GAG content, there were no significant differences when pellet collagen type II content was adjusted on the basis of DNA content.

Figure 4—
Figure 4—

Mean ± SD collagen type II protein content (A) and collagen type II protein content standardized on the basis of the total pellet DNA content (B) in MSC pellets at 14 days after initiation of monolayer expansion with 0 ng of FGF-2/mL (control treatment), 100 ng of FGF-2/mL (FGF-2), 10−7M dexamethasone (DEX), or 100 ng of FGF-2/mL and 10−7M dexamethasone (FGF-2 + DEX). Notice that the scale on the y-axis differs between portions of the figure. *Value differs significantly (P < 0.05) from the value for the control treatment.

Citation: American Journal of Veterinary Research 69, 8; 10.2460/ajvr.69.8.1013

Total pellet ALP activity—On day 14, mean ± SD total pellet ALP activity was significantly (P < 0.001) increased (5.5- to 8.5-fold increase) for the dexamethasone (1.48 ± 1.06 mmol/L) and FGF-2–dexamethasone (2.31 ± 1.33 mmol/L) treatments, compared with ALP activity for the control treatment (0.27 ± 0.06 mmol/L; Figure 5). After ALP activity was adjusted on the basis of DNA content, treatment with FGF-2–dexamethasone (0.038 ± 0.023 mmol/L) still caused a significant (P = 0.004) increase (2.3-fold increase) in ALP activity, compared with the adjusted ALP activity for the control treatment (0.016 ± 0.015 mmol/L).

Figure 5—
Figure 5—

Mean ± SD ALP activity (A) and ALP activity standardized on the basis of the total pellet DNA content (B) in MSC pellets at 14 days after initiation of monolayer expansion with the control treatment, FGF-2, dexamethasone, or FGF-2–dexamethasone. Notice that the scale on the y-axis differs between portions of the figure. See Figure 4 for remainder of key.

Citation: American Journal of Veterinary Research 69, 8; 10.2460/ajvr.69.8.1013

Pellet mRNA expression—Real-time reverse-transcription PCR analysis revealed a significant (P = 0.04) increase in aggrecan mRNA expression for treatments FGF-2, dexamethasone, and FGF-2–dexamethasone, compared with aggrecan mRNA expression for the control treatment (Figure 6). Although the response for collagen type II mRNA expression had similar increases, the values did not differ significantly (P = 0.164) among treatments. Treatment with dexamethasone or FGF-2–dexamethasone significantly (P = 0.048) increased ALP mRNA expression, compared with ALP mRNA expression for the control treatment.

Figure 6—
Figure 6—

Mean ± SD fold-difference in aggrecan mRNA (A), collagen type II mRNA (B), and ALP mRNA (C) in MSC pellets at 14 days after initiation of monolayer expansion with FGF-2, dexamethasone, or FGF-2–dexamethasone, compared with results for the control treatment. Results represent the fold-difference in mRNA after contents were standardized on the basis of mRNA expression of elongation factor-1α and logarithmically transformed. Notice that the scale on the y-axis differs among portions of the figure. See Figure 4 for remainder of key.

Citation: American Journal of Veterinary Research 69, 8; 10.2460/ajvr.69.8.1013

Histologic examination—We detected small variations in pellet size, which were dependent on aspirate. We did not measure size of the pellets; instead, pellets were only evaluated for evidence of proteoglycan production by use of toluidine blue staining. Subjectively, pellets treated with FGF-2, dexamethasone, or FGF-2–dexamethasone had more proteoglycan staining throughout the pellet matrix (Figure 7).

Figure 7—
Figure 7—

Photomicrographs of sections of MSC pellets obtained on day 14 after initiation of monolayer expansion for MSC cells cultured in a control treatment (A), FGF-2 (B), dexamethasone (C), or FGF-2–dexamethasone (D). Toluidine blue stain; bar = 0.5 mm.

Citation: American Journal of Veterinary Research 69, 8; 10.2460/ajvr.69.8.1013

Discussion

Results of the study reported here were determined on the basis of total BM-MSC pellet content, which is consistent with tissue engineering goals whereby the composition and total tissue produced are important outcome variables. In this study, a set number of BMMSCs (200,000 cells/aspirate) were used for each pellet in the treatment groups at the onset of 3-dimensional chondrogenic differentiation. Despite having similar cell numbers at the onset of differentiation, supplementation with FGF-2 in monolayer phase and dexamethasone in the 3-dimensional pellet culture phase or dexamethasone alone had substantial benefits on maintaining DNA content and enhancing the chondrogenic differentiation of the cells.

Similar to results of another study30 conducted by our laboratory group that involved the use of equine BM-MSCs, treatment with FGF-2 in the study reported here did not increase the cell numbers during monolayer expansion. This may be an equine-specific response and is in contrast to results for studies31,35 in other species that have revealed FGF-2 expansion increases numbers of BM-MSCs. In the study reported here, we did not evaluate expansion cell numbers for short time periods or by use of thymidine labeling, which has often been used in other studies31,35; thus, a difference in early replication prior to MSC monolayer confluence could have been missed. Alternatively, all MSC monolayers were supplemented with 10% serum and MSC expansion could have been maximally stimulated by the serum, which left minimal ability for FGF-2 to stimulate further enhancement of cell numbers. In addition, the power of our statistical analysis of cell number was extremely low (0.20) because of small differences in the overall means with large differences in SDs.

In this study, pellets were collected on days 1, 7, and 14 for measurement of DNA content to determine whether BM-MSCs increased in number or were lost over time in response to treatment. Analysis of these results revealed conclusively that the MSC pellets did not have an increase in DNA content once the 3-dimensional culture conditions were applied to BM-MSCs to induce chondrogenic differentiation. Instead, DNA content significantly decreased over time in the untreated control group. No other treatment group had a significant decrease in DNA content over time. In fact, treatment with dexamethasone and FGF-2–dexamethasone ameliorated this loss in DNA content during the pellet culture phase of the study. Total pellet GAG content reflects the sum of the anabolic and catabolic activity of the cells. Over time, the MSC pellets accumulated an increased amount of total matrix content despite little change in cell number. This was supported by histologic evaluation, in that our FGF-2–dexamethasone treatment had a high matrix-to-cellular ratio with increased amounts of toluidine blue stain. In essence, some of the matrix laid down during the early phase of pellet remodeling could have been protected to contribute to the increase in total pellet GAG content over time. Therefore, the increases in pellet content with dexamethasone or FGF-2–dexamethasone treatments could be attributed to differences in retention of the DNA content of the pellets as well as a gradual retention of GAG in the matrix over time. In addition, aggrecan mRNA was increased for the FGF-2, dexamethasone, or FGF-2–dexamethasone treatments. Results of this study and those of another study30 conducted by our laboratory group underline the importance of retaining the DNA content of the BM-MSCs for future matrix production in 3-dimensional systems.

In contrast to results of our previous study,30 FGF-2 supplementation alone during the monolayer phase was not sufficient to significantly increase the DNA content, compared with DNA content for the untreated control cells, during the pellet culture phase of the study reported here. However, in the present study, supplementation with FGF-2 did prevent a significant decrease in DNA content that was evident in the control samples. Differences between results of the 2 studies may have been attributable to an increase in comparison groups in the present study because FGF-2 had a slight but nonsignificant (P = 0.068) effect to increase the DNA content in the study reported here. Regardless, from a tissue engineering perspective, FGF-2 monolayer expansion of equine BM-MSCs in both studies had substantial postexpansion benefits by increasing the future chondrogenic matrix production of BM-MSCs in a 3-dimensional culture system. These beneficial postexpansion effects of FGF-2 may be extremely useful for tissue engineering protocols in which there is a minimal period of in vitro tissue culture.

The chondrogenic capacity of BM-MSCs was increased to a similar extent by supplementation with FGF-2, dexamethasone, or FGF-2–dexamethasone. This was evident by an increase in proteoglycan content of the pellet by day 7 and increases in proteoglycan content and collagen type II by day 14. Part of the increase in proteoglycans may have been mediated by an increase in mRNA expression, as was evident for aggrecan mRNA after treatment with FGF-2, dexamethasone, or FGF-2–dexamethasone. Although the increases in chondrogenic pellet matrix by dexamethasone and FGF-2–dexamethasone treatments could be partially attributed to retention in pellet DNA content, this was not as clear for treatment with FGF-2 alone. It is possible that the increase in collagen type II content and proteoglycan matrix production may have been partly attributed to the ability of FGF-2 to retain the MSCs self-renewal capacity and allow MSCs to remain undifferentiated during the monolayer expansion phase.31,32,35 Maintenance of these stem cell properties could have enhanced the ability of chondrogenic media to differentiate the population of MSCs to a chondrocytic phenotype.31 Other studies19,31 have revealed similar increases in proteoglycan and collagen type II production in human BM-MSC pellets treated with TGF-β1 and dexamethasone.

As mentioned, the production of high amounts of ALP stands out as a hallmark that distinguishes osteogenic cells in culture.44 The importance of ALP in bone formation is based on its ability to regulate mineralization in bone matrix.45 In the study reported here, only equine BM-MSCs treated with dexamethasone or FGF-2–dexamethasone had significant increases in ALP activity and ALP mRNA. When the BM-MSCs were adjusted on the basis of DNA content, only the combination of FGF-2–dexamethasone still had a significant increase in ALP activity. These findings are similar to those of studies26–29 of human MSCs in which dexamethasone treatment was required to stimulate ALP formation along osteogenic lineages. Our equine MSCs were similar to human BM-MSCs stimulated along chondrogenic cell lines, in that concurrent treatment with TGF-β1 and dexamethasone significantly increased chondrogenesis and ALP as a marker of bone formation in human BM-MSCs.19 Results of our present study and results of other studies26–28 suggest that treatment with dexamethasone may cause BM-MSCs to adopt a chondrogenic phenotype that is more likely to progress toward bone.

In the study reported here, bone marrow was harvested only from horses < 2 years of age. This age group is unlikely to represent the mean age of the population of horses that could benefit from cartilage repair. At this time, it is unknown whether these young horses will accurately represent a population of older horses. Additional studies will be necessary to determine whether the effects detected in this study are comparable to those involving BM-MSCs obtained from older horses. In this study, there was no functional testing to determine the mechanical capacity of the produced tissue and our outcome measures were fairly limited in scope and nature. Additional in-depth evaluations that use BM-MSCs from older horses would be necessary to evaluate the challenges of the use of BM-MSCs in repair of articular cartilage defects of mature horses.

After standardization of results on the basis of DNA content, only the FGF-2–dexamethasone treatment had a significant increase in ALP activity. This suggests a synergistic effect of the combination of FGF-2 and dexamethasone that was not evident with either treatment alone. In contrast to our hypothesis, FGF-2 expansion of MSCs actually increased markers of endochondral ossification (ie, ALP activity and ALP mRNA) with sequential dexamethasone treatment. In light of these findings, dexamethasone and FGF-2 may not be specific chondrogenic factors, as indicated by their ability to induce multiple phenotypes. On the basis of results for this and other studies,27,28,46 effects of dexamethasone are more likely to be mediated through cell preservation and upregulation of known chondrogenic factors, such as ALP, bone sialoprotein, Runx2, and Noggin. In contrast, expansion stimulated by FGF-2 may be more likely to be mediated through MSC preservation of self-renewal properties32,33,36 that allow an enhanced response to a subsequent differentiation stimulus.

In the study reported here, an obvious effect of dexamethasone supplementation during the 3-dimensional culture phase was to retain cellular viability of equine BM-MSCs. However, supplementation with dexamethasone also increased ALP activity and ALP mRNA expression in the pellets to be more consistent with those of a hypertrophic chondrocyte, rather than a phenotypically stable articular chondrocyte. This supported our hypothesis that supplementation with dexamethasone would stimulate an endochondral ossification phenotype. Supplementation with FGF-2 alone during the monolayer expansion phase significantly increased proteoglycan and collagen type II contents to amounts similar to those for dexamethasone supplementation but without any concurrent increases in ALP activity. Similarly, supplementation with FGF-2 during the monolayer phase and dexamethasone during the 3-dimensional phase of the study had substantial beneficial effects on the chondrogenic differentiation of the BM-MSC pellets. However, in contrast to our original hypothesis, it was determined that supplementation with FGF-2 during the monolayer phase did not prevent dexamethasone from stimulating equine BM-MSCs to develop an endochondral phenotype. On the basis of results of the study reported here and results of another study30 conducted by our laboratory group, supplementation of equine BM-MSC monolayers with FGF-2 may have substantial benefits for future tissue engineering of articular cartilage.

ABBREVIATIONS

ALP

Alkaline phosphatase

BM-MSC

Bone marrow–derived mesenchymal stem cell

FGF-2

Fibroblast growth factor-2

GAG

Glycosaminoglycan

TGF-β

Transforming growth factor-β

a.

Jamshidi bone marrow–biopsy needle, Cardinal Health, Dublin, Ohio.

b.

DMEM, Mediatech Inc, Herndon, Va.

c.

Fibroblast growth factor-2, R&D Systems Inc, Minneapolis, Minn.

d.

Trypan blue, Sigma Chemical Co, St Louis, Mo.

e.

Transforming growth factor-β1, R & D Systems, Minneapolis, Minn.

f.

Vedco Inc, St Joseph, Mo.

g.

Polypropylene conical tube, BD Biosciences, Franklin Lakes, NJ.

h.

Hoechst 33258, Sigma Chemical Co, St Louis, Mo.

i.

Papain, Sigma Chemical Co, St Louis, Mo.

j.

Native type II collagen detection kit, Chondrex Inc, Redmond, Wash.

k.

Quantichrom alkaline phosphatase assay kit, BioAssay Systems, Hayward, Calif.

l.

RNeasy Mini Kit, Qiagen, Valencia, Calif.

m.

Superscript II, Invitrogen, Carlsbad, Calif.

n.

iQ SYBR Green Supermix, Bio-Rad Laboratories, Hercules, Calif.

o.

iCycler iQ real-time PCR detection system, Bio-Rad Laboratories, Hercules, Calif.

p.

Sigma Stat, Systat Software Inc, San Jose, Calif. cine: cranial cruciate ligament injury repair in the dog. Vet Surg 2005;34:93–98.

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Contributor Notes

Supported by the Veterinary Orthopedic Society through the Hohn-Johnson Research Award.

Address correspondence to Dr. Stewart.